WorldWideScience

Sample records for light chain mutations

  1. Mutations in specific structural regions of immunoglobulin light chains are associated with free light chain levels in patients with AL amyloidosis.

    Directory of Open Access Journals (Sweden)

    Tanya L Poshusta

    Full Text Available BACKGROUND: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL, each patient has a unique monoclonal immunoglobulin light chain (LC that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains. METHODOLOGY/PRINCIPAL FINDINGS: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition. CONCLUSIONS: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.

  2. A Novel Myosin Essential Light Chain Mutation Causes Hypertrophic Cardiomyopathy with Late Onset and Low Expressivity

    Directory of Open Access Journals (Sweden)

    Paal Skytt Andersen

    2012-01-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is caused by mutations in genes encoding sarcomere proteins. Mutations in MYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT 21 mm, a resting left ventricular outflow gradient of 36 mm Hg, and left atrial dilation (54 mm. Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, in MYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion, MYL3 mutations can present with low expressivity and late onset.

  3. Differences in potential for amino acid change after mutation reveals distinct strategies for kappa and lambda light-chain variation.

    Science.gov (United States)

    Hershberg, Uri; Shlomchik, Mark J

    2006-10-24

    B cells generate varied yet functional clones under high rates of mutation of their V genes. It has been proposed that as a result of the opposing demands of diversification and preservation of integrity, the V genes of heavy and light chains have evolved to overexpress codons prone to amino acid change in their complementarity determining regions (CDR) compared with the framework (FW) regions. We have analyzed the germ-line V genes of heavy and light chains (both kappa and lambda), comparing codons of CDR and FW of the germ-line V regions both to each other and to control regions. We found that in both germ-line heavy chains and lambda chains, CDR codons are prone to replacement mutations, whereas in the FW, the opposite is true. Furthermore, the difference between CDR and FW in heavy chains and lambda chains is based on codons that are prone to nonconservative changes of amino acid. In contrast, in germ-line kappa chains, the codons in both CDR and FW are more prone to replacement mutations. We also demonstrated that negative selection during immune responses is more sensitive to nonconservative amino acid substitutions than overall amino acid change, demonstrating the applicability of our analysis to real-time process of selection in the immune system. The differences in germ-line kappa and lambda light chains' potential reaction to mutation suggests that via these two differently evolved light-chain types, the B cell repertoire encompasses two different strategies to balance diversity and stability in an immune response.

  4. Remodeling of the heart in hypertrophy in animal models with myosin essential light chain mutations

    Directory of Open Access Journals (Sweden)

    Katarzyna eKazmierczak

    2014-09-01

    Full Text Available Cardiac hypertrophy represents one of the most important cardiovascular problems yet the mechanisms responsible for hypertrophic remodeling of the heart are poorly understood. In this report we aimed to explore the molecular pathways leading to two different phenotypes of cardiac hypertrophy in transgenic mice carrying mutations in the human ventricular myosin essential light chain (ELC. Mutation-induced alterations in the heart structure and function were studied in two transgenic (Tg mouse models carrying the A57G (alanine to glycine substitution or lacking the N-terminal 43 amino acid residues (Δ43 from the ELC sequence. The first model represents an HCM disease as the A57G mutation was shown to cause malignant HCM outcomes in humans. The second mouse model is lacking the region of the ELC that was shown to be important for a direct interaction between the ELC and actin during muscle contraction. Our earlier studies demonstrated that >7 month old Tg-Δ43 mice developed substantial cardiac hypertrophy with no signs of histopathology or fibrosis. Tg mice did not show abnormal cardiac function compared to Tg-WT expressing the full length human ventricular ELC. Previously reported pathological morphology in Tg-A57G mice included extensive disorganization of myocytes and interstitial fibrosis with no abnormal increase in heart mass observed in >6 month-old animals. In this report we show that strenuous exercise can trigger hypertrophy and pathologic cardiac remodeling in Tg-A57G mice as early as 3 months of age. In contrast, no exercise-induced changes were noted for Tg-Δ43 hearts and the mice maintained a non-pathological cardiac phenotype. Based on our results, we suggest that exercise-elicited heart remodeling in Tg-A57G mice follows the pathological pathway leading to HCM, while it induces no abnormal response in Tg-Δ43 mice.

  5. Mutations of the Drosophila myosin regulatory light chain affect courtship song and reduce reproductive success.

    Science.gov (United States)

    Chakravorty, Samya; Vu, Hien; Foelber, Veronica; Vigoreaux, Jim O

    2014-01-01

    The Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2(Δ2-46) (Ext), an N-terminal extension truncation; (ii) Dmlc2(S66A,S67A) (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2(Δ2-46;S66A,S67A) (Dual), expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2 (+) rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight.

  6. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

    Science.gov (United States)

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J; Spears, Danna A; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R; Stainier, Didier Y R; Gollob, Michael H

    2016-04-12

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect.

  7. Serum Free Light Chains

    Science.gov (United States)

    ... or of one of its component parts – a kappa or lambda light chain, or rarely, a heavy chain. Traditionally, plasma cell ... protein (M-protein) production and to calculate a kappa/lambda free light chain ratio. If the protein electrophoresis test is abnormal, ...

  8. A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D; Ortiz, E; Rudiño-Piñera, E; Sánchez-López, R; Horjales, E; Becerril, B; Rodríguez-Romero, A

    2010-01-01

    Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.

  9. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    Science.gov (United States)

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  10. Light chain nephropathy

    Directory of Open Access Journals (Sweden)

    Sihem Darouich

    2015-01-01

    Full Text Available Light chain deposition disease (LCDD is characterized by the tissue deposition of monotypic immunoglobulin light chains of either kappa or lambda isotype. It is the archetypal systemic disease that is most frequently diagnosed on a kidney biopsy, although the deposits may involve several other organs. This brief review focuses on the clinicopathological features of LCDD-associated nephropathy with an emphasis on the diagnostic and therapeutic difficulties related to this elusive condition.

  11. Neurofilament light chain

    OpenAIRE

    Lu, CH; Macdonald-Wallis, C.; Gray, E; Pearce, N; Petzold, A; Norgren, N.; Giovannoni, G; Fratta, P.; Sidle, K.; Fish, M.; Orrell, R; Howard, R; Talbot, K.; Greensmith, L.; Kuhle, J

    2015-01-01

    OBJECTIVE: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). METHODS: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals...

  12. The normal counterpart of IgD myeloma cells in germinal center displays extensively mutated IgVH gene, Cmu-Cdelta switch, and lambda light chain expression.

    Science.gov (United States)

    Arpin, C; de Bouteiller, O; Razanajaona, D; Fugier-Vivier, I; Brière, F; Banchereau, J; Lebecque, S; Liu, Y J

    1998-04-20

    Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre-B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased lambda light chain expression and a Cmu-Cdelta isotype switch. Using surface markers, we have previously isolated a population of surface IgM-IgD+CD38+ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased lambda light chain expression and a C&mu-Cdelta isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.

  13. Neurofilament light chain

    Science.gov (United States)

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens

    2015-01-01

    Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98–7.94, p NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. PMID:25934855

  14. Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

    Science.gov (United States)

    Tey, Shelisa; Ahmad-Annuar, Azlina; Drew, Alexander P; Shahrizaila, Nortina; Nicholson, Garth A; Kennerson, Marina L

    2014-10-01

    The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

  15. Mutations induced by ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Pfeifer, Gerd P. [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)]. E-mail: gpfeifer@coh.org; You, Young-Hyun [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Besaratinia, Ahmad [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)

    2005-04-01

    The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms

  16. The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

    DEFF Research Database (Denmark)

    Juul, L; Hougs, L; Andersen, V

    1997-01-01

    The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were...

  17. Light chain immunofluorescence in various nephropathies

    Directory of Open Access Journals (Sweden)

    Megha S Uppin

    2011-01-01

    Full Text Available Context: Light chain immunofluoresence (IF in renal biopsy is routinely used in the diagnosis of light chain deposition disease (LCDD, amyloidosis and cast nephropathy. Light chain predominance has also been reported in certain glomerulopathies like IgA nephropathy. However, pathogenesis of this pattern of deposition in various glomerulopathies is uncertain. Aim: To discuss the pathogenesis and utility of light chain IF in nephropathies. Setting and Design: Retrospective study. Materials and Methods: The pattern of light chain IF and light microscopic diagnosis in 306 cases of various nephropathies was reviewed. Direct IF was done in all these cases with commercial fluorescence (Fluoresciene Isothiocynate conjugated polyclonal rabbit anti-human antisera against IgM, IgG, IgA, C3, C1q, kappa and lambda light chains. Results: Light chain deposits were seen in 240 (78.43% cases. In IgA nephropathy, lupus nephritis and post-infectious glomerulonephritis (PIGN, lambda positivity was more as compared to kappa. Light chain deposits in LCDD and membranous nephropathy were more kappa type. The IF pattern in amyloidosis was not consistent. Conclusion: The pathogenesis of light chain predominance in glomerulopathies is not clear and it depends on isoelectric point and size of the immune complex. Light chain IF should be performed routinely in all the renal biopsies.

  18. Lymphoplasmacytic lymphoma causing light chain cast nephropathy.

    Science.gov (United States)

    Pérez, Nuria S; Garcia-Herrera, Adriana; Rosiñol, Laura; Palos, Lily; Santiago, Evelyn; Espinosa, Gerard; Solé, Manel; Campistol, Josep M; Quintana, Luis F

    2012-01-01

    Plasma cell dyscrasias are frequently associated with kidney disease through the production of monoclonal immunoglobulin but with a diverse set of pathologic renal patterns. While almost all patients with a renal biopsy showing a cast nephropathy have myeloma, kidney involvement associated with pathological immunoglobulin light chains and lymphoma is rare. To our knowledge, this is the first report of a cast nephropathy associated with lymphoplasmacytic lymphoma. We emphasize the relation between light chain deposition and renal dysfunction in this disease with production of light chains. A therapeutic approach that decreases light chain production appears to be warranted in these patients.

  19. Localized Lymph Node Light Chain Amyloidosis

    Directory of Open Access Journals (Sweden)

    Binod Dhakal

    2015-01-01

    Full Text Available Immunoglobulin-derived light chain amyloidosis can occasionally be associated with localized disease. We present a patient with localized lymph node light chain amyloidosis without an underlying monoclonal protein or lymphoproliferative disorder and review the literature of lymph node amyloidosis discussing work-up and risk factors for systemic progression.

  20. The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

    DEFF Research Database (Denmark)

    Juul, L; Hougs, L; Andersen, V

    1997-01-01

    The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were....... V genes from the Jkappa-proximal duplication unit of the kappa locus were almost exclusively used. A total of 65% of the sequences could be assigned to four or five genes: A27 (humkv325), L6 (Vg), L2 (humkv328), and A3 and/or A19. N additions and P nucleotides were quite common and found in 32...

  1. Polyclonal light chains in cerebrovascular disease

    Directory of Open Access Journals (Sweden)

    Patrizia Fiori

    2010-08-01

    Full Text Available Patrizia Fiori1, Maria Giannetti Luigi2, Linda Iurato1, Carminantonio Tammaro3, Gigliola Esposito3, Antonio Monaco11Central Operative Unit of Neurology (Dir. A Monaco, 2Infantile Neuropsychiatry and Social Service (Dir. LM Giannetti, 3Laboratory (Dir. CA Tammaro, ASL AV, Civil Hospital of Ariano Irpino, University of Naples, ItalyAbstract: Altered membrane permeability is a hallmark of inflammation and ischemia with systemic spreading. Renal dysfunction is a risk factor for cardiovascular, cerebrovascular, and metabolic diseases. The aim of the present study was to assess proteinuria and urinary polyclonal light chains in acute stroke and chronic cerebrovascular disease compared with other neurologic diseases. Our results showed significantly increased levels of urinary polyclonal light chains in cerebrovascular disease compared with other neurologic diseases. The highest values of urinary polyclonal κ chains were found in acute stroke compared with chronic cerebrovascular disease and other neurologic diseases, while the level of λ chains was mainly increased in chronic cerebrovascular diseases. The shift to chronic renal failure seems to be signaled by a decreased polyclonal light chain/creatinemia ratio. The absence of a significant correlation with blood pressure and other seric parameters suggests that polyclonal light chains are an early marker of reversible vascular impairment with renal dysfunction before progression to irreversible renal failure and need for dialysis and/or intensive care.Keywords: polyclonal light chains, cerebrovascular disease, renal failure

  2. Markov chain for estimating human mitochondrial DNA mutation pattern

    Science.gov (United States)

    Vantika, Sandy; Pasaribu, Udjianna S.

    2015-12-01

    The Markov chain was proposed to estimate the human mitochondrial DNA mutation pattern. One DNA sequence was taken randomly from 100 sequences in Genbank. The nucleotide transition matrix and mutation transition matrix were estimated from this sequence. We determined whether the states (mutation/normal) are recurrent or transient. The results showed that both of them are recurrent.

  3. Structural repertoire of immunoglobulin λ light chains

    KAUST Repository

    Chailyan, Anna

    2011-03-01

    The immunoglobulin λ isotype is present in nearly all vertebrates and plays an important role in the human immune system. Despite its importance, few systematic studies have been performed to analyze the structural conformation of its variable regions, contrary to what is the case for κ and heavy chains. We show here that an analysis of the structures of λ chains allows the definition of a discrete set of recurring conformations (canonical structures) of their hypervariable loops and, most importantly, the identification of sequence constraints that can be used to predict their structure. We also show that the structural repertoire of λ chains is different and more varied than that of the κ chains, consistently with the current view of the involvement of the two major light-chain families in complementary strategies of the immune system to ensure a fine tuning between diversity and stability in antigen recognition. © 2011 Wiley-Liss, Inc.

  4. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Wall, Jonathan S. [Departments of Radiology and Medicine, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN (United States); González Andrade, Martín [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Sánchez-López, Rosana [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos C.P. 62210 (Mexico); Rodríguez-Ambriz, Sandra L. [Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos C.P. 62731 (Mexico); Pérez Carreón, Julio I. [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  5. Systemic Light Chain Amyloidosis Mimicking Rheumatic Disorders

    Science.gov (United States)

    2016-01-01

    Secondary amyloidosis can complicate chronic inflammatory autoimmune diseases. However, the clinical findings of primary amyloidosis may mimic those of primary rheumatologic disorders. We present the case of a 53-year-old woman who presented with dystrophic nail changes, dry eyes, bilateral carpal tunnel syndrome, Raynaud's phenomenon, and high titer positive nucleolar pattern antinuclear antibody. She was initially misdiagnosed as having Undifferentiated Connective Tissue Disease (UCTD). On further workup, she was eventually diagnosed with lambda light chain systemic amyloidosis by abdominal fat pad biopsy. Her symptoms completely resolved after autologous stem cell transplantation. With this case, we would like to highlight the similarities in the clinical features between light chain amyloidosis and rheumatological disorders. We would also like to emphasize the importance of the prompt recognition of the clinical features of amyloidosis which are crucial to triggering appropriate diagnostic procedures, since early diagnosis is a key to improving outcomes in this disease with an otherwise poor prognosis. PMID:28042297

  6. Systemic Light Chain Amyloidosis Mimicking Rheumatic Disorders

    Directory of Open Access Journals (Sweden)

    Rohit R. Rao

    2016-01-01

    Full Text Available Secondary amyloidosis can complicate chronic inflammatory autoimmune diseases. However, the clinical findings of primary amyloidosis may mimic those of primary rheumatologic disorders. We present the case of a 53-year-old woman who presented with dystrophic nail changes, dry eyes, bilateral carpal tunnel syndrome, Raynaud’s phenomenon, and high titer positive nucleolar pattern antinuclear antibody. She was initially misdiagnosed as having Undifferentiated Connective Tissue Disease (UCTD. On further workup, she was eventually diagnosed with lambda light chain systemic amyloidosis by abdominal fat pad biopsy. Her symptoms completely resolved after autologous stem cell transplantation. With this case, we would like to highlight the similarities in the clinical features between light chain amyloidosis and rheumatological disorders. We would also like to emphasize the importance of the prompt recognition of the clinical features of amyloidosis which are crucial to triggering appropriate diagnostic procedures, since early diagnosis is a key to improving outcomes in this disease with an otherwise poor prognosis.

  7. Immunoglobulin Free Light Chain Dimers in Human Diseases

    Directory of Open Access Journals (Sweden)

    Batia Kaplan

    2011-01-01

    Full Text Available Immunoglobulin free light chain (FLC kappa (κ and lambda (λ isotypes exist mainly in monomeric and dimeric forms. Under pathological conditions, the level of FLCs as well as the structure of monomeric and dimeric FLCs and their dimerization properties might be significantly altered. The abnormally high fractions of dimeric FLCs were demonstrated in the serum of patients with multiple myeloma (MM and primary systemic amyloidosis (AL, as well as in the serum of anephric patients. The presence of tetra- and trimolecular complexes formed due to dimer-dimer and dimer-monomer interactions was detected in the myeloma serum. Analysis of the amyloidogenic light chains demonstrated mutations within the dimer interface, thus raising the possibility that these mutations are responsible for amyloidogenicity. Increased κ monomer and dimer levels, as well as a high κ/λ monomer ratio, were typically found in the cerebrospinal fluid from patients with multiple sclerosis (MS. In many MS cases, the elevation of κ FLCs was accompanied by an abnormally high proportion of λ dimers. This review focuses on the disease-related changes of the structure and level of dimeric FLCs, and raises the questions regarding their formation, function, and role in the pathogenesis and diagnosis of human diseases.

  8. Myeloma light chains are ligands for cubilin (gp280).

    Science.gov (United States)

    Batuman, V; Verroust, P J; Navar, G L; Kaysen, J H; Goda, F O; Campbell, W C; Simon, E; Pontillon, F; Lyles, M; Bruno, J; Hammond, T G

    1998-08-01

    Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

  9. Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants.

    Directory of Open Access Journals (Sweden)

    Sean M O'Rourke

    2007-08-01

    Full Text Available Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.

  10. Immunoglobulin K light chain deficiency: A rare, but probably underestimated, humoral immune defect.

    Science.gov (United States)

    Sala, Pierguido; Colatutto, Antonio; Fabbro, Dora; Mariuzzi, Laura; Marzinotto, Stefania; Toffoletto, Barbara; Perosa, Anna R; Damante, Giuseppe

    2016-04-01

    Human immunoglobulin molecules are generated by a pair of identical heavy chains, which identify the immunoglobulin class, and a pair of identical light chains, Kappa or Lambda alternatively, which characterize the immunoglobulin type. In normal conditions, Kappa light chains represent approximately 2/3 of the light chains of total immunoglobulins, both circulating and lymphocyte surface bound. Very few cases of immunoglobulin Kappa or Lambda light chain defects have been reported. Furthermore, the genetic basis of this defect has been extensively explored only in a single case. We report a case of a patient suffering of serious recurrent bacterial infections, which was caused by a very rare form of immunoglobulin disorder, consisting of a pure defect of Kappa light chain. We evaluated major serum immunoglobulin concentrations, as well as total and free Kappa and Lambda light chain concentrations. Lymphocyte phenotyping was also performed and finally we tested the Kappa chain VJ rearrangement as well as the constant Kappa region sequence. Studies performed on VJ rearrangement showed a polyclonal genetic arrangement, whereas the gene sequencing for the constant region of Kappa chain showed a homozygous T to G substitution at the position 1288 (rs200765148). This mutation causes a substitution from Cys to Gly in the protein sequence and, therefore, determines the abnormal folding of the constant region of Kappa chain. We suggest that this defect could lead to an effective reduction of the variability of total antibody repertoire and a consequent defect of an apparently normal immunoglobulin response to common antigens.

  11. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    Science.gov (United States)

    Vincent, N D; Cummins, P

    1985-04-01

    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  12. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  13. Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma.

    Science.gov (United States)

    Dejoie, Thomas; Attal, Michel; Moreau, Philippe; Harousseau, Jean-Luc; Avet-Loiseau, Herve

    2016-03-01

    Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. (Clinical Trials Register.eu identifier: 2007-005204-40).

  14. Increased Serum Free Light Chains Precede the Presentation of Immunoglobulin Light Chain Amyloidosis

    Science.gov (United States)

    Weiss, Brendan M.; Hebreo, Joseph; Cordaro, Daniel V.; Roschewski, Mark J.; Baker, Thomas P.; Abbott, Kevin C.; Olson, Stephen W.

    2014-01-01

    Purpose Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. Patients and Methods We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. Results An M-Ig was detected in 100% of cases and 0% of controls (P diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P diff was greater than 23 mg/L in 85% of cases and 0% of controls (P diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Conclusion Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. PMID:25024082

  15. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes.

    Science.gov (United States)

    Shamblott, M J; Litman, G W

    1989-06-01

    Antibody to Heterodontus francisci (horned shark) immunoglobulin light chain was used to screen a spleen cDNA expression library, and recombinant clones encoding light chain genes were isolated. The complete sequences of the mature coding regions of two light chain genes in this phylogenetically distant vertebrate have been determined and are reported here. Comparisons of the sequences are consistent with the presence of mammalian-like framework and complementarity-determining regions. The predicted amino acid sequences of the genes are more related to mammalian lambda than to kappa light chains. The nucleotide sequences of the genes are most related to mammalian T-cell antigen receptor beta chain. Heterodontus light chain genes may reflect characteristics of the common ancestor of immunoglobulin and T-cell antigen receptors before its evolutionary diversification.

  16. Recurrence of light-chain deposition disease after renal transplantation

    DEFF Research Database (Denmark)

    Larsen, Thomas; Hammer, Anne; Jørgensen, Kaj Anker

    2008-01-01

    A 51-year-old male with a history of chronic renal disease received a renal allograft, in which disease recurred. Light-chain deposition disease was confirmed through biopsies of the native kidney and graft, and detection of free kappa light chains in serum. Udgivelsesdato: 2007-Sep-6...

  17. Antibody elbow angles are influenced by their light chain class

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R; Zemla, A; Wilson, I; Rupp, B

    2006-01-12

    We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa-chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195{sup o}) elbow angles. This apparent hyperflexibility of lambda-chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is also described.

  18. Increased serum free light chains precede the presentation of immunoglobulin light chain amyloidosis.

    Science.gov (United States)

    Weiss, Brendan M; Hebreo, Joseph; Cordaro, Daniel V; Roschewski, Mark J; Baker, Thomas P; Abbott, Kevin C; Olson, Stephen W

    2014-09-01

    Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. An M-Ig was detected in 100% of cases and 0% of controls (P < .001). The M-Ig was present in 100%, 80%, and 42% of cases at less than 4 years, 4 to 11 years, and more than 11 years before diagnosis, respectively. The median FLC differential (FLC-diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P < .001), 4 to 11 years (65.1 v 2.2 mg/L; P < .001), and more than 11 years (4.5 v 0.4 mg/L; P = .03) before diagnosis. The FLC-diff was greater than 23 mg/L in 85% of cases and 0% of controls (P < .001). The FLC-diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. © 2014 by American Society of Clinical Oncology.

  19. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    KAUST Repository

    Ghiotto, Fabio

    2011-01-01

    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

  20. P53 Mutations Change Phosphatidylinositol Acyl Chain Composition

    Directory of Open Access Journals (Sweden)

    Adam Naguib

    2015-01-01

    Full Text Available Phosphatidylinositol phosphate (PIP second messengers relay extracellular growth cues through the phosphorylation status of the inositol sugar, a signal transduction system that is deregulated in cancer. In stark contrast to PIP inositol head-group phosphorylation, changes in phosphatidylinositol (PI lipid acyl chains in cancer have remained ill-defined. Here, we apply a mass-spectrometry-based method capable of unbiased high-throughput identification and quantification of cellular PI acyl chain composition. Using this approach, we find that PI lipid chains represent a cell-specific fingerprint and are unperturbed by serum-mediated signaling in contrast to the inositol head group. We find that mutation of Trp53 results in PIs containing reduced-length fatty acid moieties. Our results suggest that the anchoring tails of lipid second messengers form an additional layer of PIP signaling in cancer that operates independently of PTEN/PI3-kinase activity but is instead linked to p53.

  1. Flow cytometric detection of immunoglobulin light chain in hematolymphoid immunophenotyping

    Institute of Scientific and Technical Information of China (English)

    Xu Dongsheng

    2011-01-01

    During B cell development and maturation, the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH)and light chain genes,rearrange to associate one of a number of variable, diverse, and joining gene segments. A single mature Bcell expresses an IgH chain and either a kappa or lambda light chain, which is known as allelic or isotypic exclusion. In normal or reactive conditions, lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression. The norreal proportion of kappa to lambda (κ/λ ratio) is within the range of 0. 5 - 3. 0 in peripheral blood or bone marrow and 1.2 2.7 in lymph nodes[1].The most useful feature for diagnosing mature B cell neoplasm is light chain restriction or monotypic staining with κ or λ light chain. Currently, it is a common assumption that demonstration of light chain restriction in a B lymphocyte population is generally considered proof of monoclonality and indicates malignancy although monotypic B cell populations have been infrequently demonstrated in patients with no definitive evidence ofB cell malignancy[2-4]. The most common flow cytometric analysis for determining B cell monotype is the percent κ and λimmunoglobulin light chains.Because of the importance of light chain restriction in the diagnosis of B cell neoplasm, anti immunoglobulin antibodies (e. g. anti-κ and anti-λ) are vital tools in the detection of monotypic B cell populations. Accurate determination of surface light chain expression depends on many factors, such as proper washing procedure, lysing solution, type of antibody used, specimen type or lymphoma type[5]. This article will discuss some common problems encountered in flow cytometric(FCM)deterruination of surface immunoglobulin light chain expression inhematolymphoid immunophenotyping.%@@ During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of

  2. Tetany and osteolysis in light chain myeloma with amyloidosis.

    Science.gov (United States)

    Bhakri, H. L.; Cundy, T. F.; Pettingale, K. W.

    1983-01-01

    A patient is described with light chain myeloma and amyloidosis in whom 2 unusual complications occurred, diffuse osteolytic lesions and tetany. These observations extend the previously recognized clinical spectrum of this disorder. Images Fig. 1 PMID:6413962

  3. Neurofilament light chain: a biomarker for genetic frontotemporal dementia.

    Science.gov (United States)

    Meeter, Lieke H; Dopper, Elise G; Jiskoot, Lize C; Sanchez-Valle, Raquel; Graff, Caroline; Benussi, Luisa; Ghidoni, Roberta; Pijnenburg, Yolande A; Borroni, Barbara; Galimberti, Daniela; Laforce, Robert Jr; Masellis, Mario; Vandenberghe, Rik; Ber, Isabelle Le; Otto, Markus; van Minkelen, Rick; Papma, Janne M; Rombouts, Serge A; Balasa, Mircea; Öijerstedt, Linn; Jelic, Vesna; Dick, Katrina M; Cash, David M; Harding, Sophie R; Jorge Cardoso, M; Ourselin, Sebastien; Rossor, Martin N; Padovani, Alessandro; Scarpini, Elio; Fenoglio, Chiara; Tartaglia, Maria C; Lamari, Foudil; Barro, Christian; Kuhle, Jens; Rohrer, Jonathan D; Teunissen, Charlotte E; van Swieten, John C

    2016-08-01

    To evaluate cerebrospinal fluid (CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters. In this multicenter case-control study, we investigated CSF NfL in 174 subjects (48 controls, 40 presymptomatic carriers and 86 patients with microtubule-associated protein tau (MAPT), progranulin (GRN), and chromosome 9 open reading frame 72 (C9orf72) mutations), and serum NfL in 118 subjects (39 controls, 44 presymptomatic carriers, 35 patients). In 55 subjects both CSF and serum was determined. In two subjects CSF was available before and after symptom onset (converters). Additionally, NfL levels were correlated with clinical parameters, survival, and regional brain atrophy. CSF NfL levels in patients (median 6762 pg/mL, interquartile range 3186-9309 pg/mL) were strongly elevated compared with presymptomatic carriers (804 pg/mL, 627-1173 pg/mL, P NfL correlated highly with CSF NfL (r s = 0.87, P NfL after disease onset. Additionally, NfL levels in patients correlated with disease severity, brain atrophy, annualized brain atrophy rate and survival. NfL in both serum and CSF has the potential to serve as a biomarker for clinical disease onset and has a prognostic value in genetic FTD.

  4. A mutation specific polymerase chain reaction for detecting hepatitis B virus genome mutations at nt551

    Institute of Scientific and Technical Information of China (English)

    Chun-Ling Ma; De-Xing Fang; Hua-Biao Chen; Fa-Qing Li; Hui-Ying Jin; Su-Qin Li; Wei-Guo Tan

    2003-01-01

    AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the "a" determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution. This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551.METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS,P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3' terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification. These 16 HBV S DNAs were detected by using the msPCR method.RESULTS: When the annealing temperature was raised to 71 °C, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS; At 72 °C and 5 pmole of the primers (each) in reaction of 25 μl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551TPPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A, one was positive for nt

  5. Preliminary research on myosin light chain kinase in rabbit liver

    Institute of Scientific and Technical Information of China (English)

    Bin Ren; Hua-Qing Zhu; Zhao-Feng Luo; Qing Zhou; Yuan Wang; Yu-Zhen Wang

    2001-01-01

    AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction (RT-PCR);the MLCK was obtained from rabbit liver, and its activity was analyzed by γ-32P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg-L-1, the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.

  6. Myosin light chain genes in the turkey (Meleagris gallopavo).

    Science.gov (United States)

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  7. Light scattering of thin azobenzene side-chain polyester layers

    DEFF Research Database (Denmark)

    Kerekes, Á.; Lörincz, E.; Ramanujam, P.S.

    2002-01-01

    characteristics than the liquid crystalline polyester. The amorphous samples have negligible polarization part orthogonal to the incident beam. the liquid crystalline samples have relative high orthogonal polarization part in light scattering, The light scattering results can be used to give a lower limit...... for the domain size in thin liquid crystalline polyester layers being responsible for the dominant light scattering. The characteristic domain Sizes obtained from the Fourier transformation of polarization microscopic Pictures confirm these values.......Light scattering properties of liquid crystalline and amorphous azobenzene side-chain polyester layers used for optical data storage were examined by means of transmissive scatterometry. Comparative experiments show that the amorphous polyester has significantly lower light scattering...

  8. The immunoglobulin light chain locus of the turkey, Meleagris gallopavo.

    Science.gov (United States)

    Bao, Yonghua; Wu, Sun; Zang, Yunlong; Wang, Hui; Song, Xiangfeng; Xu, Chunyang; Xie, Bohong; Guo, Yongchen

    2012-06-15

    To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.

  9. Immunoglobulin heavy chain/light chain pairs (HLC, Hevylite™) assays for diagnosing and monitoring monoclonal gammopathies.

    Science.gov (United States)

    Kraj, Maria

    2014-01-01

    Immunofixation (IFE) is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin (Ig) heavy chain/light chain immunoassays - HLC, HevyliteTM. HLC separately measures in pairs light chain types of each intact Ig class generating ratios of monoclonal Ig/uninvolved polyclonal Ig concentrations. Studies have shown that HLC and IFE are complementary methods. HLC assays quantify monoclonal proteins and identify monoclonality. It is possible to predict prognosis in multiple myeloma and to monitor response to treatment using HLC ratio. HLC ratio may serve as a parameter for myeloma induced immunoparesis and serve as a new marker for validating remission depth and relapse probabilities.

  10. A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

    Science.gov (United States)

    Lee, Chingwei V; Koenig, Patrick; Fuh, Germaine

    2014-01-01

    A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

  11. Diagnostic RAS mutation analysis by polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Ian A. Cree

    2016-06-01

    Full Text Available RAS mutation analysis is an important companion diagnostic test. Treatment of colorectal cancer with anti-Epidermal Growth Factor Receptor (EGFR therapy requires demonstration of RAS mutation status (both KRAS and NRAS, and it is good practice to include BRAF. In Non-Small Cell Lung Cancer (NSCLC and melanoma, assessment of RAS mutation status can be helpful in triaging patient samples for more extensive testing. This mini-review will discuss the role of PCR methods in providing rapid diagnostic information for cancer patients.

  12. Neurofilament light chain: a biomarker for genetic frontotemporal dementia

    OpenAIRE

    Meeter, Lieke H.; Dopper, Elise G.; Jiskoot, Lize C.; Sanchez-Valle, Raquel; Graff, Caroline; Benussi, Luisa; Ghidoni, Roberta; Pijnenburg, Yolande A; Borroni, Barbara; Galimberti, Daniela; Laforce, Robert Jr; Masellis, Mario; Vandenberghe, Rik; Le Ber, Isabelle; Otto, Markus

    2016-01-01

    Abstract Objective To evaluate cerebrospinal fluid (CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters. Methods In this multicenter case?control study, we investigated CSF NfL in 174 subjects (48 controls, 40 presymptomatic carriers and 86 patients with microtubule?as...

  13. CSF neurofilament light chain reflects corticospinal tract degeneration in ALS

    OpenAIRE

    Menke, Ricarda A.L.; Gray, Elizabeth; Lu, Ching-Hua; Kuhle, Jens; Talbot, Kevin; Malaspina, Andrea; Turner, Martin R.

    2015-01-01

    Objective Diffusion tensor imaging (DTI) is sensitive to white matter tract pathology. A core signature involving the corticospinal tracts (CSTs) has been identified in amyotrophic lateral sclerosis (ALS). Raised neurofilament light chain protein (NfL) in cerebrospinal fluid (CSF) is thought to reflect axonal damage in a range of neurological disorders. The relationship between these two measures was explored. Methods CSF and serum NfL concentrations and DTI acquired at 3?Tesla on the same da...

  14. Tertiary structure of human {Lambda}6 light chains.

    Energy Technology Data Exchange (ETDEWEB)

    Pokkuluri, P. R.; Solomon, A.; Weiss, D. T.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center/Graduate School of Medicine

    1999-01-01

    AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein

  15. Light chain (AL amyloidosis: update on diagnosis and management

    Directory of Open Access Journals (Sweden)

    Rosenzweig Michael

    2011-11-01

    Full Text Available Abstract Light chain (AL amyloidosis is a plasma cell dyscrasia characterized by the pathologic production of fibrillar proteins comprised of monoclonal light chains which deposit in tissues and cause organ dysfunction. The diagnosis can be challenging, requiring a biopsy and often specialized testing to confirm the subtype of systemic disease. The goal of treatment is eradication of the monoclonal plasma cell population and suppression of the pathologic light chains which can result in organ improvement and extend patient survival. Standard treatment approaches include high dose melphalan (HDM followed by autologous hematopoietic stem cell transplantation (SCT or oral melphalan with dexamethasone (MDex. The use of novel agents (thalidomide, lenalidomide and bortezomib alone and in combination with steroids and alkylating agents has shown efficacy and continues to be explored. A risk adapted approach to SCT followed by novel agents as consolidation reduces treatment related mortality with promising outcomes. Immunotherapeutic approaches targeting pathologic plasma cells and amyloid precursor proteins or fibrils are being developed. Referral of patients to specialized centers focusing on AL amyloidosis and conducting clinical trials is essential to improving patient outcomes.

  16. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    Science.gov (United States)

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  17. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... the reagents used to measure by immunochemical techniques both kappa and lambda types of light chain... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin (light chain specific... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  18. Familial mutations in fibrinogen Aα (FGA) chain identified in renal amyloidosis increase in vitro amyloidogenicity of FGA fragment.

    Science.gov (United States)

    Sivalingam, Vishwanath; Patel, Basant K

    2016-08-01

    Amyloidoses are clinical disorders where deposition of β-sheet rich, misfolded protein aggregates called amyloid occurs in vital organs like brain, kidney, liver or heart etc. Aggregation of several proteins such as immunoglobulin light chain, fibrinogen Aα chain (FGA) and lysozyme have been found to be associated with renal amyloidosis. Fibrinogen amyloidosis (AFib) is predominantly familial and is associated with the deposition of mutant FGA amyloid, primarily in kidneys. Over ten substitution and frame-shift mutations in FGA have been identified from AFib patients. Whether wild-type FGA is also involved in AFib is yet unknown. The affected tissues from AFib patients usually show ∼10 kDA peptide from C-terminal 80 amino acid residues of mutant FGA. Notably, this region also encompasses all known disease-related mutations. Whether these point mutations increase the amyloidogenicity of FGA leading to disease progression, have not been studied yet. Here, we have investigated the role of two disease-related mutations in affecting amyloidogenic propensity of an FGA(496-581) fragment. We found that at physiological pH, the wild-type FGA(496-581) fragment remains monomeric, whereas its E540V mutant forms amyloid-like fibrils as observed by AFM. Also, FGA(496-581) harbouring another familial mutation, R554L, converts in vitro into globular, β-sheet rich aggregates, showing amyloid-like properties. These findings suggest that familial mutations in FGA may have role in renal amyloidosis via enhanced amyloid formation.

  19. Russell body duodenitis with immunoglobulin kappa light chain restriction.

    Science.gov (United States)

    Munday, William R; Kapur, Lucy Harn; Xu, Mina; Zhang, Xuchen

    2015-01-16

    Russell bodies are eosinophilic intracytoplasmic globules which are likely the result of disturbed secretion of immunoglobulins that accumulate within the plasma cell. Russell body collections have been identified within the stomach, known as Russell body gastritis. Similar lesions within the duodenum are referred to as Russell body duodenitis, which is rare. Several Russell body gastritis case reports are associated with Helicobacter pylori. However, the etiology of Russell body duodenitis remains unclear. Here we report the first case of Russell body duodenitis with immunoglobulin light chain restriction in a background of peptic duodenitis.

  20. Super-light pearl-chain arch vaults

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Halding, Philip Skov

    2014-01-01

    in the vault. This means that the structure can sustain 4 hours standard fire. That it has a very high level of sound insulation, efficient sound damping properties, optimized production, a low production of CO2 that has already given rise to several awards, and easy transport of plane elements instead of what......Arch vaults are known as optimal and impressive structures, but due to their curved shapes they are extremely costly to produce in countries, where the cost of labor is high. By means of super-light pearl-chain technology arch vaults can be constructed from equal plane prefabricated elements, which...... are assembled on the erection site by means of post-tensioning and lifted in place in buildings and bridges. The super-light SL-deck element is developed by the company Abeo Ltd and The Technical University of Denmark for plane floor structures in buildings. However, this deck element is designed in such a way...

  1. Inferring Sequential Order of Somatic Mutations during Tumorgenesis based on Markov Chain Model.

    Science.gov (United States)

    Kang, Hao; Cho, Kwang-Hyun; Zhang, Xiaohua Douglas; Zeng, Tao; Chen, Luonan

    2015-01-01

    Tumors are developed and worsen with the accumulated mutations on DNA sequences during tumorigenesis. Identifying the temporal order of gene mutations in cancer initiation and development is a challenging topic. It not only provides a new insight into the study of tumorigenesis at the level of genome sequences but also is an effective tool for early diagnosis of tumors and preventive medicine. In this paper, we develop a novel method to accurately estimate the sequential order of gene mutations during tumorigenesis from genome sequencing data based on Markov chain model as TOMC (Temporal Order based on Markov Chain), and also provide a new criterion to further infer the order of samples or patients, which can characterize the severity or stage of the disease. We applied our method to the analysis of tumors based on several high-throughput datasets. Specifically, first, we revealed that tumor suppressor genes (TSG) tend to be mutated ahead of oncogenes, which are considered as important events for key functional loss and gain during tumorigenesis. Second, the comparisons of various methods demonstrated that our approach has clear advantages over the existing methods due to the consideration on the effect of mutation dependence among genes, such as co-mutation. Third and most important, our method is able to deduce the ordinal sequence of patients or samples to quantitatively characterize their severity of tumors. Therefore, our work provides a new way to quantitatively understand the development and progression of tumorigenesis based on high throughput sequencing data.

  2. Super-light concrete with pearl-chains

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl

    2009-01-01

    half that of similar structures in concrete and steel. They are heat-insulating, fire-resistant and they open up the possibility for large spans and the creation of advanced shapes. Fields of application include roofs, shells, beams, columns, walls, façades, offshore structures, tunnels and structures......-scale applications, such as façade elements, crash barriers and secondary structures. One special small-scale application of the principle is in frame building, where super-light frames with pearl-chain-reinforced components have some of the same advantages as timber-frame structures with regard to stability, heat...... insulation, economy and simple erection processes, but without the disadvantages of fire and rot....

  3. Intrathecal synthesis of free immunoglobulin light chains in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, M; Schaldemose Nielsen, H; Jensen, J

    1998-01-01

    OBJECTIVE: The detection of oligoclonal immunoglobulin free light chains (FLC) in the diagnosis of multiple sclerosis (MS) was compared to IgG isoelectric focusing. MATERIAL AND METHODS: Cerebrospinal fluid and serum samples from 69 patients with possible first attacks of MS, 50 patients...... with clinically definite MS (CDMS), and 118 patients with other neurological diseases (OND) were analyzed. IgG and FLC oligoclonal bands were detected by isoelectric focusing and immunoperoxidase staining. RESULTS: Intrathecal synthesis of IgG, kappa FLC, and lambda FLC oligoclonal bands, respectively, was seen...... in 92%, 92%, and 86% of MS patients; in 61%, 62%, and 64% of patients with possible first attacks of MS; and in 3%, 3%, and 8% of the patients with OND. In control patients without IgG synthesis intrathecal lambda FLC synthesis was more common than kappa FLC synthesis (P=0.03). CONCLUSION: Kappa FLC...

  4. Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains.

    Science.gov (United States)

    del Pozo Yauner, Luis; Ortiz, Ernesto; Sánchez, Rosalba; Sánchez-López, Rosana; Güereca, Leopoldo; Murphy, Charles L; Allen, Amy; Wall, Jonathan S; Fernández-Velasco, D Alejandro; Solomon, Alan; Becerril, Baltazar

    2008-08-01

    Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

  5. Enhancing lean supply chain through traffic light quality management system

    Directory of Open Access Journals (Sweden)

    Md. Mazharul Islam

    2013-03-01

    Full Text Available Lean is a continuous journey to grow and excel the company. Any company want to develop and cope with the world pace must adopt lean. However, in most of the organizations the management culture or people’s mentality is not so good to embrace change. They have predestined mind set where no change is normally allowed. Lean is a cooperative way of working that involves all departments and all personnel to work together in a team for the betterment of the entire company. Without providing fixed solution of any problem it suggests the best way that people willingly accept to do. Lean normally deals with highest quality, shorter lead time and lowest cost. In Bangladesh, most of the garment manufacturing companies are experiencing a massive quality problem. We describe a case where traffic light, a tool of lean quality system was adopted to a garment manufacturing company in Bangladesh. We also provide the charts to contrast the before and after scenario in detail, in order to illustrate the company benefits. After the traffic light system being implemented, the quality status was improved, production capacity was increased; significant days were saved that enhanced the lead time and thus strengthen the supply chain.

  6. Amino terminal sequence of heavy and light chains from ratfish immunoglobulin.

    Science.gov (United States)

    De Ioannes, A E; Aguila, H L

    1989-01-01

    The ratfish, Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (Mr 960,000), composed of heavy (Mr 71,000), light (Mr 22,500), and J (Mr 16,000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark, Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.

  7. Measurement of diversification in the immunoglobulin light chain gene of DT40 cells.

    Science.gov (United States)

    Sale, Julian E

    2012-01-01

    The immunoglobulin loci of the genetically tractable chicken B cell line DT40 provide a unique opportunity to study the cellular response to endogenously generated DNA damage in a chromosomal context. Abasic sites generated by the concerted action of Activation-Induced Deaminase (AID) and Uracil DNA Glycosylase result in both homologous recombination-dependent gene conversion and translesion synthesis-dependent point mutations. The system has provided important insights into both the early stages of AID-dependent immunoglobulin gene diversification and into the relationship between pathways of DNA damage bypass. Here we describe the assays that can be employed to monitor the rate and pattern of immunoglobulin gene diversification at the light chain locus of DT40.

  8. Verification of serum reference intervals for free light chains in a local South African population.

    Science.gov (United States)

    Zemlin, Annalise E; Rensburg, Megan A; Ipp, Hayley; Germishuys, Jurie J; Erasmus, Rajiv T

    2013-11-01

    Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3-20.6 mg/L for κ free light chains, 8.7-25.9 mg/L for λ free light chains and 0.46-1.23 for free light chain ratio. Most of the values fell within the manufacturer's recommended limits and therefore could be used for our population.

  9. Judging disease activity in rheumatoid arthritis by serum free kappa and lambda light chain levels.

    Science.gov (United States)

    Ye, Yun; Li, Su-Liang; Xie, Ming; Jiang, Ping; Liu, Kai-Ge; Li, Ya-Jun

    2013-10-01

    The study aimed to evaluate the levels of serum free kappa (κ) and lambda (λ) light chains in patients with rheumatoid arthritis (RA) as well as exploring the association between serum free κ and λ light chains and activity of RA. For this purpose, healthy individuals and patients with active RA and RA in remission were enrolled, and their serum levels of free κ and λ light chains were measured using rate nephelometry. The diagnostic accuracy of serum free κ and λ light chains was evaluated by receiver operating characteristic curves and 95% confidence intervals for areas under the curve (AUC). The results obtained indicated that the levels of serum free κ and λ light chains in patients with active RA were significantly higher than those of patients in remission and of healthy controls (p light chain and 0.781 for free λ light chain. When the optimal cut-off point for serum κ light chain was 8.02 g/L, the maximum sensitivity and specificity were 82.5% and 82.5%, respectively, and when the optimal cut-off point for serum λ light chain was 3.57 g/L, the maximum sensitivity and specificity were 80% and 82.5%, respectively. It was thus found that serum levels of free κ and λ light chains were positively correlated with disease activity in RA, the Disease Activity Score 28 (DAS28), and values for C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count (PLT), rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) (p light chains in patients with active RA are closely correlated with disease activity parameters including DAS28, CRP, ESR, PLT, RF, and ACPA. Thus, the above-mentioned levels of serum free κ and λ light chains may be used as important indicators of activity of RA.

  10. B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

    OpenAIRE

    J. Chen(Florida State University, Tallahassee, U.S.A.); Trounstine, M; Kurahara, C.; Young, F.; Kuo, C C; Y. Xu; Loring, J.F.; Alt, F W; Huszar, D

    1993-01-01

    We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, ...

  11. Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny.

    Science.gov (United States)

    Rast, J P; Anderson, M K; Ota, T; Litman, R T; Margittai, M; Shamblott, M J; Litman, G W

    1994-01-01

    The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.

  12. Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.

    Science.gov (United States)

    Matheson, Louise S; Osborn, Michael J; Smith, Jennifer A; Corcos, Daniel; Hamon, Maureen; Chaouaf, Rima; Coadwell, John; Morgan, Geoff; Oxley, David; Brüggemann, Marianne

    2009-08-01

    Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

  13. A Mouse Neurodegenerative Dynein Heavy Chain Mutation Alters Dynein Motility and Localization in Neurospora crassa

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2013-01-01

    Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems. PMID:22991199

  14. Novel Mutation in the α-Myosin Heavy Chain Gene Is Associated With Sick Sinus Syndrome

    OpenAIRE

    Ishikawa, Taisuke; Jou, Chuanchau J.; Nogami, Akihiko; Kowase, Shinya; Arrington, Cammon B.; Barnett, Spencer M.; Harrell, Daniel T.; Arimura, Takuro; Tsuji, Yukiomi; Kimura, Akinori; Makita, Naomasa

    2015-01-01

    Recent genome-wide association studies have demonstrated an association between MYH6, the gene encoding α-myosin heavy chain (α-MHC), and sinus node function in the general population. Moreover, a rare MYH6 variant, R721W, predisposing susceptibility to sick sinus syndrome has been identified. However, the existence of disease-causing MYH6 mutations for familial sick sinus syndrome and their underlying mechanisms remain unknown. Methods and Results-We screened 9 genotype-negative probands wit...

  15. CSF neurofilament light chain reflects corticospinal tract degeneration in ALS

    Science.gov (United States)

    Menke, Ricarda A L; Gray, Elizabeth; Lu, Ching-Hua; Kuhle, Jens; Talbot, Kevin; Malaspina, Andrea; Turner, Martin R

    2015-01-01

    Objective Diffusion tensor imaging (DTI) is sensitive to white matter tract pathology. A core signature involving the corticospinal tracts (CSTs) has been identified in amyotrophic lateral sclerosis (ALS). Raised neurofilament light chain protein (NfL) in cerebrospinal fluid (CSF) is thought to reflect axonal damage in a range of neurological disorders. The relationship between these two measures was explored. Methods CSF and serum NfL concentrations and DTI acquired at 3 Tesla on the same day were obtained from ALS patients (n = 25 CSF, 40 serum) and healthy, age-similar controls (n = 17 CSF, 25 serum). Within-group correlations between NfL and DTI measures of microstructural integrity in major white matter tracts (CSTs, superior longitudinal fasciculi [SLF], and corpus callosum) were performed using tract-based spatial statistics. Results NfL levels were higher in patients compared to controls. CSF levels correlated with clinical upper motor neuron burden and rate of disease progression. Higher NfL levels were significantly associated with lower DTI fractional anisotropy and increased radial diffusivity in the CSTs of ALS patients, but not in controls. Interpretation Elevated CSF and serum NfL is, in part, a result of CST degeneration in ALS. This highlights the wider potential for combining neurochemical and neuroimaging-based biomarkers in neurological disease. PMID:26273687

  16. Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

    Science.gov (United States)

    El Naggar, Amel A; El-Naggar, Mostafa; Mokhamer, El-Hassan; Avad, Mona W

    2015-01-01

    The measurement of serum free light chain (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. The aim of the present work was to study the prognostic value of sFLC in multiple myeloma in relation to other serum biomarkers, response to therapy and survival. Forty five newly diagnosed patients with MM were included in the study. Patients were divided into responders and non-responders groups according to response to therapy. sFLC and serum Amyloid A (SAA) were measured by immunonephelometry. The non-responders group showed a statistically significant higher kappa/lambda or lambda/kappa ratio and higher β2 microglobulin level, but lower albumin level at presentation, as compared to the responders group (P < 0.001). However, no statistically significant difference was detected between the two groups regarding SA A or calcium levels. Comparison between sFLC ratio obtained before and after therapy revealed significant decrease after treatment in the responders group (P = 0.05). Survival was significantly inferior in patients with an FLC ratio of ≥ 2.6 or ≤ 0.56 compared with those with an FLC ratio that was between 0.56 and 2.6 (P = 0.002).

  17. Surface-mediated light transmission in metal nanoparticle chains

    NARCIS (Netherlands)

    Compaijen, P. Jasper; Malyshev, Victor A.; Knoester, Jasper

    2013-01-01

    We study theoretically the efficiency of the transmission of optical signals through a linear chain consisting of identical and equidistantly spaced silver metal nanoparticles. Two situations are compared: the transmission efficiency through an isolated chain and through a chain in close proximity o

  18. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    Science.gov (United States)

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  19. Serum Immunoglobulin Free Light Chain Assessment in IgG4-Related Disease

    Directory of Open Access Journals (Sweden)

    Aurélie Grados

    2013-01-01

    Full Text Available Immunoglobulin free light chains are produced in excess during normal antibody synthesis. Their evaluation is commonly used in case of a monoclonal gammopathy. In polyclonal hypergammaglobulinemia related to the Sjögren syndrome or systemic lupus, erythematosus serum free light chain levels are increased and could correlate with disease activity. We show here that the κ ( and λ ( free light chains and the κ : λ ratio ( are increased in sixteen patients with IgG4-related disease when compared to healthy controls. The increase of κ and λ free light chains probably reflects the marked polyclonal B cell activation of the disease. We could not assess in this small cohort of patients a significative correlation of serum free light chain levels and disease activity or extension.

  20. Neurofilament light chain: A prognostic biomarker in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens; Turner, Martin R; Malaspina, Andrea

    2015-06-02

    To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale-Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan-Meier analysis. CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98-7.94, p NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. © 2015 American Academy of Neurology.

  1. Plasma neurofilament light chain predicts progression in progressive supranuclear palsy.

    Science.gov (United States)

    Rojas, Julio C; Karydas, Anna; Bang, Jee; Tsai, Richard M; Blennow, Kaj; Liman, Victor; Kramer, Joel H; Rosen, Howard; Miller, Bruce L; Zetterberg, Henrik; Boxer, Adam L

    2016-03-01

    Blood-based biomarkers for neurodegenerative conditions could improve diagnosis and treatment development. Neurofilament light chain (NfL), a marker of axonal injury, is elevated in cerebrospinal fluid (CSF) of patients with progressive supranuclear palsy (PSP). The goal of this study was to determine the diagnostic and prognostic value of plasma NfL in patients with PSP. Plasma NfL was measured with ultrasensitive digital immunoassay-based technology at baseline and 1-year follow-up in a pilot cohort of 15 PSP patients and 12 healthy controls, and a validation cohort of 147 PSP patients. Mixed linear models tested the ability of plasma NfL to predict neurological, cognitive and functional decline, and brain atrophy. Baseline mean plasma NfL levels were elevated in PSP patients (31 ± 4 pg/mL, vs. control, 17.5 ± 1 pg/mL, P NfL levels had more severe neurological (PSPRS, -36.9% vs. -28.9%, P = 0.04), functional (SEADL, -38.2% vs. -20%, P = 0.03), and neuropsychological (RBANS, -23.9% vs. -12.3%, P = 001) deterioration over 1 year. Higher baseline NfL predicted greater whole-brain and superior cerebellar peduncle volume loss. Plasma and CSF NfL were significantly correlated (r = 0.74, P = 0.002). Plasma NfL is elevated in PSP and could be of value as a biomarker both to assist clinical diagnosis and to monitor pharmacodynamic effects on the neurodegenerative process in clinical trials.

  2. Light-chain nephropathy in patient with renal carcinoma.

    OpenAIRE

    Enia, G.; Maringhini, S; L'Abbate, A; Zoccali, C.; Maggiore, Q

    1981-01-01

    Paraneoplastic syndromes are often associated with renal parenchymal tumours. This report describes a case of renal-cell carcinoma with kappa-chain nephropathy. The patient, a 60-year-old man, had renal tubular dysfunction, shown by low serum concentrations of urate and phosphate. Kappa-chains were found in both serum and urine, but no lambda-chains were found. Investigations showed a clear-cell carcinoma, and the patient underwent a radical nephrectomy. Two years after operation serum phosph...

  3. Frequent lambda light chain gene rearrangement and expression in a Ly-1 B lymphoma with a productive kappa chain allele.

    OpenAIRE

    Hardy, R R; Dangl, J L; Hayakawa, K.; Jager, G.; Herzenberg, L.A.

    1986-01-01

    We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continu...

  4. Antigen nature and complexity influence human antibody light chain usage and specificity.

    Science.gov (United States)

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

  5. On the codon assignment of chain termination signals and the minimization of the effects of frameshift mutations

    CERN Document Server

    Jestin, J L

    1997-01-01

    It has been suggested that the minimization of the probability for lethal mutations is a major constraint shaping the genetic code, with the finding that the genetic code is highly protective against transition mutations. Here, we show that recent data on polymerase-induced frameshifts provide a rationale for the codon assignment of chain termination signals (CTS).

  6. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases.

    Directory of Open Access Journals (Sweden)

    Johanna Gaiottino

    Full Text Available Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL in blood samples, as a biofluid source easily accessible for longitudinal studies.We developed and applied a highly sensitive electrochemiluminescence (ECL based immunoassay for quantification of NfL in blood and CSF.Patients with Alzheimer's disease (AD (30.8 pg/ml, n=20, Guillain-Barré-syndrome (GBS (79.4 pg/ml, n=19 or amyotrophic lateral sclerosis (ALS (95.4 pg/ml, n=46 had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients and healthy controls (HC (3.3 pg/ml, n=67, p<0.0001. Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033, GBS (r=0.79, p<0.0001 and ALS (r=0.70, p<0.0001, but not in CP (r=0.11, p=0.3739. The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%.We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfL(Umea47:3; levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

  7. Spectroscopic properties of light-chain derivatives of murine MOPC-315 immunoglobulin A

    DEFF Research Database (Denmark)

    Zidovetzki, R; Farver, O; Pecht, I

    1981-01-01

    Three light-chain derivatives of the homogeneous IgA, secreted by the mouse myeloma MOPC-315, were studied employing circular dichroism and thermal-perturbation spectroscopy: (a) the light-chain dimer with intact native inter-chain disulfide bond, L2,cov; (b) the light-chain dimer with this bond...... reduced and alkylated, L2,ncov; and (c) the dimer of only the variable regions of the light chains, (VL)2. Comparison of the well resolved circular dichroism spectra of these derivatives allowed the assignments of the bands above 290 nm to the following chromophores: Trp-35L and Trp-91L in the variable...... are found between these light-chain derivatives ant the light chain--heavy chain associates, namely the intact protein M-315 and FV fragment. The comparison between the CD spectra of the free and the hapten-bound L2,cov, L2,ncov and (VL)2 directly demonstrates the existence of the conformational transitions...

  8. Mutation directional selection sheds light on prion pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Liang [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo 255049 (China); Ji, Hong-Fang, E-mail: jhf@sdut.edu.cn [Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo 255049 (China)

    2011-07-01

    Highlights: {yields} Most pathogenic mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. {yields} Mutation-induced changes may strengthen the interactions between PrP and facilitating factors. {yields} The findings also have significant implications for exploring potential regions involved in the conformational transition from PrP{sup C} to PrP{sup Sc}. -- Abstract: As mutations in the PRNP gene account for human hereditary prion diseases (PrDs), it is crucial to elucidating how these mutations affect the central pathogenic conformational transition of normal cellular prion protein (PrP{sup C}) to abnormal scrapie isoform (PrP{sup Sc}). Many studies proposed that these pathogenic mutations may make PrP more susceptible to conformational change through altering its structure stability. By evaluating the most recent observations regarding pathogenic mutations, it was found that the pathogenic mutations do not exert a uniform effect on the thermodynamic stability of the human PrP's structure. Through analyzing the reported PrDs-related mutations, we found that 25 out of 27 mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. Based on the triggering role reported by previous studies of facilitating factors in PrP{sup C} conversion, e.g., lipid and polyanion, we proposed that the mutation-induced changes may strengthen the interaction between PrP and facilitating factors, which will accelerate PrP conversion and cause PrDs.

  9. Exact Markov chain and approximate diffusion solution for haploid genetic drift with one-way mutation.

    Science.gov (United States)

    Hössjer, Ola; Tyvand, Peder A; Miloh, Touvia

    2016-02-01

    The classical Kimura solution of the diffusion equation is investigated for a haploid random mating (Wright-Fisher) model, with one-way mutations and initial-value specified by the founder population. The validity of the transient diffusion solution is checked by exact Markov chain computations, using a Jordan decomposition of the transition matrix. The conclusion is that the one-way diffusion model mostly works well, although the rate of convergence depends on the initial allele frequency and the mutation rate. The diffusion approximation is poor for mutation rates so low that the non-fixation boundary is regular. When this happens we perturb the diffusion solution around the non-fixation boundary and obtain a more accurate approximation that takes quasi-fixation of the mutant allele into account. The main application is to quantify how fast a specific genetic variant of the infinite alleles model is lost. We also discuss extensions of the quasi-fixation approach to other models with small mutation rates.

  10. Light chain editors of anti-DNA receptors in human B cells.

    Science.gov (United States)

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André; Weigert, Martin

    2014-02-10

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.

  11. Fragments of the constant region of immunoglobulin light chains are constituents of AL-amyloid proteins

    DEFF Research Database (Denmark)

    Olsen, K E; Sletten, K; Westermark, Per

    1998-01-01

    Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin...... light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously...

  12. Super-light Structures with Pearl-chain Reinforcement

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Bagger, Anne

    2010-01-01

    of prefabricated segments of a strong material, assembled by one or more prestressing wires. By means of pearl-chain reinforcement, it becomes affordable to provide structures with a wide variety of shapes. This allows a more resource-economical design, because it removes practical hindrances for application...... of compression zones with optimal curved shapes. Some benefits of SLS with pearl-chains are: considerable savings on materials, energy, and CO2, reduction of costs, no scaffolding, cheaper moulds, faster assembly on site, improved durability, good thermal insulation, good indoor-climate, user friendly operation...

  13. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    Energy Technology Data Exchange (ETDEWEB)

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta (IIT); (Iowa); (Miami-MED)

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  14. Biased Immunoglobulin Light Chain Gene Usage in the Shark.

    Science.gov (United States)

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-10-15

    This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing.

  15. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

    Science.gov (United States)

    Hohman, V S; Schluter, S F; Marchalonis, J J

    1992-01-01

    A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

  16. DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF INTRATHECAL SYNTHESIS OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN MULTIPLE SCLEROSIS

    Directory of Open Access Journals (Sweden)

    V. D. Nazarov

    2015-01-01

    Full Text Available Increased intrathecal synthesis and oligoclonal banding of immunoglobulins (Ig in cerebrospinal fluid (CSF are major immunological findings in multiple sclerosis. Free light chains are fragments of Ig molecules that are produced in parallel to oligoclonal Ig, and their production reflects total Ig synthesis within central nervous system. Assessment of intrathecal kappaand lambda free light chain production within CNS compartment will help to improve diagnosis and prediction of outcomes in multiple sclerosis. Concentrations of kappa and lambda free light chains and clonality of immunoglobulin pattern synthesis were studied in paired CSF and serum samples of 151 patients, including 92 patients with multiple sclerosis, and 33 patients with clinically isolated syndrome which later was transformed into the definite multiple sclerosis. A control group consisted of 26 patients with other inflammatory diseases of central nervous system. Several Ig parameters have been determined in the patients, i.e., Ig clonality in CSF; concentrations of free light chains (both kappaand lambda- in CSF; as well as their indexes and ratios. It was established that synthesis of kappa free light chains were significantly elevated in patients with multiple sclerosis. Moreover, the amounts of free light Ig chains in patients with positive oligoclonality test were significantly higher than in cases without intrathecal oligoclonal synthesis. With respect to diagnostic significance, the kappa quotient proved the best available option for diagnosis of multiple sclerosis. Its combination with Ig oligoclonality assays caused a decrease in false-negative diagnostics by 42%. Diagnostic benefit of the kappa quotient could be also confirmed by the data of ROC analysis. Also concentration of lambda free light chains in cerebrospinal fluid showed a negative correlation with conversion terms of clinically isolated syndrome to evident multiple sclerosis. Therefore, the indexes of free Ig

  17. The dimeric Aalpha chain composition of dysfibrinogenemic molecules with mutations at Aalpha 16.

    Science.gov (United States)

    Meh, D A; Siebenlist, K R; Galanakis, D K; Bergtrom, G; Mosesson, M W

    1995-06-15

    In the last stage of fibrinogen synthesis, two Aalpha-Bbeta-gamma half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aalpha16 R-->H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA*, FPA*) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aalpha16 R-->H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R-->C (four) or R-->H (three) Aalpha16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot ('N-DSK') cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve 'N-DSK' components with two FPA*'s (N-DSK homodimer), one FPA* (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aalpha chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aalpha chain are produced, both Aalpha chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.

  18. Immunoglobulin light chain immunohistochemistry revisited, with emphasis on reactive follicular hyperplasia versus follicular lymphoma.

    Science.gov (United States)

    Weiss, Lawrence M; Loera, Sofia; Bacchi, Carlos E

    2010-05-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet to reliably perform it on formalin-fixed paraffin sections is often difficult. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chains that are reactive in paraffin sections. In reactive lymphoid tissues, polytypic staining was noted in greater than 95% of cases, with strong staining of plasma cells, moderate staining of the follicular dendritic cell network, and weak staining of mantle zone cells. Strong staining of the appropriate light chain was seen in each of the 7 cases of multiple myeloma. In a series of 58 cases of B-cell lymphoma, correlation between the results of immunohistochemistry and flow cytometry was obtained in 36 cases (62%), including 32 cases (21 kappa and 11 lambda) in which a single light chain was expressed. Monotypic staining was also seen in 6 additional cases (10%) in which flow cytometry was negative. Thirty of 46 cases (65%) of follicular lymphoma showed monotypic light chain expression, in contrast to 64 of 67 cases (95%) of reactive lymphoid hyperplasia, which showed polytypic light chain expression. These antibodies may provide an effective adjunct to the diagnosis of B-cell lymphoma in routine diagnostic work.

  19. Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Xu Q

    2015-06-01

    Full Text Available Qing Xu,1,* Yazhen Zhu,2,* Yali Bai,1 Xiumin Wei,1 Xirun Zheng,2 Mao Mao,1 Guangjuan Zheng21Translational Bioscience and Diagnostics, WuXi AppTec, Shanghai, 2Department of Pathology, Guangdong Provincial Hospital of TCM, Guangzhou University of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, People’s Republic of China*These authors contributed equally to this workBackground: Two types of epidermal growth factor receptor (EGFR mutations in exon 19 and exon 21 (ex19del and L858R are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR method in detecting the three EGFR mutations in patients with lung cancer.Methods: Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR.Results: The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect

  20. Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation

    Institute of Scientific and Technical Information of China (English)

    Xiao-Mou Peng; Lin Gu; Xue-Juan Chen; Jian-Guo Li; Yang-Su Huang; Zhi-Liang Gao

    2005-01-01

    AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control.To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe ndent of the amount of templates and the number of PCRcycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

  1. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain...... pigments, polycyclic aromatic hydrocarbons (PAHs) which can function as light harvesting systems1. Thus, mixed membranes formed by single chain amphiphile and PAHs could represent simple primitive energy uptake systems which transduce light into chemical energy that can power an internalized catalytic...

  2. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  3. Light-switching-light optical transistor based on metallic nanoparticle cross-chains geometry incorporating Kerr nonlinearity

    Energy Technology Data Exchange (ETDEWEB)

    AbdelMalek, Fathi; Aroua, Walid [National Institute of Applied Science and Technology, University of Carthage, Tunis (Tunisia); Haxha, Shyqyri [Computer Science and Technology Department, Bedfordshire University, Luton (United Kingdom); Flint, Ian [Selex ES Ltd, Luton, Bedfordshire (United Kingdom)

    2016-08-15

    In this research work, we propose all-optical transistor based on metallic nanoparticle cross-chains geometry. The geometry of the proposed device consists of two silver nanoparticle chains arranged along the x- and z-axis. The x-chain contains a Kerr nonlinearity, the source beam is set at the left side of the later, while the control beam is located at the top side of the z-chain. The control beam can turn ON and OFF the light transmission of an incoming light. We report a theoretical model of a very small all-optical transistor proof-of-concept made of optical 'light switching light' concept. We show that the transmission efficiency strongly depends on the control beam and polarization of the incoming light. We investigate the influence of a perfect reflector and reflecting substrate on the transmission of the optical signal when the control beam is turned ON and OFF. These new findings make our unique design a potential candidate for future highly-integrated optical information processing chips. (copyright 2016 by WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  4. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  5. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland

    Directory of Open Access Journals (Sweden)

    Maria Kraj

    2015-01-01

    Full Text Available We report three cases of light chain escape (LCE at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS that satisfied the criteria for progression to light chain multiple myeloma (LCMM with a rapid rise in serum free light chain (FLC levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR. In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC and retrospective heavy/light chain analysis (HLC detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients.

  6. Super-light concrete with pearl-chains

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl

    2009-01-01

    half that of similar structures in concrete and steel. They are heat-insulating, fire-resistant and they open up the possibility for large spans and the creation of advanced shapes. Fields of application include roofs, shells, beams, columns, walls, façades, offshore structures, tunnels and structures...... the applicability and to reveal any potential hidden problems. Continuing research will aim to reduce the weight and thereby the resource consumption further. In addition, the paper introduces pearl-chain reinforcement, which is a new principle for creating compression and tension zones. A small number of simple...

  7. Decoding Warburg's hypothesis: tumor-related mutations in the mitochondrial respiratory chain.

    Science.gov (United States)

    Garcia-Heredia, Jose M; Carnero, Amancio

    2015-12-08

    Otto Warburg observed that cancer cells derived their energy from aerobic glycolysis by converting glucose to lactate. This mechanism is in opposition to the higher energy requirements of cancer cells because oxidative phosphorylation (OxPhos) produces more ATP from glucose. Warburg hypothesized that this phenomenon occurs due to the malfunction of mitochondria in cancer cells. The rediscovery of Warburg's hypothesis coincided with the discovery of mitochondrial tumor suppressor genes that may conform to Warburg's hypothesis along with the demonstrated negative impact of HIF-1 on PDH activity and the activation of HIF-1 by oncogenic signals such as activated AKT. This work summarizes the alterations in mitochondrial respiratory chain proteins that have been identified and their involvement in cancer. Also discussed is the fact that most of the mitochondrial mutations have been found in homoplasmy, indicating a positive selection during tumor evolution, thereby supporting their causal role.

  8. Markov Chain-Like Quantum Biological Modeling of Mutations, Aging, and Evolution

    Directory of Open Access Journals (Sweden)

    Ivan B. Djordjevic

    2015-08-01

    Full Text Available Recent evidence suggests that quantum mechanics is relevant in photosynthesis, magnetoreception, enzymatic catalytic reactions, olfactory reception, photoreception, genetics, electron-transfer in proteins, and evolution; to mention few. In our recent paper published in Life, we have derived the operator-sum representation of a biological channel based on codon basekets, and determined the quantum channel model suitable for study of the quantum biological channel capacity. However, this model is essentially memoryless and it is not able to properly model the propagation of mutation errors in time, the process of aging, and evolution of genetic information through generations. To solve for these problems, we propose novel quantum mechanical models to accurately describe the process of creation spontaneous, induced, and adaptive mutations and their propagation in time. Different biological channel models with memory, proposed in this paper, include: (i Markovian classical model, (ii Markovian-like quantum model, and (iii hybrid quantum-classical model. We then apply these models in a study of aging and evolution of quantum biological channel capacity through generations. We also discuss key differences of these models with respect to a multilevel symmetric channel-based Markovian model and a Kimura model-based Markovian process. These models are quite general and applicable to many open problems in biology, not only biological channel capacity, which is the main focus of the paper. We will show that the famous quantum Master equation approach, commonly used to describe different biological processes, is just the first-order approximation of the proposed quantum Markov chain-like model, when the observation interval tends to zero. One of the important implications of this model is that the aging phenotype becomes determined by different underlying transition probabilities in both programmed and random (damage Markov chain-like models of aging, which

  9. Markov Chain-Like Quantum Biological Modeling of Mutations, Aging, and Evolution.

    Science.gov (United States)

    Djordjevic, Ivan B

    2015-08-24

    Recent evidence suggests that quantum mechanics is relevant in photosynthesis, magnetoreception, enzymatic catalytic reactions, olfactory reception, photoreception, genetics, electron-transfer in proteins, and evolution; to mention few. In our recent paper published in Life, we have derived the operator-sum representation of a biological channel based on codon basekets, and determined the quantum channel model suitable for study of the quantum biological channel capacity. However, this model is essentially memoryless and it is not able to properly model the propagation of mutation errors in time, the process of aging, and evolution of genetic information through generations. To solve for these problems, we propose novel quantum mechanical models to accurately describe the process of creation spontaneous, induced, and adaptive mutations and their propagation in time. Different biological channel models with memory, proposed in this paper, include: (i) Markovian classical model, (ii) Markovian-like quantum model, and (iii) hybrid quantum-classical model. We then apply these models in a study of aging and evolution of quantum biological channel capacity through generations. We also discuss key differences of these models with respect to a multilevel symmetric channel-based Markovian model and a Kimura model-based Markovian process. These models are quite general and applicable to many open problems in biology, not only biological channel capacity, which is the main focus of the paper. We will show that the famous quantum Master equation approach, commonly used to describe different biological processes, is just the first-order approximation of the proposed quantum Markov chain-like model, when the observation interval tends to zero. One of the important implications of this model is that the aging phenotype becomes determined by different underlying transition probabilities in both programmed and random (damage) Markov chain-like models of aging, which are mutually

  10. Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease

    DEFF Research Database (Denmark)

    Pecci, A.; Panza, E.; Pujol-Moix, N.

    2008-01-01

    MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness...

  11. Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

    Science.gov (United States)

    DiBella, Linda M.; Gorbatyuk, Oksana; Sakato, Miho; Wakabayashi, Ken-ichi; Patel-King, Ramila S.; Pazour, Gregory J.; Witman, George B.; King, Stephen M.

    2005-01-01

    Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum. PMID:16195342

  12. Prognostic value of serum free light chain ratio at diagnosis in multiple myeloma.

    Science.gov (United States)

    Kyrtsonis, Marie-Christine; Vassilakopoulos, Theodoros P; Kafasi, Nicoletta; Sachanas, Sotirios; Tzenou, Tatiana; Papadogiannis, Argiroula; Galanis, Zacharoula; Kalpadakis, Christina; Dimou, Maria; Kyriakou, Elias; Angelopoulou, Maria K; Dimopoulou, Maria N; Siakantaris, Marina P; Dimitriadou, Evangelia M; Kokoris, Styliani I; Panayiotidis, Panayiotis; Pangalis, Gerassimos A

    2007-05-01

    The prognostic value of baseline serum free light chain ratio (sFLCR) was investigated in 94 multiple myeloma (MM) patients. sFLCR was calculated as kappa/lambda or lambda/kappa, depending on the patients' dominating monoclonal light chain. Median baseline sFLCR was 3.57 in kappa-MM patients, 45.09 in lambda-MM. 'High' sFLCR (> or = the observed median value for kappa- and lambda-MM respectively) correlated with elevated serum creatinine and lactate dehydrogenase, extensive marrow infiltration and light chain type MM. The 5-year disease-specific survival was 82% and 30% in patients with sFLCR lower than and equal or greater than the median, respectively (P = 0.0001). sFLCR was an independent prognostic factor.

  13. Cast nephropathy and light-chain deposition disease in Waldenström macroglobulinemia.

    Science.gov (United States)

    Gnemmi, Viviane; Leleu, Xavier; Provot, François; Moulonguet, Florence; Buob, David

    2012-09-01

    Waldenström macroglobulinemia is a rare low-grade hematologic malignancy due to clonal proliferation of B lymphocytes responsible for immunoglobulin M (IgM) monoclonal gammopathy secreted in serum. This disease is characterized by lymphoplasmacytic tumoral infiltration of bone marrow and various organs, especially the liver and spleen. Kidney involvement in Waldenström macroglobulinemia has been described previously with reports of various forms of glomerular injury: large intracapillary IgM pseudothrombi, cryoglobulinemia-associated membranoproliferative glomerulonephritis, or amyloidosis. Interstitial infiltration by tumoral B lymphocytes is another classic pattern. Conversely, tubular involvement in the form of myeloma-like casts or basement membrane deposition of monoclonal light chain (light-chain deposition disease) is unusual. We report the occurrence of cast nephropathy associated with light-chain deposition disease in 2 patients with Waldenström macroglobulinemia, which resulted in severe and prolonged kidney failure.

  14. Lambda Light Chain Myeloma with Oliguric Cast Nephropathy and Remission with Bortezomib, Doxorubicin and Dexamethasone

    Directory of Open Access Journals (Sweden)

    R Hada

    2012-12-01

    We report the clinical course and outcome of a patient presenting with acute kidney injury and oliguria, preceded by acute gastroenteritis and intake of Chinese medications and dialysis dependent state for eight weeks. Kidney biopsy revealed cast nephropathy with lambda light chain restriction and severe tubular injury. Serum protein electrophoresis was normal with no “M spike” but serum free light chain ratio was altered with very high lambda and normal kappa light chain levels. Bone marrow biopsy showed >85% atypical plasma cells. Haemodialysis was continued and chemotherapy with bortezomib, doxorubicin and dexamethasone was started. Kidney function gradually improved with discontinuation of dialysis after 1 month and complete remission of acute kidney injury and myeloma in 4 months of chemotherapy. Keywords: acute kidney injury; bortezomib; cast nephropathy; multiple myeloma.

  15. Higher Serum Levels of Free ĸ plus λ Immunoglobulin Light Chains Ameliorate Survival of Hemodialysis Patients

    DEFF Research Database (Denmark)

    Thilo, Florian; Caspari, Christina; Scholze, Alexandra;

    2011-01-01

    Background/Aims: Impaired immune function is common in patients with chronic renal failure. Now, we determined whether serum levels of free immunoglobulin light chains predict mortality in patients with chronic kidney disease stage 5 on hemodialysis. Methods: We performed a prospective cohort study...... of 160 hemodialysis patients with a median follow-up of 15 months (interquartile range, 3-44 months). Serum levels of free κ and λ immunoglobulin light chains were measured at the start of the study. The primary end point was mortality from any cause. Results: In survivors, median serum levels of free κ....../l (χ(2) = 5.91; p = 0.015 by log-rank, Mantel-Cox, test). We performed univariate and multivariate regression analysis showing that older age and lower serum levels of free κ plus λ immunoglobulin light chains predicted mortality in hemodialysis patients. Conclusion: Higher serum levels of free κ plus...

  16. Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain

    Science.gov (United States)

    Espinoza-Fonseca, L. Michel; Colson, Brett A.; Thomas, David D.

    2014-01-01

    We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylatedRLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

  17. Primary CNS Nonamyloidogenic Light Chain Deposition Disease: Case Report and Brief Review.

    Science.gov (United States)

    Mercado, Juan Jose; Markert, James M; Meador, William; Chapman, Philip; Perry, Arie; Hackney, James R

    2017-06-01

    The true incidence of light chain deposition disease (LCDD) restricted to the central nervous system (CNS) is unknown. To our knowledge only 7 cases of LCDD restricted to the brain have been previously reported. We herein describe an unusual example. A 44-year-old man presented with a history of ischemic retinopathy in 2004 and left lower extremity hypoesthesia in 2007 that progressed gradually to left-sided weakness and numbness in the 2 years prior to his hospitalization in 2015. A stereotactic brain biopsy was performed, displaying nonspecific hyaline deposits of amorphous "amyloid-like" material involving deep brain white matter and vessels. These were Congo red negative and were accompanied by a sparse lymphoplasmacytic infiltrate. Plasma cells demonstrated kappa light chain class restriction by chromogenic in situ hybridization (CISH). There was patchy reactivity with kappa immunohistochemistry in the amorphous deposits. A diagnosis of light chain deposition disease was made. Subsequent systemic myeloma and lymphoma workups were negative. Previously reported cases have included men and women, spanning the ages of 19 and 72 years, often presenting with hemiparesis, hypoesthesia, or seizures. Deposits have been reported in the cerebrum and cerebellum. T2/FLAIR (fluid attenuation inversion recovery) changes are usual, but lesions may or may not produce contrast enhancement. The light chain deposition may be of kappa or lambda class. Most lesions have been accompanied by local lymphoid and/or plasma cell infiltrates exhibiting light chain restriction of the same class as the deposits. In summary, LCDD limited to the CNS is a rare lesion consisting of deposition of amyloid-like, but Congo red-negative monotypic light chain usually produced by local lymphoplasmacytic infiltrates.

  18. Mutation in the alpha 5(IV) collagen chain in juvenile-onset Alport syndrome without hearing loss or ocular lesions

    DEFF Research Database (Denmark)

    Zhou, J; Hertz, Jens Michael; Tryggvason, K

    1992-01-01

    -specific probes confirmed the inheritance of the mutation with the phenotype. The mutation changed the GGC codon for glycine-1143 to GAC for aspartate. Substitution of glycine-1143, located in the collagenous domain of the alpha 5(IV) chain, for any other amino acid can be expected to interfere......A single base mutation was identified in the type IV collagen alpha 5 chain gene (COL4A5) of a Danish kindred with Alport syndrome. The 27-year-old male proband developed hematuria in childhood and terminal renal failure at the age of 25 years. He has no hearing loss or ocular lesions. Electron...... gradient gel electrophoresis analysis suggested a sequence variant in the proband and his mother. This was confirmed by sequencing of the PCR-amplified exon 14 region of the hemizygous proband, which demonstrated the base change G----A abolishing an MspI restriction site. Hybridization analysis with allele...

  19. Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Xiao-Mou Peng; Xue-Juan Chen; Jian-Guo Li; Lin Gu; Yang-Su Huang; Zhi-Liang Gao

    2003-01-01

    AIM: Point mutation, one of the commonest gene mutations,is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple.For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice.METHODS: Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3′ end except for last 2base pairs and a different non-complemented region in 5′end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube.The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR.RESULTS: CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 102-108copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 102-104copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of

  20. Prognostic value of depressed midwall systolic function in cardiac light-chain amyloidosis.

    Science.gov (United States)

    Perlini, Stefano; Salinaro, Francesco; Musca, Francesco; Mussinelli, Roberta; Boldrini, Michele; Raimondi, Ambra; Milani, Paolo; Foli, Andrea; Cappelli, Francesco; Perfetto, Federico; Palladini, Giovanni; Rapezzi, Claudio; Merlini, Giampaolo

    2014-05-01

    Cardiac amyloidosis represents an archetypal form of restrictive heart disease, characterized by profound diastolic dysfunction. As ejection fraction is preserved until the late stage of the disease, the majority of patients do fulfill the definition of diastolic heart failure, that is, heart failure with preserved ejection fraction (HFpEF). In another clinical model of HFpEF, that is, pressure-overload hypertrophy, depressed midwall fractional shortening (mFS) has been shown to be a powerful prognostic factor. To assess the potential prognostic role of mFS in cardiac light-chain amyloidosis with preserved ejection fraction, we enrolled 221 consecutive untreated patients, in whom a first diagnosis of cardiac light-chain amyloidosis was concluded between 2008 and 2010. HFpEF was present in 181 patients. Patients in whom cardiac involvement was excluded served as controls (n = 121). Prognosis was assessed after a median follow-up of 561 days. When compared with light-chain amyloidosis patients without myocardial involvement, cardiac light-chain amyloidosis was characterized by increased wall thickness (P chain amyloidosis with normal ejection fraction, depressed circumferential mFS, a marker of myocardial contractile dysfunction, is a powerful predictor of survival.

  1. Two mutational hotspots in the interleukin-2 receptor {gamma} chain gene causing human X-linked severe combined immunodeficiency

    Energy Technology Data Exchange (ETDEWEB)

    Pepper, A.E.; Puck, J.M. [National Institutes of Health, Bethesda, MD (United States); Buckley, R.H. [and others

    1995-09-01

    Human severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immunity, is most commonly caused by mutations in the X-linked gene for interleukin-2 (IL-2) receptor {gamma} chain (IL2RG). For mutational analysis of IL2RG in males with SCID, SSCP screening was followed by DNA sequencing. Of 40 IL2RG mutations found in unrelated SCID patients, 6 were point mutations at the CpG dinucleotide at cDNA 690-691, encoding amino acid R226. This residue lies in the extracellular domain of the protein in a region not previously recognized to be significantly conserved in the cytokine receptor gene family, 11 amino acids upstream from the highly conserved WSXWS motif. Three additional instances of mutation at another CpG dinucleotide at cDNA 879 produced a premature termination signal in the intracellular domain of IL2RG, resulting in loss of the SH2-homologous intracellular domain known to be essential for signaling from the IL-2 receptor complex. Mutations at these two hotspots constitute >20% of the X-linked SCID mutations found by our group and a similar proportion of all reported IL2RG mutations. 41 refs., 5 figs., 1 tab.

  2. A prospective study of nutritional status in immunoglobulin light chain amyloidosis

    DEFF Research Database (Denmark)

    Sattianayagam, Prayman T; Lane, Thirusha; Fox, Zoe;

    2013-01-01

    Weight loss is common in systemic immunoglobulin light chain amyloidosis but there are limited data on the impact of nutritional status on outcome. Using the Patient-Generated Subjective Global Assessment (PG-SGA) score, we prospectively examined nutritional status in 110 consecutive newly......-diagnosed, treatment-naïve patients with immunoglobulin light chain amyloidosis attending the UK National Amyloidosis Centre. At study entry, 72 of 110 (66%) patients had a PG-SGA score of 4 or over, indicating malnutrition requiring specialist nutritional intervention. Number of amyloidotic organs, elevated alkaline...... phosphatase, presence of autonomic neuropathy and advanced Mayo disease stage were independently associated with poor nutritional status (P...

  3. Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects.

    Science.gov (United States)

    Bannwarth, Sylvie; Procaccio, Vincent; Paquis-Flucklinger, Veronique

    2005-06-01

    Molecular analysis of mitochondrial DNA (mtDNA) is a critical step in diagnosis and genetic counseling of respiratory chain defects. No fast method is currently available for the identification of unknown mtDNA point mutations. We have developed a new strategy based on complete mtDNA PCR amplification followed by digestion with a mismatch-specific DNA endonuclease, Surveyor Nuclease. This enzyme, a member of the CEL nuclease family of plant DNA endonucleases, cleaves double-strand DNA at any mismatch site including base substitutions and small insertions/deletions. After digestion, cleavage products are separated and analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. Although this method allows the analysis of 2 kb mtDNA amplicons and the detection of multiple mutations within the same fragment, it does not lead to the identification of homoplasmic base substitutions. Homoplasmic pathogenic mutations have been described. Nevertheless, most homoplasmic base substitutions are neutral polymorphisms while deleterious mutations are typically heteroplasmic. Here, we report that this method can be used to detect mtDNA mutations such as m.3243A>G tRNA(Leu) and m.14709T>C tRNA(Glu) even when they are present at levels as low as 3% in DNA samples derived from patients with respiratory chain defects. Then, we tested five patients suffering from a mitochondrial respiratory chain defect and we identified a variant (m.16189T>C) in two of them, which was previously associated with susceptibility to diabetes and cardiomyopathy. In conclusion, this method can be effectively used to rapidly and completely screen the entire human mitochondrial genome for heteroplasmic mutations and in this context represents an important advance for the diagnosis of mitochondrial diseases.

  4. Clinical features and mutations in seven Chinese patients with very long chain acyl-CoA dehydrogenase deficiency

    Institute of Scientific and Technical Information of China (English)

    Rui-Nan Zhang; Yi-Fan Li; Wen-Juan Qiu; Jun Ye; Lian-Shu Han; Hui-Wen Zhang; Na Lin; Xue-Fan Gu

    2014-01-01

    Background: Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is an inherited metabolic disease caused by deleterious mutations in the ACADVL gene that encodes very long chain acyl-CoA dehydrogenase (VLCAD), and which can present as cardiomyopathy in neonates, as hypoketotic hypoglycemia in infancy, and as myopathy in late-onset patients. Although many ACADVL mutations have been described, no prevalent mutations in the ACADVL gene have been associated with VLCADD. Herein, we report the clinical course of the disease and explore the genetic mutation spectrum in seven Chinese patients with VLCADD. Methods: Seven Chinese patients, from newborn to 17 years old, were included in this study. Tandem mass spectrometry was performed to screen for VLCAD defi ciency. All exons and fl anking introns of the ACADVL gene were analyzed using polymerase chain reaction and direct sequencing. Online analysis tools were used to predict the impact of novel mutations. Results: All cases had elevated serum levels of tetradecanoylcarnitine (C14:1) which is the characteristic biomarker for VLCADD. The phenotype of VLCADD is heterogeneous. Two patients were hospitalized for hypoactivity and hypoglycemia shortly after birth. Three patients showed hepatomegaly and hypoglycemia in infancy. The other two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Three of the patients died at the age of 6-8 months. Eleven different mutations in the ACADVL gene in the 7 patients were identified, including seven reported mutations (p.S22X, p.W427X, p.A213T, p.G222R, p.R450H, c.296- 297delCA, c.1605+1G>T) and four novel mutations (p.S72F, p.Q100X, p.M437T, p.D466Y). The p.R450H and p.D466Y (14.28%, 2/14 alleles) mutations were identifi ed in two alleles respectively. Conclusions: The clinical manifestations were heterogeneous and ACADVL gene mutations were heterozygous in the seven VLCADD Chinese patients. R450H may be a relatively common mutation in Asian

  5. Clinical features and mutations in seven Chinese patients with very long chain acyl-CoA dehydrogenase deficiency.

    Science.gov (United States)

    Zhang, Rui-Nan; Li, Yi-Fan; Qiu, Wen-Juan; Ye, Jun; Han, Lian-Shu; Zhang, Hui-Wen; Lin, Na; Gu, Xue-Fan

    2014-05-01

    Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is an inherited metabolic disease caused by deleterious mutations in the ACADVL gene that encodes very long chain acyl-CoA dehydrogenase (VLCAD), and which can present as cardiomyopathy in neonates, as hypoketotic hypoglycemia in infancy, and as myopathy in late-onset patients. Although many ACADVL mutations have been described, no prevalent mutations in the ACADVL gene have been associated with VLCADD. Herein, we report the clinical course of the disease and explore the genetic mutation spectrum in seven Chinese patients with VLCADD. Seven Chinese patients, from newborn to 17 years old, were included in this study. Tandem mass spectrometry was performed to screen for VLCAD deficiency. All exons and flanking introns of the ACADVL gene were analyzed using polymerase chain reaction and direct sequencing. Online analysis tools were used to predict the impact of novel mutations. All cases had elevated serum levels of tetradecanoylcarnitine (C14:1) which is the characteristic biomarker for VLCADD. The phenotype of VLCADD is heterogeneous. Two patients were hospitalized for hypoactivity and hypoglycemia shortly after birth. Three patients showed hepatomegaly and hypoglycemia in infancy. The other two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Three of the patients died at the age of 6-8 months. Eleven different mutations in the ACADVL gene in the 7 patients were identified, including seven reported mutations (p.S22X, p.W427X, p.A213T, p.G222R, p.R450H, c.296-297delCA, c.1605+1G>T) and four novel mutations (p.S72F, p.Q100X, p.M437T, p.D466Y). The p.R450H and p.D466Y (14.28%, 2/14 alleles) mutations were identified in two alleles respectively. The clinical manifestations were heterog-eneous and ACADVL gene mutations were heterozygous in the seven VLCADD Chinese patients. R450H may be a relatively common mutation in Asian populations. The genotype and phenotype had a

  6. Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer

    Science.gov (United States)

    2015-09-01

    Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer PRINCIPAL...Caused by UVB Light in Skin Cancer 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Monica Ransom, PhD Betty Diamond 5d. PROJECT NUMBER...cells, Skin Cancer 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a

  7. Detection of three nonsense mutations and one missense mutation in the interleukin-2 receptor [gamma] chain gene in SCIDX1 that differently affect the mRNA processing

    Energy Technology Data Exchange (ETDEWEB)

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de (INSERM, Paris (France)); Subtil, A.; Dautry-Varsat, A. (Institut Pasteur, Paris (France))

    1994-05-01

    The interleukin-2 receptor [gamma] (IL-2R[gamma]) chain gene encodes a 64-kDa protein that not only composes the high-affinity form of the IL-2 binding receptor in association with the 2R [alpha] and [beta] chains, but also participates in at least the IL-4 and IL-7 receptor complexes. Mutations in this gene have recently been shown to cause X-linked severe combined immunodeficiency (SCIDX1). This disease of the immune system results from an early block of T lymphocyte and natural killer (NK) cell differentiation, which leads to a severe cellular and humoral immune defect that is lethal unless treated by bone marrow transplantation. Analysis of the IL-2R[gamma] gene in SCIDX1 patients has revealed the presence of heterogeneous mutations principally located in the extracellular domain of the molecule. We report here three intraexonic mutations and one deletion in the IL-2R[gamma] gene in four SCIDX1 patients. These mutations appear to differentially affect RNA processing, either by decreasing IL-2R[gamma] mRNA level or by the skipping of a constitutive exon. 16 refs., 1 fig.

  8. Light, nutrients, and food-chain length constrain planktonic energy transfer efficiency across multiple trophic levels.

    Science.gov (United States)

    Dickman, Elizabeth M; Newell, Jennifer M; González, María J; Vanni, Michael J

    2008-11-25

    The efficiency of energy transfer through food chains [food chain efficiency (FCE)] is an important ecosystem function. It has been hypothesized that FCE across multiple trophic levels is constrained by the efficiency at which herbivores use plant energy, which depends on plant nutritional quality. Furthermore, the number of trophic levels may also constrain FCE, because herbivores are less efficient in using plant production when they are constrained by carnivores. These hypotheses have not been tested experimentally in food chains with 3 or more trophic levels. In a field experiment manipulating light, nutrients, and food-chain length, we show that FCE is constrained by algal food quality and food-chain length. FCE across 3 trophic levels (phytoplankton to carnivorous fish) was highest under low light and high nutrients, where algal quality was best as indicated by taxonomic composition and nutrient stoichiometry. In 3-level systems, FCE was constrained by the efficiency at which both herbivores and carnivores converted food into production; a strong nutrient effect on carnivore efficiency suggests a carryover effect of algal quality across 3 trophic levels. Energy transfer efficiency from algae to herbivores was also higher in 2-level systems (without carnivores) than in 3-level systems. Our results support the hypothesis that FCE is strongly constrained by light, nutrients, and food-chain length and suggest that carryover effects across multiple trophic levels are important. Because many environmental perturbations affect light, nutrients, and food-chain length, and many ecological services are mediated by FCE, it will be important to apply these findings to various ecosystem types.

  9. Human lambda light-chain constant region gene CMor lambda: the primary structure of lambda VI Bence Jones protein Mor.

    OpenAIRE

    Frangione, B.; Moloshok, T; Prelli, F; Solomon, A

    1985-01-01

    Serologic, structural, and genetic analyses have shown that the constant (C) region of human kappa light chains is encoded by a single gene, whereas that of lambda chains is encoded by multiple genes. We have determined the complete C region amino acid sequence of two monoclonal lambda VI light chains, Bence Jones proteins Sut and Mor. The C region of lambda chains Sut and Mor consists of 105 residues, as is characteristic for human lambda light chains, of which 102 are identical in sequence....

  10. λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    Science.gov (United States)

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  11. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    Science.gov (United States)

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

  12. Early Prognostic Value of Monitoring Serum Free Light Chain in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation.

    Science.gov (United States)

    Özkurt, Zübeyde Nur; Sucak, Gülsan Türköz; Akı, Şahika Zeynep; Yağcı, Münci; Haznedar, Rauf

    2017-03-16

    We hypothesized the levels of free light chains obtained before and after autologous stem cell transplantation can be useful in predicting transplantation outcome. We analyzed 70 multiple myeloma patients. Abnormal free light chain ratios before stem cell transplantation were found to be associated early progression, although without any impact on overall survival. At day +30, the normalization of levels of involved free light chain related with early progression. According to these results almost one-third reduction of free light chain levels can predict favorable prognosis after autologous stem cell transplantation.

  13. N332-Directed broadly neutralizing antibodies use diverse modes of HIV-1 recognition: inferences from heavy-light chain complementation of function.

    Directory of Open Access Journals (Sweden)

    Marie Pancera

    Full Text Available Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332 is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type was generally consistent (58 ± 10 µg/ml. In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.

  14. Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Andresen, B S; Jensen, T G; Bross, P

    1994-01-01

    spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11......Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985......), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot...

  15. Immunoglobulin free light chains: new insights in mast cell activation and immunology

    NARCIS (Netherlands)

    Thio, M.

    2011-01-01

    In this thesis, several studies are described that elaborate on the biological properties of immunoglobulin free light chains (Ig-fLC) related to the activation of mast cells and effects on other cells. Mast cell degranulation through Ig-fLC requires two events. At first, mast cell-bound Ig-fLCs sho

  16. Bone marrow lambda-type light chain crystalline structures associated with multiple myeloma.

    Science.gov (United States)

    Schvartz, H; Bonhomme, P; Caulet, S; Beorchia, A; Patey, M; Caulet, T

    1985-01-01

    A 58-year-old man showed bone marrow crystalline structures associated with a lambda light chain producing multiple myeloma. Analysis and processing of electron images clearly displayed the periodic structure of the crystals. Immunochemistry suggested that they contained the whole or a fragmented constant portion of immunoglobulin.

  17. Constant region of a kappa III immunoglobulin light chain as a major AL-amyloid protein

    DEFF Research Database (Denmark)

    Engvig, J P; Olsen, K E; Gislefoss, R E

    1998-01-01

    and the corresponding AL protein as a kappa III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence...

  18. Light-induced circular birefringence in cyanoazobenzene side-chain liquid-crystalline polyester films

    DEFF Research Database (Denmark)

    Naydenova, I; Nikolova, L; Ramanujam, P.S.;

    1999-01-01

    We report the inducement of large circular birefringence (optical activity) in films of a cyanoazobenzene side-chain liquid-crystalline polyester on illumination with circularly polarized light. The polyester has no chiral groups and is initially isotropic. The induced optical rotation is up to 5...

  19. Diagnostic performance of measuring free light chains in fat tissue of patients with AL amyloidosis

    NARCIS (Netherlands)

    Bijzet, J.; Van Gameren, I.I.; Limburg, P.C.; Bos, R.; Vellenga, E.; Hazenberg, B.P.C.

    2010-01-01

    Background: The diagnostic performance of measuring immunoglobulin free light chains in abdominal subcutaneous fat tissue was studied for detection and characterisation of AL amyloidosis. Methods: Fat tissue specimens of 107 consecutive patients with AL amyloidosis (23 k and 84 l) and 95 controls (3

  20. Quantitative Measurement of Immunoglobulins and Free Light Chains Using Mass Spectrometry

    NARCIS (Netherlands)

    VanDuijn, M.M.; Jacobs, J.F.M.; Wevers, R.A.; Engelke, U.F.H.; Joosten, I.; Luider, T.M.

    2015-01-01

    Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the stren

  1. A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

    Science.gov (United States)

    Mali, Bhupesh C; Badgujar, Shamkant B; Shukla, Kunal K; Bhanushali, Paresh B

    2017-02-01

    We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

  2. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    Science.gov (United States)

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear.

  3. Spectroscopic properties of light-chain derivatives of murine MOPC-315 immunoglobulin A

    DEFF Research Database (Denmark)

    Zidovetzki, R; Farver, O; Pecht, I

    1981-01-01

    domains, and Trp-148L, Trp-185L and the disulfide of Cys-214L in the constant domains. The differences in the spectral characteristics of L2,cov as compared to those of L2,ncov and (VL)2 illustrate the significant influence of the disulfide bridge on the conformation of the L2,cov. Pronounced differences...... are found between these light-chain derivatives ant the light chain--heavy chain associates, namely the intact protein M-315 and FV fragment. The comparison between the CD spectra of the free and the hapten-bound L2,cov, L2,ncov and (VL)2 directly demonstrates the existence of the conformational transitions...

  4. Immunoglobulin heavy chain/light chain pair measurement is associated with survival in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Jardin, Fabrice; Delfau-Larue, Marie Hélène; Molina, Thierry Jo; Copie-Bergman, Christiane; Brière, Josette; Petrella, Tony; Canioni, Danielle; Fabiani, Bettina; Jais, Jean-Philippe; Figeac, Martin; Leroy, Karen; Mareschal, Sylvain; Salles, Gilles André; Coiffier, Bertrand; Delarue, Richard; Peyrade, Frédéric; Bosly, André; André, Marc; Ketterer, Nicolas; Haioun, Corinne; Tilly, Hervé

    2013-09-01

    Elevated serum free light chains (FLCs) have been associated with an unfavorable prognosis in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to determine the clinical relevance of a quantitative assessment of intact circulating immunoglobulin (Ig), using serum Ig heavy chain/light chain pair (HLC) measurements in patients with DLBCL. FLC and HLC were measured in 409 serum samples of patients with DLBCL included in the LNH03-B clinical trial program of the Groupe d'Etudes des Lymphomes de l'Adulte (GELA). Patients with an abnormal IgMκ/IgMλ ratio or an abnormal FLC ratio more frequently displayed adverse clinical characteristics. Patients with abnormal IgMκ/IgMλ ratios had inferior progression-free survival (PFS) and overall survival (OS) as compared to patients with a normal ratio in the overall cohort (5-year PFS 44.9% vs. 69.3%, p = 0.0003 and 5-year OS 50.8% vs. 78.1%, p = 0.0003) and in the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) cohort (5-year OS 43.5% vs. 70.3%, p = 0.003). In multivariate analysis, including elevated FLC/HLC and International Prognostic Index (IPI), an abnormal IgMκ/IgMλ ratio (hazard ratio [HR] = 1.54, 95% confidence interval [CI] 1.03-2.3, p = 0.03) remained predictive of shorter progression-free survival. Gene expression profile experiments and immunohistochemistry indicate that this measurement is at least partially related to tumor cell secretion. Both elevated serum FLCs and an abnormal IgMκ/IgMλ ratio are associated with unfavorable outcomes in patients with DLBCL treated by R-CHOP.

  5. Value of serum free light chain testing for the diagnosis and monitoring of monoclonal gammopathies in hematology.

    Science.gov (United States)

    Jagannath, Sundar

    2007-09-01

    The automated quantification of serum free kappa and lambda light chain concentrations provides a highly sensitive tool for the diagnosis and monitoring of monoclonal gammopathies. An abnormal kappa:lambda ratio supports the presence of clonal plasma cell expansion and requires further investigation. More than 94% of myeloma, light chain myeloma, and AL amyloidosis and, likewise, a majority of patients with light chain deposition disease are detectable with this technology. Importantly, these assays identify M-proteins in most patients with oligosecretory disease and permit their recruitment into clinical trials from which they have been previously excluded. Combining serum free light chain testing with traditional electrophoresis provides > 99% accuracy in the first-line diagnosis of monoclonal gammopathies and eliminates the need for urine testing in most instances. One third of patients with monoclonal gammopathy of undetermined significance have an abnormal free light chain ratio, and these patients harbor a greater risk of progression to plasma cell dyscrasia. For monitoring response to therapy, the international uniform response criteria define a normal free light chain ratio as an essential element of the "stringent complete response" category. Because the half-life of free light chains is chain measurements at short sampling intervals allow real-time measurement of treatment-induced tumor kill, and provide prompt indications of chemosensitivity, dose adequacy, need for alternative approaches, and even prognosis, as demonstrated in AL amyloidosis if the involved free light chain concentration normalizes. Clinical applications of these assays will likely increase as their utility is more widely explored.

  6. Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

    Science.gov (United States)

    Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

    2014-11-07

    We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

  7. Microscopic theory of light-induced deformation in amorphous side-chain azobenzene polymers.

    Science.gov (United States)

    Toshchevikov, V; Saphiannikova, M; Heinrich, G

    2009-04-16

    We propose a microscopic theory of light-induced deformation of side-chain azobenzene polymers taking into account the internal structure of polymer chains. Our theory is based on the fact that interaction of chromophores with the polarized light leads to the orientation anisotropy of azobenzene macromolecules which is accompanied by the appearance of mechanical stress. It is the first microscopic theory which provides the value of the light-induced stress larger than the yield stress. This result explains a possibility for the inscription of surface relief gratings in glassy side-chain azobenzene polymers. For some chemical architectures, elongation of a sample demonstrates a nonmonotonic behavior with the light intensity and can change its sign (a stretched sample starts to be uniaxially compressed), in agreement with experiments. Using a viscoplastic approach, we show that the irreversible strain of a sample, which remains after the light is switched off, decreases with increasing temperature and can disappear at certain temperature below the glass transition temperature. This theoretical prediction is also confirmed by recent experiments.

  8. Light-assisted, templated self-assembly of gold nanoparticle chains.

    Science.gov (United States)

    Jaquay, Eric; Martínez, Luis Javier; Huang, Ningfeng; Mejia, Camilo A; Sarkar, Debarghya; Povinelli, Michelle L

    2014-09-10

    We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS) to trap and assemble 200 nm diameter gold nanoparticles. We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. Unlike our previous demonstration of LATS with polystyrene particles, we find that the interparticle interactions play a significant role in the resulting particle patterns. Despite a two-dimensionally periodic intensity profile in the slab, the particles form one-dimensional chains whose orientations can be controlled by the incident polarization of the light. The formation of chains can be understood in terms of a competition between the gradient force due to the excitation of the mode in the slab and optical binding between particles.

  9. Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

    Science.gov (United States)

    Torán, J L; Sánchez-Pulido, L; Kremer, L; del Real, G; Valencia, A; Martínez-A, C

    2001-01-01

    We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

  10. De Novo and Inherited Mutations in COL4A2, Encoding the Type IV Collagen α2 Chain Cause Porencephaly

    Science.gov (United States)

    Yoneda, Yuriko; Haginoya, Kazuhiro; Arai, Hiroshi; Yamaoka, Shigeo; Tsurusaki, Yoshinori; Doi, Hiroshi; Miyake, Noriko; Yokochi, Kenji; Osaka, Hitoshi; Kato, Mitsuhiro; Matsumoto, Naomichi; Saitsu, Hirotomo

    2012-01-01

    Porencephaly is a neurological disorder characterized by fluid-filled cysts or cavities in the brain that often cause hemiplegia. It has been suggested that porencephalic cavities result from focal cerebral degeneration involving hemorrhages. De novo or inherited heterozygous mutations in COL4A1, which encodes the type IV α1 collagen chain that is essential for structural integrity for vascular basement membranes, have been reported in individuals with porencephaly. Most mutations occurred at conserved Gly residues in the Gly-Xaa-Yaa repeats of the triple-helical domain, leading to alterations of the α1α1α2 heterotrimers. Here we report on two individuals with porencephaly caused by a heterozygous missense mutation in COL4A2, which encodes the type IV α2 collagen chain. Mutations c.3455G>A and c.3110G>A, one in each of the individuals, cause Gly residues in the Gly-Xaa-Yaa repeat to be substituted as p.Gly1152Asp and p.Gly1037Glu, respectively, probably resulting in alterations of the α1α1α2 heterotrimers. The c.3455G>A mutation was found in the proband's mother, who showed very mild monoparesis of the left upper extremity, and the maternal elder uncle, who had congenital hemiplegia. The maternal grandfather harboring the mutation is asymptomatic. The c.3110G>A mutation occurred de novo. Our study confirmed that abnormalities of the α1α1α2 heterotrimers of type IV collagen cause porencephaly and stresses the importance of screening for COL4A2 as well as for COL4A1. PMID:22209246

  11. Exempting homologous pseudogene sequences from polymerase chain reaction amplification allows genomic keratin 14 hotspot mutation analysis

    NARCIS (Netherlands)

    Hut, PHL; van der Vlies, P; Jonkman, MF; Verlind, E; Shimizu, H; Buys, CHCM; Scheffer, H

    In patients with the major forms of epidermolysis bullosa simplex, either of the keratin genes KRT5 or KRT14 is mutated. This causes a disturbance of the filament network resulting in skin fragility and blistering. For KRT5, a genomic mutation detection system has been described previously. Mutation

  12. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans

    Science.gov (United States)

    Wojtovich, Andrew P.; Wei, Alicia Y.; Sherman, Teresa A.; Foster, Thomas H.; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

  13. Distribution of kappa and lambda light chain isotypes among human blood immunoglobulin-secreting cells after vaccination with pneumococcal polysaccharides

    DEFF Research Database (Denmark)

    Heilmann, C; Barington, T

    1989-01-01

    The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pok...

  14. Co-deposition of amyloidogenic immunoglobulin light and heavy chains in localized pulmonary amyloidosis.

    Science.gov (United States)

    Kaplan, Batia; Martin, Brian M; Boykov, Olga; Gal, Rivka; Pras, Mordechai; Shechtman, Itzhak; Saute, Milton; Kramer, Mordechai R

    2005-10-01

    Localized pulmonary amyloidosis is a rare condition whose pathogenesis is insufficiently understood. In the present study, we report a case of localized pulmonary amyloidosis associated with lung-restricted lymphoplasmacytoid lymphoma, monoclonal for immunoglobulin (Ig) G lambda (lambda). Biochemical microtechniques have been applied for extraction, purification, and characterization of amyloid proteins. Surprisingly, chemical analysis of these proteins revealed a not-previously-described case of combined deposits containing Ig fragments of gamma heavy chain (variable domain) and lambda light chain (constant domain). In view of the absence of circulating monoclonal Ig, this case supports the hypothesis that localized amyloid is formed by local plasmacytoid cells.

  15. Deposition of kappa and lambda light chains in amyloid filaments of dialysis-related amyloidosis.

    Science.gov (United States)

    Brancaccio, D; Ghiggeri, G M; Braidotti, P; Garberi, A; Gallieni, M; Bellotti, V; Zoni, U; Gusmano, R; Coggi, G

    1995-10-01

    beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

  16. Homozygosity for a novel mutation in the C1q C chain gene in a Turkish family with hereditary C1q deficiency

    DEFF Research Database (Denmark)

    Gulez, N; Genel, F; Atlihan, F;

    2010-01-01

    . Immunologic studies revealed absence of classic pathway hemolytic activity and undetectable levels of Clq. Exon-specific amplification of genomic DNA by polymerase chain reaction followed by direct sequence analysis revealed a novel homozygous missense mutation at codon 48 in the C1q C gene causing a glycine......-to-arginine substitution affecting the collagen-like region of C1q. No changes were seen in the exons of the A and B chains. The mutation affected both the formation and the secretion of C1q variant molecules. We describe a novel mutation in the C1q C chain gene that leads to an interchange in amino acids resulting...

  17. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  18. Three cases of congenital dysfibrinogenemia in unrelated Chinese families: heterozygous missense mutation in fibrinogen alpha chain Argl6His.

    Science.gov (United States)

    Luo, Meiling; Deng, Donghong; Xiang, Liqun; Cheng, Peng; Liao, Lin; Deng, Xuelian; Yan, Jie; Lin, Faquan

    2016-09-01

    Congenital dysfibrinogenemia (CD) is a qualitative fibrinogen disorder caused by an abnormal fibrinogen molecule structure, leading to dysfunctional blood coagulation. This study describes 3 cases of dysfibrinogenemia identified in the unrelated Chinese pedigrees.Routine coagulation screening tests were performed on the probands and their families. The antigens and functionality of fibrinogen was measured using an immunoturbidimetry assay and the Clauss method, respectively. To identify the genetic mutation responsible for these dysfibrinogens, genomic DNA extracted from the blood was analyzed using PCR amplification and direct sequencing. The presence of the mutant chains was determined using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy. Purified plasma fibrinogen of 3 probands was analyzed using SDS-PAGE, fibrinogen clottability, fibrin polymerization, fibrinopeptide release, and scanning electron microscopy (SEM).The 3 probands had a long thrombin time. Levels of functional fibrinogen were found to be very low, while the fibrinogen antigen was within the normal range. DNA sequencing revealed a heterozygous Arg16His substitution in the fibrinogen Aα chain (FGA). The mutant chains were found to be expressed using MALDI-TOF mass spectroscopy. SDS-PAGE did not reveal any difference in the molecular weights of 3 polypeptide chains between normal and abnormal fibrinogens. Fibrinogen clottability showed a slower fibrin clot formation than the healthy control. Fibrin polymerization, after addition of thrombin, showed a prolonged lag phase and decreased final turbidity. The kinetics of fibrinopeptides release revealed a decreased amount of the released fibrinopeptide A. SEM of the patient's fibrin clot was found to be abnormal.Results indicate that the 3 probands with dysfibrinogenemia were caused by mutations of Aα chain Arg16His. Mutation of this fibrinogen induced dysfunction of plasma fibrinogen.

  19. IMMUNOMETRIC ASSAY TO DETERMINE FREE LIGHT CHAIN CONCENTRATIONS OF HUMAN IMMUNOGLOBULINS

    Directory of Open Access Journals (Sweden)

    M. P. Samoylovich

    2016-01-01

    Full Text Available Detection of total free light chains (FLC of immunoglobulins and their ratio (kappa/lambda quotient are used in diagnostics and monitoring of multiple myeloma and other gammapathies, primary amyloidosis and multiple sclerosis. Previously described immunoassays with monoclonal antibodies (Mabs against cryptic and constantly exposed epitopes of FLC failed to recognize rare variants of lambda Bence-Jones proteins and a significant proportion of lambda chains excreted with urine. Aiming to improve this approach, a novel murine Mab (IgG2b coded as 1C8 was employed, which specifically binds free lambda chains but doesn’t interact with native IgA, IgG, and IgM. The novel Mab recognized an epitope exposed at free lambda chains in peripheral blood of healthy donors and patients with multiple myeloma. It is not destroyed or masked upon renal filtration.The aim of this study was to determine basic features of improved assay system, and to estimate its potential in diagnostics of monoclonal gammapathies. The mixtures of three Bence-Jones proteins of either kappa- or lambda- types purified from the urine of multiple myeloma patients were used as calibrator samples.Improved immunometric assay is able to detect free kappa and lambda chains in serum and urine at a scale of 1 to 100 ng/ml, thus being three orders more sensitive than, e.g., detection levels of Freelite method based on polyclonal antibodies.A novel assay allows to detect free kappa and lambda chains at comparable levels in serum or urine, and to deduce kappa/lambda ratio. The proposed assay is able to detect FLC in 10,000-fold excess of whole IgG molecules. The calibrating plots for both antigens are linear on log-log scales, with very similar slopes. Detection thresholds for kappa or lambda chains proved to be 5 and 3 ng/ml, respectively. Mean concentrations of free kappa chains in sera of healthy donors were 6.7±2.1, in urine, 4.2±3.8 mcg/ml. Mean concentrations of free lambda chains were 4

  20. Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease

    Directory of Open Access Journals (Sweden)

    Timo Friedrich

    2012-03-01

    Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt, a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD, a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

  1. Power-Law Behavior in Signal Scattering Process in Vertical Granular Chain with Light Impurities

    Institute of Scientific and Technical Information of China (English)

    XU Ai-Guo

    2001-01-01

    We investigate the scattering process of impulse in vertical granular chain with light impurities. When the perturbation is weak, the quantities describing the reflection rate exhibit power-law behavior with the impurity depth. The exponent is nearly independent of vi. When the perturbation is very strong, the vertical chain shows similar behavior to that of the horizontal chain, so the exponent is nearly zero. Our numerical investigation begins from the weak perturbation region and extends to the nonlinear region and found a peak of the exponent. The difficulty in extending the numerical investigation to a stronger perturbation region is analyzed.PACS numbers: 45.70.-n, 43.25.+y, 46.40.Cd

  2. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody...

  3. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib...

  4. Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens.

    Science.gov (United States)

    Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

    2015-12-01

    Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.

  5. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  6. Light-chain deposition disease of the kidney: a case report.

    Science.gov (United States)

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Maiz, Hedi Ben

    2012-04-01

    A 41-year-old man was admitted for evaluation of nephrotic syndrome associated with microhematuria, hypertension, and moderate renal failure. In serum and urine samples, monoclonal IgG-lambda was detected. Bone marrow examination showed normal representation of all cell lines with normal range of plasma cells. Renal biopsy demonstrated diabetes-like nodular glomerulosclerosis. Immunofluorescence failed to demonstrate the presence of kappa or lambda light chains in the kidney. Electron microcopy showed granular electron-dense deposits along the glomerular basement membranes and in the mesangial nodules. The patient was diagnosed as having light-chain deposition disease (LCDD) without evidence of plasma cell dyscrasia. This report was designed to stress the significant challenges that remain in the diagnosis of LCDD-related glomerulopathy. The salient morphological features that help in making an accurate diagnosis are discussed.

  7. Unique morphology of intratubular light chain casts in multiple myeloma: The amyloid cast nephropathy

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2014-01-01

    Full Text Available Cast nephropathy is the most frequent pattern of renal involvement in multiple myeloma characterized by presence of tubular casts with characteristic morphology that are composed of monotypic (either kappa or lambda light chains as seen by immunofluorescence microscopy. Rarely these casts may show evidence of amyloidogenesis and assume a unique morphology, which needs to be appreciated for arriving at accurate diagnosis. We present the case of an elderly male presenting with features of acute kidney injury and detected with extensive inspissation of intratubular casts with lambda light chain restriction and a unique morphology with spiculated congophilic periphery. Further investigations confirmed the presence of systemic myeloma. Presence of intratubular amyloid casts is a rare occurrence which needs to be recognized by the pathologist and forms a vital element in timely diagnosis of the systemic disease which often presents with renal involvement.

  8. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers

    OpenAIRE

    Das, Sabyasachi; Nikolaidis, Nikolas; Klein, Jan; Nei, Masatoshi

    2008-01-01

    The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (κ, λ, and σ isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess thr...

  9. Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

    Science.gov (United States)

    2013-04-01

    Synaptic Research will develop dichain hybrids consisting of Clostridium botulinum toxin light chains (LCs) from serotypes A (long-lived) and E...provided this plasmid to the McNutt laboratory that will use a modified version of this virus to produce transgenic mice expressing the designer...interactions via FRET using 24 transgenic lines that co-express fluorescently tagged LCs and target proteins. After optimization of pull-down

  10. 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite

    Directory of Open Access Journals (Sweden)

    Guest Editor: A.R. Bradwell

    2011-08-01

    Full Text Available The 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite was held in Bath, UK on 23rd and 24th September 2010. This Symposium included measurements in monoclonal gammopathies, myeloma kidney and inflammatory diseases; data from the Multi-centre, Phase III, Randomised control trial on extended haemodialysis with the Gambro protein-leaking HCO 1100 dialyser; and presentations on Hevylite (intact immunoglobulin kappa/lambda ratios in a variety of clinical situations.

  11. 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite)

    OpenAIRE

    Guest Editor: A.R. Bradwell

    2011-01-01

    The 6th International Symposium on Clinical Applications of Serum Free Light Chain Analysis (plus Hevylite) was held in Bath, UK on 23rd and 24th September 2010. This Symposium included measurements in monoclonal gammopathies, myeloma kidney and inflammatory diseases; data from the Multi-centre, Phase III, Randomised control trial on extended haemodialysis with the Gambro protein-leaking HCO 1100 dialyser; and presentations on Hevylite (intact immunoglobulin kappa/lambda ratios) in a variety ...

  12. Spinon and bound-state excitation light cones in Heisenberg XXZ chains

    Science.gov (United States)

    de Paula, A. L.; Bragança, H.; Pereira, R. G.; Drumond, R. C.; Aguiar, M. C. O.

    2017-01-01

    We investigate the out-of-equilibrium dynamics after a local quench that connects two spin-1/2 XXZ chains prepared in the ground state of the Hamiltonian in different phases, one in the ferromagnetic phase and the other in the critical phase. We analyze the time evolution of the on-site magnetization and bipartite entanglement entropy via adaptive time-dependent density matrix renormalization group. In systems with short-range interactions, such as the one we consider, the velocity of information transfer is expected to be bounded, giving rise to a light-cone effect. Interestingly, our results show that, when the anisotropy parameter of the critical chain is sufficiently close to that of the isotropic ferromagnet, the light cone is determined by the velocity of spin-wave bound states that propagate faster than single-particle ("spinon") excitations. Furthermore, we investigate how the system approaches equilibrium in the inhomogeneous ground state of the connected system, in which the ferromagnetic chain induces a nonzero magnetization in the critical chain in the vicinity of the interface.

  13. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    Science.gov (United States)

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

  14. [The value of serum free light chain in differential diagnosis of monoclonal gammopathy of renal significance].

    Science.gov (United States)

    Li, C; Wen, Y B; Li, H; Su, W; Li, J; Cai, J F; Chen, L M; Li, X M; Li, X W

    2017-08-08

    Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS (n=32) and monoclonal gammopathy of undetermined significance (MGUS) (n=17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis (n=24, 75.0%), light chain deposition disease (n=7, 21.9%), and fibrillary glomerulopathy (n=1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy (n=10), focal segmental glomerulosclerosi (FSGS) (n=3), diabetic glomerulopathy (n=1), Henoch-Schonlein purpura nephritis (n=1), anti-GBM disease concurrent with membranous nephropathy (n=1) and glomerulomegaly (n=1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE (Pvalue for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

  15. Computational design of a specific heavy chainlight chain interface for expressing fully IgG bispecific antibodies.

    Science.gov (United States)

    Froning, K J; Leaver-Fay, A; Wu, X; Phan, S; Gao, L; Huang, F; Pustilnik, A; Bacica, M; Houlihan, K; Chai, Q; Fitchett, J R; Hendle, J; Kuhlman, B; Demarest, S J

    2017-07-20

    The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use. © 2017 The Protein Society.

  16. Homozygosity for the E526V Mutation in Fibrinogen A Alpha-Chain Amyloidosis: The First Report

    Directory of Open Access Journals (Sweden)

    Isabel Tavares

    2015-01-01

    Full Text Available Systemic hereditary amyloidoses are autosomal dominant diseases associated with mutations in genes encoding ten different proteins. The clinical phenotype has implications on therapeutic approach, but it is commonly variable and largely dependent on the type of mutation. Except for rare cases involving gelsolin or transthyretin, patients are heterozygous for the amyloidogenic variants. Here we describe the first patient identified worldwide as homozygous for a nephropathic amyloidosis, involving the fibrinogen variant associated with the fibrinogen alpha-chain E526V (p.Glu545Val mutation. In 1989, a 44-year-old woman presented with hypertension, hepatosplenomegaly, nephrotic syndrome, and renal failure. She started hemodialysis in 1990 and 6 years later underwent isolated kidney transplantation from a deceased donor. Graft function and clinical status were unremarkable for 16 years, despite progressively increased left ventricular mass on echocardiography. In 2012, 4 months before death, she deteriorated rapidly with severe heart failure, precipitated by Clostridium difficile colitis and urosepsis. Affected family members developed nephropathy, on average, nearly three decades later, which may be explained by the gene dosage effects on the phenotype of E526V (p.Glu545Val fibrinogen A alpha-chain amyloidosis.

  17. Spontaneous mutation 7B-1 in tomato impairs blue light-induced stomatal opening.

    Science.gov (United States)

    Hlavinka, Jan; Nauš, Jan; Fellner, Martin

    2013-08-01

    It was reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L.), an ABA overproducer, is defective in blue light (BL) signaling leading to BL-specific resistance to abiotic and biotic stresses. In this work, we examine responses of stomata to blue, red and white lights, fusicoccin, anion channel blockers (anthracene-9-carboxylic acid; 9-AC and niflumic acid; NIF) and ABA. Our results showed that the aperture of 7B-1 stomata does not increase in BL, suggesting that 7B-1 mutation impairs an element of BL signaling pathway involved in stomatal opening. Similar stomatal responses of 7B-1 and wild type (WT) to fusicoccin or 9-AC points out that activity of H(+)-ATPase and 9-AC-sensitive anion channels per se is not likely affected by the mutation. Since 9-AC restored stomatal opening of 7B-1 in BL, it seems that 9-AC and BL could block similar type of anion channels. The stomata of both genotypes did not respond to NIF neither in darkness nor in any light conditions tested. In light, 9-AC but not NIF restored stomatal opening inhibited by ABA in WT and 7B-1. We suggest that in comparison to WT, the activity of S-type anion channels in 7B-1 is more promoted by increased ABA content, and less reduced by BL, because of the mutant resistance to BL.

  18. Joint inference of microsatellite mutation models, population history and genealogies using transdimensional Markov Chain Monte Carlo.

    Science.gov (United States)

    Wu, Chieh-Hsi; Drummond, Alexei J

    2011-05-01

    We provide a framework for Bayesian coalescent inference from microsatellite data that enables inference of population history parameters averaged over microsatellite mutation models. To achieve this we first implemented a rich family of microsatellite mutation models and related components in the software package BEAST. BEAST is a powerful tool that performs Bayesian MCMC analysis on molecular data to make coalescent and evolutionary inferences. Our implementation permits the application of existing nonparametric methods to microsatellite data. The implemented microsatellite models are based on the replication slippage mechanism and focus on three properties of microsatellite mutation: length dependency of mutation rate, mutational bias toward expansion or contraction, and number of repeat units changed in a single mutation event. We develop a new model that facilitates microsatellite model averaging and Bayesian model selection by transdimensional MCMC. With Bayesian model averaging, the posterior distributions of population history parameters are integrated across a set of microsatellite models and thus account for model uncertainty. Simulated data are used to evaluate our method in terms of accuracy and precision of estimation and also identification of the true mutation model. Finally we apply our method to a red colobus monkey data set as an example.

  19. Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme

    DEFF Research Database (Denmark)

    Bross, P; Jespersen, C; Jensen, T G

    1995-01-01

    We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding...

  20. Dilated cardiomyopathy mutation (R134W in mouse cardiac troponin T induces greater contractile deficits against α-myosin heavy chain than against β-myosin heavy chain

    Directory of Open Access Journals (Sweden)

    Sampath K Gollapudi

    2016-10-01

    Full Text Available Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM in humans. However, it remains unclear whether a single determinant — such as myofilament Ca2+ sensitivity — is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT, which is known to modulate crossbridge (XB recruitment dynamics in a myosin heavy chain (MHC-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W of the human DCM mutation (R131W, we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC and transgenic mice (β-MHC. TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units than in β-MHC fiber bundles (0.08 pCa units. Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

  1. Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

    Science.gov (United States)

    Shamblott, M J; Litman, G W

    1989-01-01

    The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving. Images PMID:2511000

  2. A novel fibrinogen B beta chain frameshift mutation causes congenital afibrinogenaemia.

    Science.gov (United States)

    Zhang, Jian; Zhao, Xiaojuan; Wang, Zhaoyue; Yu, Ziqiang; Cao, Lijuan; Zhang, Wei; Bai, Xia; Ruan, Changgeng

    2013-07-01

    Congenital afibrinogenaemia is a rare autosomal recessive disorder caused by various mutations within the fibrinogen genes FGA, FGB and FGG. Ins/del mutations in FGB are extremely rare. We report a patient with afibrinogenaemia who suffered from umbilical cord bleeding and repeated bleeding episodes. His plasma fibrinogen levels could not be detected using the Clauss method and immunological methods. Molecular analyses revealed homozygosity in a novel four bases insertion in codon 40 of FGB exon 2 (g. 2833_2834 ins GTTT), which resulted in a truncated 50-residue polypeptide that contained 11 exceptional abnormal residues. In the transient expression experiments, mutant fibrinogen could be detected at higher level than wild-type fibrinogen in COS-7 cell lysates but not in culture media. These results suggest that the homozygous mutation in FGB could be responsible for congenital afibrinogenaemia in this patient. This frameshift mutation could impair fibrinogen assembly and secretion without influencing the protein synthesis.

  3. Identifikation und Charakterisierung von Medium Chain Acyl-CoA Dehydrogenase und Myosin Light Chain 4 als neuartige Interaktionspartner des Östrogenrezeptor alpha

    OpenAIRE

    Schanz, Miriam A.

    2012-01-01

    Estrogens (E2) are key regulators of growth, differentiation and physiological processes in various target tissues, including the human heart. E2 exerts its effects mainly through its cognate receptors, e.g.estrogen receptor (ER) α. ERα acts in concert with other interaction partners to mediate estrogenic effects. So far, only few interaction partners of ERα are known in the human myocardium. This project identified Medium Chain Acyl-CoA Dehydrogenase (MCAD) and atrial Myosin Light Chain ...

  4. Mutation in the alpha 5(IV) collagen chain in juvenile-onset Alport syndrome without hearing loss or ocular lesions

    DEFF Research Database (Denmark)

    Zhou, J; Hertz, Jens Michael; Tryggvason, K

    1992-01-01

    A single base mutation was identified in the type IV collagen alpha 5 chain gene (COL4A5) of a Danish kindred with Alport syndrome. The 27-year-old male proband developed hematuria in childhood and terminal renal failure at the age of 25 years. He has no hearing loss or ocular lesions. Electron.......9-kb fragments present in control DNA but the presence of a 2.2-kb variant fragment, indicating the loss of an MspI restriction site in the 3' end of the gene. The mother had all three fragments, indicating heterozygosity. PCR amplification of exon 14 (counted from the 3' end) and subsequent denaturing...

  5. [Aspect of brain MRI in mitochondrial respiratory chain deficiency. A diagnostic algorithm of the most common mitochondrial genetic mutations].

    Science.gov (United States)

    Devaux-Bricout, M; Grévent, D; Lebre, A-S; Rio, M; Desguerre, I; De Lonlay, P; Valayannopoulos, V; Brunelle, F; Rötig, A; Munnich, A; Boddaert, N

    2014-05-01

    Mitochondrial diseases are due to deficiency of the respiratory chain and are characterized by a broad clinical and genetic heterogeneity that makes diagnosis difficult. Some clinical presentations are highly suggestive of given gene mutations, allowing rapid genetic diagnosis. However, owing to the wide pattern of symptoms in mitochondrial disorders and the constantly growing number of disease genes, their genetic diagnosis is frequently difficult and genotype/phenotype correlations remain elusive. For this reason, brain MRI appears as a useful tool for genotype/phenotype correlations. Here, we report the most frequent neuroradiological signs in mitochondrial respiratory chain deficiency and we propose a diagnostic algorithm based on neuroimaging features, so as to direct molecular genetic tests in patients at risk of mitochondrial respiratory chain deficiency. This algorithm is based on the careful analysis of five areas on brain MRI: (1) basal ganglia (hyperintensities on T2 or calcifications); (2) cerebellum (hyperintensities on T2 or atrophy); (3) brainstem (hyperintensities on T2 or atrophy); (4) white matter (leukoencephalopathy); (5) cortex (sub-tentorial atrophy); (6) stroke-like episodes. We believe that the combination of brain MRI features is of value to support respiratory chain deficiency and direct molecular genetic tests.

  6. Role of single-point mutations and deletions on transition temperatures in ideal proteinogenic heteropolymer chains in the gas phase.

    Science.gov (United States)

    Olivares-Quiroz, L

    2016-07-01

    A coarse-grained statistical mechanics-based model for ideal heteropolymer proteinogenic chains of non-interacting residues is presented in terms of the size K of the chain and the set of helical propensities [Formula: see text] associated with each residue j along the chain. For this model, we provide an algorithm to compute the degeneracy tensor [Formula: see text] associated with energy level [Formula: see text] where [Formula: see text] is the number of residues with a native contact in a given conformation. From these results, we calculate the equilibrium partition function [Formula: see text] and characteristic temperature [Formula: see text] at which a transition from a low to a high entropy states is observed. The formalism is applied to analyze the effect on characteristic temperatures [Formula: see text] of single-point mutations and deletions of specific amino acids [Formula: see text] along the chain. Two probe systems are considered. First, we address the case of a random heteropolymer of size K and given helical propensities [Formula: see text] on a conformational phase space. Second, we focus our attention to a particular set of neuropentapeptides, [Met-5] and [Leu-5] enkephalins whose thermodynamic stability is a key feature on their coupling to [Formula: see text] and [Formula: see text] receptors and the triggering of biochemical responses.

  7. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Tsuda, Takashi; Izumi, Tohru; Shibata, Akira (Niigata Univ. (Japan). School of Medicine)

    1992-08-01

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51[+-]11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of [sup 201]Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author).

  8. Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis.

    Science.gov (United States)

    Boyne, J R; Yosuf, H M; Bieganowski, P; Brenner, C; Price, C

    2000-12-01

    MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G(1) and the growing bud tip during S and G(2) phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.

  9. Prognostic Significance of Serum Free Light Chains in Chronic Lymphocytic Leukemia

    OpenAIRE

    2013-01-01

    Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet ...

  10. Super-light and pearl-chain technology for support of ancient structures

    DEFF Research Database (Denmark)

    Hertz, Kristian Dahl; Schmidt, Jacob Wittrup; Goltermann, Per

    2014-01-01

    optimal and often curved paths of strong concrete assembled from smaller segments by prestressing wires, so that expensive curved moulds and supports can be avoided. Pearl-chains can provide a resistance to impact and earthquake of ancient structures. High-strength concrete and prestressed carbon fibre...... reinforcement may be applied, because the new technology solves the main problems for that, since the light aggregate concrete provides a fire protection needed for both materials and provides a stabilization of the slender cores in compression. The paper explains more about the new technology...

  11. Detection of beta-globin gene mutations among Kelantan Malay thalassaemia patients by polymerase chain reaction restriction fragment length polymorphism.

    Science.gov (United States)

    Rozitah, R; Nizam, M Z; Nur Shafawati, A R; Nor Atifah, M A; Dewi, M; Kannan, T P; Ariffin, N; Norsarwany, M; Setianingsih, I; Harahap, A; Zilfalil, B A

    2008-12-01

    Beta-thalassaemia major is an autosomal recessive disorder that results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. Beta-thalassaemia has emerged as one of the most common public health problems in Malaysia, particularly among Malaysian Chinese and Malays. This study aimed to observe the spectrum of mutations found in Kelantan Malay beta-thalassaemia major patients who attended the Paediatrics Daycare Unit, Hospital Universiti Sains Malaysia, Kelantan, Malaysia, the data of which was being used in establishing the prenatal diagnosis in this Human Genome Centre. This was a cross-sectional study conducted with 35 Kelantan Malay beta-thalassaemia major patients. DNA was extracted from the blood collected from the patients and subjected to polymerase chain reaction (PCR) amplification. Six restriction enzymes were used to digest the PCR products for the detection of mutations. Five out of the six beta-globin gene defects were detected, namely, IVS-1 nt5 (G>C), IVS-1 nt1 (G>T), codon 26 (G>A), codon 41-42 (4 bp del) and codon 19 (A>G). The mutation which was not observed in this study was in codon 15 (G>A). The two most common mutations observed were codon 26 (G>A) and IVS-1 nt5 (G>C), which was detected in 26 and 17 patients, respectively. Two patients did not show any of the six mutations. Our results added to the existing data on the common beta-globin gene defects in Kelantan Malay beta-thalassaemia patients.

  12. Effect of specimen type on free immunoglobulin light chains analysis on the Roche Diagnostics cobas 8000 analyzer.

    Science.gov (United States)

    Nelson, Louis S; Steussy, Bryan; Morris, Cory S; Krasowski, Matthew D

    2015-01-01

    The measurement of free immunoglobulin light chains is typically performed on serum; however, the use of alternative specimen types has potential benefits. Using the Freelite™ kappa and lambda free light chains assay on a Roche Diagnostics cobas 8000 c502 analyzer, we compared three specimen types (serum, EDTA-plasma and lithium heparin plasma separator gel-plasma) on 100 patients. Using Deming regression and eliminating outliers (limiting data to light chain concentrations below 400 mg/L), the three specimen types showed comparable results for kappa light chain concentration, lambda light chain concentration, and kappa/lambda ratio with slopes close to 1.0 and y-intercepts close to zero. EDTA-plasma showed slightly more positive bias relative to serum than lithium heparin. Analysis using EDTA-plasma and lithium heparin plasma showed comparable linearity, precision, and temperature stability. A single sample showing hook effect (not in the comparison set) gave comparable results using either plasma specimen type. For the Freelite™ kappa and lambda free light chains assay, both EDTA-plasma or lithium heparin-plasma can serve as acceptable substitutes for serum, at least for the Roche cobas 8000 analyzer.

  13. Fibrinogen geneva II: a new congenitally abnormal fibrinogen alpha chain (Gly17Asp) with a review of similar mutations resulting in abnormal knob A.

    Science.gov (United States)

    Casini, Alessandro; De Maistre, Emmanuel; Casini-Stuppi, Virginie; Fontana, Pierre; Neerman-Arbez, Marguerite; de Moerloose, Philippe

    2014-04-01

    Congenital dysfibrinogenemias are characterized by biosynthesis of a structurally abnormal fibrinogen molecule that exhibits reduced functional levels compared with the level of fibrinogen antigen. To date a large number of mutations have been identified in patients with dysfibrinogenemia. Mutations occurring at the thrombin cleavage site (Arg16-Gly17 in the mature alpha-chain) at the amino-terminal end of the fibrinogen alpha chain are a common cause of the disease. These mutations causing abnormal fibrin polymerization are associated with different phenotypes. Here, we report the identification of a novel heterozygous missense mutation of Glycine 17 (Gly17Asp) in a female patient with mild bleeding manifestations, and compare it with other previously reported mutations also resulting in abnormal knob A.

  14. Single-Tube Mutation Scanning of The Epidermal Growth Factor Receptor Gene Using Multiplex LATE-PCR and Lights-On/Lights-Off Probes

    Science.gov (United States)

    Tetrault, Shana M.; Rice, John E.; Wangh, Lawrence J.; Sanchez, J. Aquiles

    2014-01-01

    Background Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21. Methods The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation. Results The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies. Conclusion The LATE-PCR EGFR assay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies. PMID:25411647

  15. Reversible Addition-Fragmentation Chain Transfer Polymerization of Acrylonitrile under Irradiation of Blue LED Light

    Directory of Open Access Journals (Sweden)

    Zhicheng Huang

    2016-12-01

    Full Text Available Compared to unhealthy UV or γ-ray and high-energy-consumption thermal external stimuli, the promising light emitting diode (LED external stimulus has some outstanding technological merits such as narrow wavelength distribution, low heat generation and energy consumption, and safety for human beings. In this work, a novel reversible addition-fragmentation transfer (RAFT polymerization system for acrylonitrile (AN was developed under the irradiation of blue LED light at room temperature, using 1,2,3,5-tetrakis(carbazol-9-yl-4,6-dicyanobenzene (4CzIPN as a novel radical initiator and 2-cyanoprop-2-yl-1-dithionaphthalate (CPDN as the typical chain transfer agent. Well-defined polyacrylonitrile (PAN with a controlled molecular weight and narrow molecular weight distribution was successfully synthesized. This strategy may provide another effective method for scientific researchers or the industrial community to synthesize a PAN-based precursor of carbon fibers.

  16. Vernier zone residue of 4 mouse subgroup II kappa light chains is a critical determinant for antigen recognition

    NARCIS (Netherlands)

    Haard, de H.; Kazemier, B.; Bent, van der A.; Oudshoorn, P.; Boender, P.; Arends, J.W.; Gemen, van B.

    1999-01-01

    Background: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. Objectives: In this study

  17. Bos taurus genome sequence reveals the assortment of immunoglobulin and surrogate light chain genes in domestic cattle

    Directory of Open Access Journals (Sweden)

    Liljavirta Jenni

    2009-04-01

    Full Text Available Abstract Background The assortment of cattle immunoglobulin and surrogate light chain genes has been extracted from the version 3.1 of Bos taurus genome sequence as a part of an international effort to sequence and annotate the bovine genome. Results 63 variable lambda chain and 22 variable kappa chain genes were identified and phylogenetically assigned to 8 and 4 subgroups, respectively. The specified phylogenetic relationships are compatible with the established ruminant light chain variable gene families or subgroups. Because of gaps and uncertainties in the assembled genome sequence, the number of genes might change in the future versions of the genome sequence. In addition, three bovine surrogate light chain genes were identified. The corresponding cDNAs were cloned and the expression of the surrogate light chain genes was demonstrated from fetal material. Conclusion The bovine kappa gene locus is compact and simple which may reflect the preferential use of the lambda chain in cattle. The relative orientation of variable and joining genes in both loci are consistent with a deletion mechanism in VJ joining. The orientation of some variable genes cannot be determined from the data available. The number of functional variable genes is moderate when compared to man or mouse. Thus, post-recombinatorial mechanisms might contribute to the generation of the bovine pre-immune antibody repertoire. The heavy chains probably contribute more to recombinational immunoglobulin repertoire diversity than the light chains but the heavy chain locus could not be annotated from the version 3.1 of Bos taurus genome.

  18. Mapping a Mutation in "Caenorhabditis elegans" Using a Polymerase Chain Reaction-Based Approach

    Science.gov (United States)

    Myers, Edith M.

    2014-01-01

    Many single nucleotide polymorphisms (SNPs) have been identified within the "Caenorhabditis elegans" genome. SNPs present in the genomes of two isogenic "C. elegans" strains have been routinely used as a tool in forward genetics to map a mutation to a particular chromosome. This article describes a laboratory exercise in which…

  19. Spontaneous recurrent mutations and a complex rearrangement in the MECP2 gene in the light of current models of mutagenesis.

    Science.gov (United States)

    Todorov, Tihomir; Todorova, Albena; Motoescu, Cristina; Dimova, Petia; Iancu, Daniela; Craiu, Dana; Stoian, Daniela; Barbarii, Ligia; Bojinova, Veneta; Mitev, Vanyo

    2012-06-01

    Mutations in the methyl-CpG-binding protein 2 (MECP2) gene are associated with Rett syndrome (RTT). The MECP2 gene has some unique characteristics: (1) it is mainly affected by de novo mutations, due to recurrent independent mutational events in a defined "hot spot" regions or positions; (2) complex mutational events along a single allele are frequently found in this gene; (3) most mutations arise on paternal X chromosome. The recurrent point mutations involve mainly CpG dinucleotides, where C>T transitions are explained by methylation-mediated deamination. The complex mutational events might be explained by the genomic architecture of the region involving the MECP2 gene. The finding that most spontaneous mutations arise on paternal X-chromosome supports the higher contribution of replication-mediated mechanism of mutagenesis. We present 9 types of mutations in the MECP2 gene, detected in a group of 22 Bulgarian and 6 Romanian classical RTT patients. Thirteen patients were clarified on molecular level (46.4%). The point mutations in our sample account for 61.5%. One intraexonic deletion was detected in the present study (7.7%). One novel insertion c.321_322insGAAG, p.(Lys107_Leu108insGluAlafs2*) was found (7.7%). Large deletions and complex mutations account for 23%. A novel complex mutational event c.[584_624del41insTT; 638delTinsCA] was detected in a Romanian patient. We discuss different types of the MECP2 mutations detected in our sample in the light of the possible mechanisms of mutagenesis. Complex gene rearrangements involving a combination of deletions and insertions have always been most difficult to detect, to specify precisely and hence to explain in terms of their underlying mutational mechanisms.

  20. Immunohistochemical and morphometric analysis of immunoglobulin light-chain immunoreactive amyloid in psammoma bodies of the human choroid plexus.

    Science.gov (United States)

    Jovanović, Ivan; Ugrenović, Sladjana; Vasović, Ljiljana; Stojanović, Ivan

    2014-03-01

    The aim of this research was to establish the presence of amyloid and to quantify immunohistochemical reactions of kappa and lambda light chains of psammoma bodies of the choroid plexus. Choroid plexus tissue obtained from 14 right lateral ventricles postmortem was processed histologically and stained with Congo red, thioflavin T, and monoclonal antibodies for kappa and lambda light chains. Morphological analysis was performed with a light microscope at lens magnifications of 4×, 10×, 20×, 25×, and 40×. The morphometric characteristics of psammoma bodies that were kappa and lambda positive and negative were analyzed with ImageJ. Histological analysis showed that the psammoma bodies, stromal blood vessel walls, and some epithelial cells reacted positively with Congo red and thioflavin T. Psammoma bodies were predominantly positive for lambda light chains. Lambda positivity was detected inside some stromal blood vessels, which pointed to a probable systemic origin for these light chains. Morphometric analysis showed that the mean optical densities of lambda- and kappa-positive psammoma bodies were significantly higher than those that gave a negative reaction. The percentage of lambda-positive psammoma bodies was significantly higher than the percentage of lambda-negative psammoma bodies in 80% of the cases, while the reaction with kappa light chains was negative in the majority of the cases. Linear regression analysis showed a significant increase in the percentage of lambda-positive psammoma bodies and their mean optical density with age. Finally, it can be concluded that the positive reaction of psammoma bodies in the choroid plexus with respect to amyloid and lambda light chains may point to the presence of light-chain amyloid in their structures.

  1. Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins.

    Science.gov (United States)

    Glenney, J R

    1990-01-01

    The calpactin I complex is composed of two heavy chain (39K) and two light chain (11K) subunits. The heavy chain is a member of a protein family that includes lipocortins, endonexin and chromobindins while the light chain is a member of the S100 family (7 distinct members are known). We have found that the kidney epithelial cell line MDBK expresses four members of the heavy chain family and two members of the light chain protein family. Antibodies to the light chain of calpactin I were found to cause the precipitation of injected antibody together with the associated heavy chain without apparent effect on the distribution of related proteins. This suggests a differential targeting of various members of the calpactin heavy and light chain families even within the same cell.

  2. Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age.

    Science.gov (United States)

    Belik, J; Kerc, Ewa; Pato, Mary D

    2006-03-01

    We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

  3. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    Science.gov (United States)

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  4. Regulatory light chain phosphorylation increases eccentric contraction-induced injury in skinned fast-twitch fibers.

    Science.gov (United States)

    Childers, Martin K; McDonald, Kerry S

    2004-02-01

    During contraction, activation of Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) results in phosphorylation of myosin's regulatory light chain (RLC), which potentiates force and increases speed of force development over a wide range of [Ca(2+)]. We tested the hypothesis that RLC phosphorylation by MLCK mediates the extent of eccentric contraction-induced injury as measured by force deficit in skinned fast-twitch skeletal muscle fibers. Results indicated that RLC phosphorylation in single skinned rat psoas fibers significantly increased Ca(2+) sensitivity of isometric force; isometric force from 50 +/- 16 to 59 +/- 18 kN/m(2) during maximal Ca(2+) activation; peak absolute power output from 38 +/- 15 to 48 +/- 14 nW during maximal Ca(2+) activation; and the magnitude of contraction-induced force deficit during maximal (pCa 4.5) activation from 26 +/- 9.8 to 35 +/- 9.6%. We conclude that RLC phosphorylation increases force deficits following eccentric contractions, perhaps by increasing the number of force-generating cross-bridges.

  5. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

    Science.gov (United States)

    Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

    2014-01-01

    Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

  6. [Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis].

    Science.gov (United States)

    Miuge, N S; Tikhonov, A V; Ozerniuk, N D

    2005-01-01

    mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

  7. Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

    Science.gov (United States)

    Mitsuda, Yukie; Hifumi, Emi; Tsuruhata, Kumi; Fujinami, Hiroko; Yamamoto, Naoki; Uda, Taizo

    2004-04-20

    A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

  8. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light

  9. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    1993-01-01

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light c

  10. Mutations in the medium chain acyl-CoA dehydrogenase (MCAD) gene

    DEFF Research Database (Denmark)

    Tanaka, K; Yokota, I; Coates, P M

    1992-01-01

    Medium chain acyl-CoA dehydrogenase (MCAD) catalyzes the first reaction of the beta-oxidation cycle for 4-10-carbon fatty acids. MCAD deficiency is one of the most frequent inborn metabolic disorders in populations of northwestern European origin. In the compilation of data from a worldwide study...

  11. The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa.

    Science.gov (United States)

    Ray, A; Williams, H D

    1997-11-15

    The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.

  12. Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates.

    Science.gov (United States)

    Criscitiello, Michael F; Flajnik, Martin F

    2007-10-01

    The discovery of a fourth immunoglobulin (Ig) light (L) chain isotype in sharks has revealed the origins and natural history of all vertebrate L chains. Phylogenetic comparisons have established orthology between this new shark L chain and the unique Xenopus L chain isotype sigma. More importantly, inclusion of this new L chain family in phylogenetic analyses showed that all vertebrate L chains can be categorized into four ancestral clans originating prior to the emergence of cartilaginous fish: one restricted to elasmobranchs (sigma-cart/type I), one found in all cold-blooded vertebrates (sigma/teleost type 2/elasmobranch type IV), one in all groups except bony fish (lambda/elasmobranch type II), and one in all groups except birds (kappa/elasmobranch type III/teleost type 1 and 3). All four of these primordial L chain isotypes (sigma, sigma-cart, lambda and kappa) have maintained separate V region identities since their emergence at least 450 million years ago, suggestive of an ancient physiological distinction of the L chains. We suggest that, based upon unique, discrete sizes of complementarity determining regions 1 and 2 and other features of the V region sequences, the different L chain isotypes arose to provide different functional conformations in the Ig binding site when they pair with heavy chains.

  13. Characteristics and Long-Term Outcome of Patients with Systemic Immunoglobulin Light-Chain Amyloidosis

    DEFF Research Database (Denmark)

    Nelson, Lærke Marie; Gustafsson, Finn; Gimsing, Peter

    2014-01-01

    and predominantly originate from highly selected centers. We aimed to evaluate patient characteristics and outcomes in a cohort treated at a single center. Methods: This is a single-center retrospective study of 63 consecutive patients diagnosed with AL amyloidosis between January 2000 and December 2012. Patients......Background/Aims: Immunoglobulin light-chain (AL) amyloidosis is a systemic disorder that causes progressive organ dysfunction. The optimal treatment strategy requires accurate patient stratification with an emphasis on the extent of cardiac involvement. Reports on its prognosis are sparse...... were evaluated by treatment strategy and cardiac involvement. Results: The mean age at diagnosis was 61.4 years (±8.9), and 39 patients (62%) were male. Thirty-two (51%) patients presented with cardiac amyloid involvement (CA) and the remaining 31 (49%) had noncardiac amyloidosis (NCA). The median...

  14. Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion.

    Science.gov (United States)

    Jones, Laura A; Villemant, Cécile; Starborg, Toby; Salter, Anna; Goddard, Georgina; Ruane, Peter; Woodman, Philip G; Papalopulu, Nancy; Woolner, Sarah; Allan, Victoria J

    2014-11-24

    Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.

  15. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    Science.gov (United States)

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts.

  16. Induction therapy pre-autologous stem cell transplantation in immunoglobulin light chain amyloidosis: a retrospective evaluation.

    Science.gov (United States)

    Hwa, Yi L; Kumar, Shaji K; Gertz, Morie A; Lacy, Martha Q; Buadi, Francis K; Kourelis, Taxiarchis V; Gonsalves, Wilson I; Rajkumar, S Vincent; Go, Ronald S; Leung, Nelson; Kapoor, Prashant; Dingli, David; Kyle, Robert A; Russell, Stephen; Lust, John A; Hayman, Suzanne R; Lin, Yi; Zeldenrust, Steven; Dispenzieri, Angela

    2016-10-01

    There is no consensus on whether patients with immunoglobulin light chain amyloidosis (AL) should receive induction therapy prior to an autologous stem cell transplant (ASCT). This study investigated the relationships between baseline bone marrow plasmacytosis (BMPC), cardiac staging, and pre-transplant induction in AL patients. All patients who received ASCT for AL within 12 months of diagnosis were included. Patient characteristics and outcomes were abstracted. Univariate and multivariate modeling was performed. Among 415 AL patients, 35% had induction prior to ASCT. Post-ASCT hematologic CR plus VGPR rates were significantly higher in those with baseline BMPC ≤ 10% compared to BMPC >10% (58% versus 40%, P = 0.0013). Significant risk factors for lack of attainment of CR included attenuated dose melphalan conditioning, baseline BMPC > 10%, no induction, and male gender. The 5-year OS for the entire group was 65%. On multivariate analysis, risk factors for inferior OS included no induction therapy, advanced AL amyloid staging, BMPC > 10%, attenuated conditioning melphalan dose, and male gender. Patients with Mayo 2012 stage I-II patients with BMPC ≤ 10%, who comprised 56% of the ASCT population fared exceedingly well regardless of whether or not they received induction therapy with a 5-year OS of 81 to 83%. Induction therapy pre-ASCT may improve outcomes among AL patients due to a rapid reduction of toxic light chains or alternatively by elimination of less fit patients by "testing" their ability to tolerate chemotherapy. Prospective studies will be required to sort out these and other questions. Am. J. Hematol. 91:984-988, 2016. © 2016 Wiley Periodicals, Inc.

  17. Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish

    Science.gov (United States)

    Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C.; Connors, Lawreen H.; Merlini, Giampaolo; Falk, Rodney H.; MacRae, Calum A.

    2013-01-01

    Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies. PMID:23624626

  18. Association between Free Light Chain Levels, and Disease Progression and Mortality in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    European Uremic Toxin (EUTox Work Group

    2013-11-01

    Full Text Available Immunoglobulin free light chains (FLCs form part of the middle molecule group of uremic toxins. Accumulation of FLCs has been observed in patients with chronic kidney disease (CKD. The aim of the present study was to measure FLC levels in patients at different CKD stages and to assess putative associations between FLC levels on one hand and biochemical/clinical parameters and mortality on the other. One hundred and forty patients at CKD stages 2-5D were included in the present study. Routine clinical biochemistry assays and assays for FLC kappa (κ and lambda (λ and other uremic toxins were performed. Vascular calcification was evaluated using radiological techniques. The enrolled patients were prospectively monitored for mortality. Free light chain κ and λ levels were found to be elevated in CKD patients (especially in those on hemodialysis. Furthermore, FLC κ and λ levels were positively correlated with inflammation, aortic calcification and the levels of various uremic toxins levels. A multivariate linear regression analysis indicated that FLC κ and λ levels were independently associated with CKD stages and β2 microglobulin levels. Elevated FLC κ and λ levels appeared to be associated with mortality. However, this association disappeared after adjustment for a propensity score including age, CKD stage and aortic calcification. In conclusion, our results indicate that FLC κ and λ levels are elevated in CKD patients and are associated with inflammation, vascular calcification and levels of other uremic toxins. The observed link between elevated FLC levels and mortality appears to depend on other well-known factors.

  19. Serum levels of immunoglobulin free light chains in patients with chronic hepatitis C presenting cryoglobulinemia.

    Science.gov (United States)

    Oliveira, Isabela S; Cabral, Milena S; Jesus, Larissa S; Paraná, Raymundo; Atta, Ajax M; Sousa Atta, Maria Luiza B

    2014-01-01

    Hepatitis C virus (HCV) infects B-lymphocytes, provokes cellular dysfunction and causes lymphoproliferative diseases such as cryoglobulinemia and non-Hodgkin's B-cell lymphoma. In the present study, we investigated the serum levels of kappa and lambda free light chains (FLC) of immunoglobulins and the kappa/lambda FLC ratio in Brazilian patients with chronic HCV infection and cryoglobulinemia. We also analyzed the immunochemical composition of the cryoglobulins in these patients. Twenty-eight cryoglobulinemic HCV patients composed the target group, while 37 HCV patients without cryoglobulinemia were included as controls. The median levels of kappa and lambda FLC were higher in patients with cryoglobulinemia compared to controls (p=0.001 and p=0.003, respectively), but the kappa/lambda FLC ratio was similar in patients with and without cryoglobulinemia (p>0.05). The median FLC ratio was higher in HCV patients presenting with advanced fibrosis of the liver compared to HCV patients without fibrosis (p=0.004). Kappa and lambda FLC levels were strongly correlated with the IgA, IgG and IgM levels in the patients with cryoglobulinemia. In patients without cryoglobulinemia, the kappa FLC level was only correlated with the IgG level, whereas the lambda FLC were weakly correlated with the IgA, IgG and IgM levels. An immunochemical pattern of mixed cryoglobulins (MC), predominantly IgM, IgG, IgA and kappa light chain, was verified in these immune complexes. We concluded that HCV-infected patients presenting cryoglobulinemia have vigorous polyclonal B-lymphocyte activation due to chronic HCV infection and persistent immune stimulation.

  20. 5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

    Institute of Scientific and Technical Information of China (English)

    YANG Chunxiang; WEI Dongmei; CHEN Chen; YU Weiping; ZHU Minsheng

    2005-01-01

    Long myosin light chain kinase (L-MLCK) contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, re- spectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determina- tion of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle forma- tion caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.

  1. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  2. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    Science.gov (United States)

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha

    2014-06-01

    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  3. An outer arm dynein light chain acts in a conformational switch for flagellar motility

    Science.gov (United States)

    Patel-King, Ramila S.

    2009-01-01

    A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

  4. Amplification refractory mutation system polymerase chain reaction versus optimized polymerase chain reaction restriction-fragment length polymorphism for apolipoprotein E genotyping of majorly depressed patients.

    Science.gov (United States)

    You, Hongmin; Chen, Jin; Zhou, Jingjing; Huang, Hua; Pan, Junxi; Wang, Ziye; Lv, Lin; Zhang, Lujun; Li, Juan; Qin, Bin; Yang, Yongtao; Xie, Peng

    2015-11-01

    Major depressive disorder (MDD) is a prevalent, debilitating mood disorder that has been associated with several genetic polymorphisms. One such polymorphism, namely that of apolipoprotein E (APOE), has three allelic forms (ε2, ε3 and ε4) that encode for six unique isoforms of the APOE protein. A growing number of techniques have been developed for APOE genotyping; however, not all polymerase chain reaction (PCR)‑based genotyping techniques are equally accurate or cost‑effective. In order to find a more accurate and cost‑effective APOE genotyping method for MDD screening in large populations, the present study comparatively evaluated two genotyping methods, amplification refractory mutation system PCR (ARMS‑PCR) and optimized PCR restriction‑fragment length polymorphism (PCR‑RFLP), in blood samples taken from a population of 708 MDD patients. Although either of the two methods were able to detect all six unique APOE genotypes, comparisons of the two methods with Sanger sequencing demonstrated that ARMS‑PCR (94%) was significantly more accurate than optimized PCR‑RFLP (82%). ARMS‑PCR should prove useful in quickly verifying ambiguous results obtained by other APOE genotyping methods and can be cost-effectively performed in the setting of a small laboratory or a population-based screening program.

  5. Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I.

    Science.gov (United States)

    Smeitink, J; Loeffen, J; Smeets, R; Triepels, R; Ruitenbeek, W; Trijbels, F; van den Heuvel, L

    1998-08-01

    Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts.

  6. Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).

    Science.gov (United States)

    Guichard, C; Harricane, M C; Lafitte, J J; Godard, P; Zaegel, M; Tack, V; Lalau, G; Bouvagnet, P

    2001-04-01

    Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

  7. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  8. Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

    Science.gov (United States)

    Griffin, Laura M; Snowden, James R; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Baker, Terry S

    2014-03-01

    Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

  9. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    Science.gov (United States)

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

  10. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  11. Tay-Sachs disease mutations in HEXA target the α chain of hexosaminidase A to endoplasmic reticulum-associated degradation.

    Science.gov (United States)

    Dersh, Devin; Iwamoto, Yuichiro; Argon, Yair

    2016-12-01

    Loss of function of the enzyme β-hexosaminidase A (HexA) causes the lysosomal storage disorder Tay-Sachs disease (TSD). It has been proposed that mutations in the α chain of HexA can impair folding, enzyme assembly, and/or trafficking, yet there is surprisingly little known about the mechanisms of these potential routes of pathogenesis. We therefore investigated the biosynthesis and trafficking of TSD-associated HexA α mutants, seeking to identify relevant cellular quality control mechanisms. The α mutants E482K and G269S are defective in enzymatic activity, unprocessed by lysosomal proteases, and exhibit altered folding pathways compared with wild-type α. E482K is more severely misfolded than G269S, as observed by its aggregation and inability to associate with the HexA β chain. Importantly, both mutants are retrotranslocated from the endoplasmic reticulum (ER) to the cytosol and are degraded by the proteasome, indicating that they are cleared via ER-associated degradation (ERAD). Leveraging these discoveries, we observed that manipulating the cellular folding environment or ERAD pathways can alter the kinetics of mutant α degradation. Additionally, growth of patient fibroblasts at a permissive temperature or with chemical chaperones increases cellular Hex activity by improving mutant α folding. Therefore modulation of the ER quality control systems may be a potential therapeutic route for improving some forms of TSD.

  12. Tay–Sachs disease mutations in HEXA target the α chain of hexosaminidase A to endoplasmic reticulum–associated degradation

    Science.gov (United States)

    Dersh, Devin; Iwamoto, Yuichiro; Argon, Yair

    2016-01-01

    Loss of function of the enzyme β-hexosaminidase A (HexA) causes the lysosomal storage disorder Tay–Sachs disease (TSD). It has been proposed that mutations in the α chain of HexA can impair folding, enzyme assembly, and/or trafficking, yet there is surprisingly little known about the mechanisms of these potential routes of pathogenesis. We therefore investigated the biosynthesis and trafficking of TSD-associated HexA α mutants, seeking to identify relevant cellular quality control mechanisms. The α mutants E482K and G269S are defective in enzymatic activity, unprocessed by lysosomal proteases, and exhibit altered folding pathways compared with wild-type α. E482K is more severely misfolded than G269S, as observed by its aggregation and inability to associate with the HexA β chain. Importantly, both mutants are retrotranslocated from the endoplasmic reticulum (ER) to the cytosol and are degraded by the proteasome, indicating that they are cleared via ER-associated degradation (ERAD). Leveraging these discoveries, we observed that manipulating the cellular folding environment or ERAD pathways can alter the kinetics of mutant α degradation. Additionally, growth of patient fibroblasts at a permissive temperature or with chemical chaperones increases cellular Hex activity by improving mutant α folding. Therefore modulation of the ER quality control systems may be a potential therapeutic route for improving some forms of TSD. PMID:27682588

  13. HbS-Savaria: the anti-polymerization effect of a single mutation in human alpha-chains.

    Science.gov (United States)

    Srinivasulu, Sonati; Acharya, A Seetharama; Prabhakaran, Muthuchidambaran; Fabry, Mary E; Alami, Raouf; Fiering, Steven N; Bouhasirra, Eric E; Nagel, Ronald L

    2007-12-01

    Recombinant alpha-Savaria globin (alpha(S49R)) was assembled with beta(S) chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (alpha (2) (S49R) beta (2) (E6V) ) that exhibited normal O(2) affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O(2) affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 +/- 0.025 vs. 0.46 +/- 0.03, respectively). The C(SAT) of HbS increased from 16.7 +/- 0.8 to 27.0 +/- 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at alpha-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory alpha-chains and gene therapy of sickle cell anemia.

  14. In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

    Science.gov (United States)

    Arosio, Paolo; Owczarz, Marta; Müller-Späth, Thomas; Rognoni, Paola; Beeg, Marten; Wu, Hua; Salmona, Mario; Morbidelli, Massimo

    2012-01-01

    Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

  15. In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Paolo Arosio

    Full Text Available Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL and light chain deposition diseases (LCDD. In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m at pH 7.4. The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4(-≫Cl(->H(2PO(4(-, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

  16. Genetically engineering cyanobacteria to convert CO₂, water, and light into the long-chain hydrocarbon farnesene.

    Science.gov (United States)

    Halfmann, Charles; Gu, Liping; Gibbons, William; Zhou, Ruanbao

    2014-12-01

    Genetically engineered cyanobacteria offer a shortcut to convert CO2 and H2O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C15H24), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO2, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1 ± 1.8 μg·L(-1)·O.D.(-1)·d(-1). Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.

  17. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

    Science.gov (United States)

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  18. Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1 Are Essential for the Binding of Clathrin Light Chain

    Directory of Open Access Journals (Sweden)

    Joel A. Ybe

    2009-01-01

    Full Text Available The interaction between HIP family proteins (HIP1 and HIP12/1R and clathrin is fundamental to endocytosis. We used circular dichroism (CD to study the stability of an HIP1 subfragment (aa468-530 that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83C∘. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494. The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae, HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

  19. Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

    Science.gov (United States)

    Harrison, Alistair; Olds-Clarke, Patricia; King, Stephen M.

    1998-01-01

    The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme. PMID:9490726

  20. Comparison of two real-time quantitative polymerase chain reaction strategies for minimal residual disease evaluation in lymphoproliferative disorders: correlation between immunoglobulin gene mutation load and real-time quantitative polymerase chain reaction performance.

    Science.gov (United States)

    Della Starza, Irene; Cavalli, Marzia; Del Giudice, Ilaria; Barbero, Daniela; Mantoan, Barbara; Genuardi, Elisa; Urbano, Marina; Mannu, Claudia; Gazzola, Anna; Ciabatti, Elena; Guarini, Anna; Foà, Robin; Galimberti, Sara; Piccaluga, Pierpaolo; Gaidano, Gianluca; Ladetto, Marco; Monitillo, Luigia

    2014-09-01

    We compared two strategies for minimal residual disease evaluation of B-cell lymphoproliferative disorders characterized by a variable immunoglobulin heavy chain (IGH) genes mutation load. Twenty-five samples from chronic lymphocytic leukaemia (n = 18) or mantle cell lymphoma (n = 7) patients were analyzed. Based on IGH variable region genes, 22/25 samples carried > 2% mutations, 20/25 > 5%. In the IGH joining region genes, 23/25 samples carried > 2% mutations, 18/25 > 5%. Real-time quantitative polymerase chain reaction was performed on IGH genes using two strategies: method A utilizes two patient-specific primers, whereas method B employs one patient-specific and one germline primer, with different positions on the variable, diversity and joining regions. Twenty-three samples (92%) resulted evaluable using method A, only six (24%) by method B. Method B poor performance was specifically evident among mutated IGH variable/joining region cases, although no specific mutation load above, which the real-time quantitative polymerase chain reaction failed was found. The molecular strategies for minimal residual disease evaluation should be adapted to the B-cell receptor features of the disease investigated.

  1. Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

    DEFF Research Database (Denmark)

    O'Reilly, Linda P; Andresen, Brage S; Engel, Paul C

    2005-01-01

    Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When t...

  2. The frequency of a disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase in sudden infant death syndrome

    DEFF Research Database (Denmark)

    Banner, Jytte; Gregersen, N; Kølvraa, S

    1993-01-01

    syndrome is still a matter of controversy. The present study investigated 120 well-defined cases of sudden infant death syndrome in order to detect the frequency of the most common disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase (G985) compared with the frequency...

  3. TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

    NARCIS (Netherlands)

    Powell, C.A.; Kopajtich, R.; D'Souza, A.R.; Rorbach, J.; Kremer, L.S.; Husain, R.A.; Dallabona, C.; Donnini, C.; Alston, C.L.; Griffin, H.; Pyle, A.; Chinnery, P.F.; Strom, T.M.; Meitinger, T.; Rodenburg, R.J.; Schottmann, G.; Schuelke, M.; Romain, N.; Haller, R.G.; Ferrero, I.; Haack, T.B.; Taylor, R.W.; Prokisch, H.; Minczuk, M.

    2015-01-01

    Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, repr

  4. Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself

    National Research Council Canada - National Science Library

    McCrudden, Cian M; O'Rourke, Martin G; Cherry, Kim E; Yuen, Hiu-Fung; O'Rourke, Declan; Babur, Muhammad; Telfer, Brian A; Thomas, Huw D; Keane, Patrick; Nambirajan, Thiagarajan; Hagan, Chris; O'Sullivan, Joe M; Shaw, Chris; Williams, Kaye J; Curtin, Nicola J; Hirst, David G; Robson, Tracy

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  5. Vasoactivity of Rucaparib, a PARP-1 Inhibitor, is a Complex Process that Involves Myosin Light Chain Kinase, P2 Receptors, and PARP Itself: e0118187

    National Research Council Canada - National Science Library

    Cian M McCrudden; Martin G O'Rourke; Kim E Cherry; Hiu-Fung Yuen; Declan O'Rourke; Muhammad Babur; Brian A Telfer; Huw D Thomas; Patrick Keane; Thiagarajan Nambirajan; Chris Hagan; Joe M O'Sullivan; Chris Shaw; Kaye J Williams; Nicola J Curtin; David G Hirst; Tracy Robson

    2015-01-01

    ...), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK...

  6. A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

    Science.gov (United States)

    Rognoni, Paola; Lavatelli, Francesca; Casarini, Simona; Palladini, Giovanni; Verga, Laura; Pedrazzoli, Paolo; Valentini, Giovanna; Merlini, Giampaolo; Perfetti, Vittorio

    2013-01-01

    Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.

  7. A Strategy for Synthesis of Pathogenic Human Immunoglobulin Free Light Chains in E. coli

    Science.gov (United States)

    Rognoni, Paola; Lavatelli, Francesca; Casarini, Simona; Palladini, Giovanni; Verga, Laura; Pedrazzoli, Paolo; Valentini, Giovanna; Merlini, Giampaolo; Perfetti, Vittorio

    2013-01-01

    Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine (“free” LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders. PMID:24086679

  8. A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

    Directory of Open Access Journals (Sweden)

    Paola Rognoni

    Full Text Available Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC. Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.

  9. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    Science.gov (United States)

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  10. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

    Science.gov (United States)

    Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

    2015-03-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

  11. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

    Science.gov (United States)

    Jaskowski, Troy D; Litwin, Christine M; Hill, Harry R

    2006-02-01

    Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios.

  12. Assay interference caused by antibodies reacting with rat kappa light-chain in human sera.

    Science.gov (United States)

    Degn, Søren E; Andersen, Stig Henrik; Jensen, Lisbeth; Thiel, Steffen; Jensenius, Jens C

    2011-09-30

    The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such

  13. [The prognostic value of baseline serum free light chain in cardiac amyloidosis].

    Science.gov (United States)

    Zhao, Lei; Tian, Zhuang; Fang, Quan

    2016-03-01

    To analyze the prognostic value of baseline serum free light chain (sFLC) in light-chain (AL) cardiac amyloidosis. Twenty-seven patients with AL cardiac amyloidosis were retrospectively reviewed from January 2014 to January 2015. sFLC was measured by immuoturbidimetric assay. Baseline characteristics, echocardiographic parameters and electrocardiogram data were analyzed. According to the median baseline dFLC (involved sFLC minus uninvolved sFLC), patients were categorized into either the low dFLC(≤ 307 mg/L) or the high dFLC group (>307 mg/L). More subjects in the high dFLC group with early/late diastolic mitral velocity ratio (E/A ratio) over 2 (71.4% vs 30.8%, P=0.035), and subjects in this group had a shorter median survival time than those in the low dFLC group (3 months vs 17 months, P=0.004). A similar phenomenon for median survival time was observed when the subjects were redivided either by a new cut-off value of 180 mg/L for dFLC (low dFLC group: 17 months; high dFLC group: 4 months, P=0.014) or a κ/λ ratio, in which subjects with κ type sFLC-ratio ≤ 19.6 and λ type sFLC-ratio>0.065 were in the low sFLC-ratio group (17 months) and those with κ type sFLC-ratio > 19.6 and λ type sFLC-ratio ≤ 0.065 were in the high sFLC-ratio group (4 months, P=0.023). In multivariate analysis, dFLC and New York Heart Association (NYHA)classification of cardiac function were two risk factors associated with all-cause mortality in patients, among which the hazard ratio for higher dFLC was 4.28 (95%CI 1.55-11.8, P=0.005). The level of sFLC could be a marker for the prognosis of AL cardiac amyloidosis.

  14. The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive

    DEFF Research Database (Denmark)

    O'Reilly, Linda; Bross, Peter; Corydon, Thomas J;

    2004-01-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, ...

  15. An Association between Neutrophils and Immunoglobulin Free Light Chains in the Pathogenesis of Chronic Obstructive Pulmonary Disease

    NARCIS (Netherlands)

    Braber, Saskia; Thio, Marco; Blokhttis, Bart R.; Henricks, Paul A. J.; Koelink, Pim J.; Kormelink, Tom Groot; Bezemer, Gillina F. G.; Kerstjens, Huib A. M.; Postma, Dirkje S.; Garssen, Johan; Kraneveld, Aletta D.; Redegeld, Frank A.; Folkerts, Gert

    2012-01-01

    Rationale: Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgL

  16. Hematogones With Lambda Light Chain Restriction in a 4-Year-Old Boy With Burkitt Lymphoma: A Potential Diagnostic Pitfall.

    Science.gov (United States)

    Guillory, Tesha; Li, Shiyong; Bergsagel, Daniel J; Weinzierl, Elizabeth; Bunting, Silvia T

    2016-05-01

    Hematogones are immature normal B cell precursors with a characteristic immunophenotype profile on flow cytometry that typically do not express surface immunoglobulin light chains. In this report, we describe a case in which the hematogones exhibit light chain restriction. Our patient was a 4-year-old boy with a complicated medical history involving treatment for a presumed bilateral Wilms tumor of the kidney that on later resection was diagnosed as Burkitt lymphoma. Flow cytometry analysis of his bone marrow revealed a small distinct population of cells expressing dim cluster of differentiation (CD)10, CD19, CD22, CD38, dim CD58, human leukocyte antigen-D related (HLA-DR), and dim CD45, which are characteristic of hematogones. These cells, however, demonstrated dim surface immunoglobulin lambda light-chain restriction. Molecular study results for immunoglobulin heavy and kappa light-chain gene rearrangements were negative. We present this case to raise awareness of the potential pitfalls of working up bone marrow for involvement by B cell lymphoproliferative disorder.

  17. Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi.

    Science.gov (United States)

    Chu, W Y; Chen, J; Zhou, R X; Zhao, F L; Meng, T; Chen, D X; Nong, X X; Liu, Z; Lu, S Q; Zhang, J S

    2011-04-01

    Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

  18. Removal of free light chains in hemodialysis patients without multiple myeloma: a crossover comparison of three different dialyzers.

    Science.gov (United States)

    Donati, Gabriele; Moretti, Maria Ilaria; Baraldi, Olga; Spazzoli, Alessandra; Capelli, Irene; Comai, Giorgia; Marchetti, Antonio; Sarma, Maria; Mancini, Rita; La Manna, Gaetano

    2016-11-25

    Immunoglobulin light chains are classified as middle molecule uremic toxins able to interact with B lymphocyte membranes leading to the activation of transmembrane signaling. The ensuing impairment of neutrophil function can contribute to the chronic inflammation state of uremic patients, and the increased risk of bacterial infections or vascular calcifications. The aim of this crossover observational study was to assess the difference in free light chain removal by three different hemodialysis filters in patients not affected by multiple myeloma. Free light chain removal was compared in the polymethylmethacrylate (PMMA) membrane Filtryzer BK-F, the polyphenylene HFR17 filter and the conventional polysulfone filter F7HPS. Twenty chronic hemodialysis patients were enrolled: mean age was 67.7 ± 17.0 years, M/F = 14/6, dialysis vintage (months) 25.5 ± 32.0. The patients were randomized into two groups of treatment lasting 6 weeks each. The dialysis sessions checked were the midweek sessions and the blood was drawn at times 0, 120' and 240'. Kappa (k) and lambda (λ) light chain levels, β2microglobulin (β2M), C reactive protein (CRP) and albumin were checked. K light chain levels were 345.0 ± 100.0 mg/L, λ light chains were 121.4 ± 27.0 mg/L. The values of k light chains at times 120' and 240' were significantly lower with PMMA and HFR17 than those obtained with F7. The reduction ratio per session (RRs) for k light chains was 44.1 ± 4.3% with HFR17, 55.3 ± 3.4% with PMMA, 25.7 ± 8.3% with F7 (p = 0.018). The RRs for λ light chains was 30.3 ± 2.9% with HFR17, 37.8 ± 17.3% with PMMA, 14.0 ± 3.9% with F7 (p = 0.032). As to β2M, RRs was 42.4 ± 3.2% with HFR17 vs. 33.9 ± 2.8% with PMMA vs. 6.3 ± 1.9% with F7 (p = 0.022). The three filters tested showed no differences in CRP or albumin levels. In terms of light chain and β2M removal, the PMMA and on-line HFR filters are similar and

  19. Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species.

    Science.gov (United States)

    Ayres Sá, L; Menezes, M A; dos Santos Mermelstein, C

    2001-08-01

    Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus. Copyright 2001 Wiley-Liss, Inc.

  20. The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes

    DEFF Research Database (Denmark)

    Ghelli, Anna; Tropeano, Concetta V; Calvaruso, Maria Antonietta

    2013-01-01

    Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe...... exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However......, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating...

  1. Prolonged Cerebrospinal Fluid Neurofilament Light Chain Increase in Patients with Post-Traumatic Disorders of Consciousness.

    Science.gov (United States)

    Bagnato, Sergio; Grimaldi, Luigi M E; Di Raimondo, Giorgio; Sant'Angelo, Antonino; Boccagni, Cristina; Virgilio, Vittorio; Andriolo, Maria

    2017-08-15

    The mechanisms involved in secondary brain injury after the acute phase of severe traumatic brain injury (TBI) are largely unknown. Ongoing axonal degeneration, consequent to the initial trauma, may lead to secondary brain injury. To test this hypothesis, we evaluated the cerebrospinal fluid (CSF) level of neurofilament light chain (NF-L), a proposed marker of axonal degeneration, in 10 patients who developed a severe disorder of consciousness after a TBI, including 7 in a minimally conscious state and 3 with unresponsive wakefulness syndrome (time since brain injury, 309 ± 169 days). CSF NF-L level was measured with a commercially available NF-L enzyme-linked immunosorbent assay. CSF NF-L level was very high in all 10 patients, ranging from 2.4- to 60.5-fold the upper normal limit (median value, 4458 pg/mL; range, 695-23,000). Moreover, NF-L level was significantly higher after a severe TBI than in a reference group of 9 patients with probable Alzheimer's disease, a population with elevated levels of CSF NF-L attributed to neuronal degeneration (median value, 1173 pg/mL; range, 670-3643; p < 0.01). CSF NF-L level was correlated with time post-TBI (p = 0.04). These results demonstrate prolonged secondary brain injury, suggesting that patients exhibit ongoing axonal degeneration up to 19 months after a severe TBI.

  2. [Effect of free light chain ratio normalization after treatment on prognosis of patients with multiple myeloma].

    Science.gov (United States)

    Meng, Qing-Qi; Wang, Jian-Ning; Song, Min; Bao, Hong-Yu; Hou, Yan-Qiu; Zhang, Liu-Bo; Jiang, Su-Yu

    2014-12-01

    This study was aimed to investigate the normalization of serum free light chain ratio (sPLCR) after treatment of multiple myeloma (MM) and its influence on the prognosis of MM patients. The clinical data of 42 patients with MM were analyzed retrospectively from January 2009 to November 2013 in out department. According to sPLCR consecutive normalization for more 4 weeks or not after treatment, the patients were classified in patients with mormalized sPLCR and patients with abnormalized sPLCR, then the influence of traditional prognostic factors of MM on sPLCR and effect of sPLCR on overall survival (OS) time of MM patients were analyzed. The results showed that the influence of age, ISS stage displayed statistical difference between sFLCR normalization group and abnormalization group, the age ≥ 65 years and ISS stage III negatively impacted on sFLCR normalization (P normalized sFLCR were as follows: CR - 60%, VGPR - 38.89%; PR - 28.57%; 17 patients (40.48%) with sFLCR normalization showed superior OS, as compared with patients with sPLCR abnormalization (P normalization is the independent prognostic factor for MM, suggesting that the MM patients with sPLCR normalization after treatment have superior prognosis.

  3. Cerebrospinal fluid neurofilament light chain levels predict visual outcome after optic neuritis

    DEFF Research Database (Denmark)

    Modvig, Signe; Degn, M; Sander, B

    2016-01-01

    BACKGROUND: Optic neuritis is a good model for multiple sclerosis relapse, but currently no tests can accurately predict visual outcome. OBJECTIVE: The purpose of this study was to examine whether cerebrospinal fluid (CSF) biomarkers of tissue damage and remodelling (neurofilament light chain (NF-L...... cell layer+inner plexiform layer (GC-IPL) thicknesses. RESULTS: CSF NF-L levels at onset predicted inter-ocular differences in follow-up LCVA (β=13.8, p=0.0008), RNFL (β=5.6, p=0.0004) and GC-IPL (β=4.0, p=0.0008). The acute-phase GC-IPL thickness also predicted follow-up LCVA (β=12.9, p=0.0021 for NF-L......, β=-1.1, p=0.0150 for GC-IPL). Complete/incomplete remission was determined based on LCVA from 30 healthy controls. NF-L had a positive predictive value of 91% and an area under the curve (AUC) of 0.79 for incomplete remission. CONCLUSION: CSF NF-L is a promising biomarker of visual outcome after...

  4. Serum neurofilament light chain levels are increased in patients with a clinically isolated syndrome

    DEFF Research Database (Denmark)

    Disanto, Giulio; Adiutori, Rocco; Dobson, Ruth

    2016-01-01

    BACKGROUND: Neurofilament light chain (NfL) represents a promising biomarker for axonal injury. We present the first exploratory study on serum NfL in patients with a clinically isolated syndrome (CIS) and healthy controls. METHODS: We investigated serum NfL levels in 100 patients with CIS...... with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls. RESULTS: NfL levels were higher in FC (24.......1 pg/mL (13.5-51.8)) and NC (19.3 pg/mL (13.6-35.2)) than in healthy controls (7.9 pg/mL (5.6-17.2)) (OR=5.85; 95% CI 2.63 to 13.02; p=1.5×10(-5) and OR=7.03; 95% CI 2.85 to 17.34; p=2.3×10(-5), respectively). When grouping FC and NC, increased serum NfL concentration was also associated...

  5. Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

    Science.gov (United States)

    Xue, Song; Javor, Sacha; Hixon, Mark S; Janda, Kim D

    2014-11-04

    Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

  6. Immunoparesis status in immunoglobulin light chain amyloidosis at diagnosis affects response and survival by regimen type

    Science.gov (United States)

    Muchtar, Eli; Dispenzieri, Angela; Kumar, Shaji K.; Dingli, David; Lacy, Martha Q.; Buadi, Francis K.; Hayman, Suzanne R.; Kapoor, Prashant; Leung, Nelson; Chakraborty, Rajshekhar; Russell, Stephen; Lust, John A.; Lin, Yi; Go, Ronald S.; Zeldenrust, Steven; Kyle, Robert A.; Rajkumar, S. Vincent; Gertz, Morie A.

    2016-01-01

    Clinical tools to guide in the appropriate treatment selection in immunoglobulin light chain (AL) amyloidosis are not well developed. We evaluated the response and outcome for various regimens at first-line treatment (n=681) and first progression (n=240) stratified by the immunoparesis status at diagnosis. Immunoparesis was assessed by the average relative difference of the uninvolved immunoglobulins, classifying patients into a negative average relative difference (i.e. significant immunoparesis) or a positive average relative difference (no/modest immunoparesis). Treatment was categorized as autologous stem cell transplant and four non-transplant regimens (melphalan-based; bortezomib-based, immunomodulatory drug-based and dexamethasone alone). Patients with significant immunoparesis who underwent stem cell transplant had a significantly lower rate of very good partial response or better response (58%), progression-free survival (median 30 months) and overall survival (108 months), compared to those without significant immunoparesis (80%, 127 months, median not reached, respectively; Pcomparisons). Among the non-transplant regimens, melphalan resulted in an unfavorable progression-free survival (11 vs. 27 months; Pcompared to those without significant immunoparesis. In contrast, no significant difference in outcomes between the immunoparesis groups was seen for those treated with bortezomib or immunomodulatory drugs. At first progression, immunoparesis status did not impact response or survival of any regimen. Melphalan at first-line provided poorer outcomes for patients with significant immunoparesis, while bortezomib or immunomodulatory drugs were more likely to overcome the adverse prognosis associated with significant immunoparesis. PMID:27479823

  7. Clinical and prognostic differences among patients with light chain deposition disease, myeloma cast nephropathy and both.

    Science.gov (United States)

    Zand, Ladan; Nasr, Samih H; Gertz, Morie A; Dispenzieri, Angela; Lacy, Martha Q; Buadi, Francis K; Kumar, Shaji; Kyle, Robert A; Fervenza, Fernando C; Sethi, Sanjeev; Dingli, David; Rajkumar, S Vincent; Kapoor, Prashant; McCurdy, Arleigh; Leung, Nelson

    2015-01-01

    In some patients with light chain deposition disease (LCDD) there is also evidence of myeloma cast nephropathy (MCN) on renal biopsy. The purpose of this study was to evaluate the renal and survival outcome of patients with concomitant diagnosis of MCN and LCDD to LCDD and MCN alone. Eighty seven patients were identified and divided into LCDD (n=45), MCN (n=29), and LCDD+ MCN (n=13). Patients with LCDD+ MCN had a worse overall survival (OS) compared to patients with LCDD (p=0.03), but similar to patients with MCN (p=0.4). Death-censored renal survival was no different amongst the groups. Presenting with acute renal failure at time of renal biopsy (HR 7.2, p=0.0002) was an independent poor renal prognostic factor while older age (HR 1.06, p=0.0002), presence of osteolytic lesions (HR 4.4, p<0.0001), and requirement for dialysis or creatinine≥5 mg/dL (HR 3.2, p=0.0006) at time of renal biopsy were independent poor prognostic factors for OS.

  8. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available BACKGROUND: Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  9. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  10. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers.

    Science.gov (United States)

    Das, Sabyasachi; Nikolaidis, Nikolas; Klein, Jan; Nei, Masatoshi

    2008-10-28

    The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (kappa, lambda, and sigma isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: kappa, lambda, and sigma. Of these, two (kappa and lambda) are also found in reptiles and some mammals. The lambda isotype is found in all tetrapods tested to date, whereas the kappa isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

  11. The clinical utility and prognostic value of multiparameter flow cytometry immunophenotyping in light-chain amyloidosis.

    Science.gov (United States)

    Paiva, Bruno; Vídriales, María-Belén; Pérez, José J; López-Berges, María-Consuelo; García-Sanz, Ramón; Ocio, Enrique M; de Las Heras, Natalia; Cuello, Rebeca; García de Coca, Alfonso; Pardal, Emilia; Alonso, José; Sierra, Magdalena; Bárez, Abelardo; Hernández, José; Suárez, Lissbett; Galende, Josefina; Mateos, María-Victoria; San Miguel, Jesús F

    2011-03-31

    The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients with AL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL.

  12. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    Science.gov (United States)

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  13. Clinical Comparisons of Two Free Light Chain Assays to Immunofixation Electrophoresis for Detecting Monoclonal Gammopathy

    Directory of Open Access Journals (Sweden)

    Han-Sung Kim

    2014-01-01

    Full Text Available Free light chains (FLCs are useful biomarkers for the diagnosis and monitoring of various plasma cell dyscrasias. One hundred fifty-seven samples from 120 patients for screening or monitoring of monoclonal gammopathy (MG were included. The new N Latex FLC assays (Siemens Healthcare Diagnostics GmbH, Germany were compared with the Freelite FLC assays (The Binding Site Ltd., UK and the results were analyzed with those of immunofixation electrophoresis (IFE. The Freelite FLC assay showed significantly wider assay ranges than the N Latex FLC assay. The correlation coefficients of the two FLC kappa (κ assays, lambda (λ assays, and the κ/λ ratio were 0.9792, 0.8264, and 0.9064, respectively. The concordance rate was 84.7% for the FLC κ assays, 79.6% for FLC λ, and 89.2% for the κ/λ ratio. The clinical sensitivity and specificity of the κ/λ ratios were 72.2% and 93.6% for the Freelite assay and 64.6% and 100% for the N Latex FLC assay. Two FLC assays showed good correlations and concordance. However, the clinical sensitivity of the κ/λ ratio was higher in the Freelite FLC assays; clinical specificity was higher in the N Latex FLC assay. Both FLC assays seem to have limited clinical utility in detecting MG in certain clinical settings.

  14. Circulating antibody free light chains and risk of posttransplant lymphoproliferative disorder.

    Science.gov (United States)

    Engels, E A; Preiksaitis, J; Zingone, A; Landgren, O

    2012-05-01

    Posttransplant lymphoproliferative disorder (PTLD) is a major complication of solid-organ transplantation. With human immunodeficiency virus infection (an analogous immunosuppressive state), elevated kappa and lambda immunoglobulin free light chains (FLCs) in peripheral blood are associated with increased risk of lymphoma. To assess the role of B-cell dysfunction in PTLD, we measured circulating FLCs among Canadian transplant recipients, including 29 individuals with PTLD and 57 matched transplant recipients who were PTLD-free. Compared with controls, PTLD cases had higher kappa FLCs (median 1.53 vs. 1.07 times upper limit of normal) and lambda FLCs (1.03 vs. 0.68). Using samples obtained on average 3.5 months before PTLD diagnosis, cases were more likely to have polyclonal FLC elevations (i.e. elevated kappa and/or lambda with normal kappa/lambda ratio: odds ratio [OR] 4.2, 95%CI 1.1-15) or monoclonal elevations (elevated kappa and/or lambda with abnormal ratio: OR 3.0, 95%CI 0.5-18). Strong FLC-PTLD associations were also observed at diagnosis/selection. Among recipients with Epstein-Barr virus (EBV) DNA measured in blood, EBV DNAemia was associated with FLC abnormalities (ORs 6.2 and 3.2 for monoclonal and polyclonal elevations). FLC elevations are common in transplant recipients and associated with heightened PTLD risk. FLCs likely reflect B-cell dysfunction, perhaps related to EBV-driven lymphoproliferation.

  15. Helicobacter pylori CagA disrupts epithelial patterning by activating myosin light chain.

    Directory of Open Access Journals (Sweden)

    Jonathan B Muyskens

    Full Text Available Helicobacter pylori infection is a leading cause of ulcers and gastric cancer. We show that expression of the H. pylori virulence factor CagA in a model Drosophila melanogaster epithelium induces morphological disruptions including ectopic furrowing. We find that CagA alters the distribution and increases the levels of activated myosin regulatory light chain (MLC, a key regulator of epithelial integrity. Reducing MLC activity suppresses CagA-induced disruptions. A CagA mutant lacking EPIYA motifs (CagA(EPISA induces less epithelial disruption and is not targeted to apical foci like wild-type CagA. In a cell culture model in which CagA(EPISA and CagA have equivalent subcellular localization, CagA(EPISA is equally potent in activating MLC. Therefore, in our transgenic system, CagA is targeted by EPIYA motifs to a specific apical region of the epithelium where it efficiently activates MLC to disrupt epithelial integrity.

  16. Prognostic value of serum heavy/light chain ratios in patients with POEMS syndrome.

    Science.gov (United States)

    Wang, Chen; Su, Wei; Cai, Qian-Qian; Cai, Hao; Ji, Wei; Di, Qian; Duan, Ming-Hui; Cao, Xin-Xin; Zhou, Dao-Bin; Li, Jian

    2016-07-01

    POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. The prognostic value of diagnosing concurrent multiple myeloma in immunoglobulin light chain amyloidosis.

    Science.gov (United States)

    Dinner, Shira; Witteles, Wesley; Witteles, Ronald; Lam, Anthony; Arai, Sally; Lafayette, Richard; George, Tracy I; Schrier, Stanley L; Liedtke, Michaela

    2013-05-01

    The prevalence and prognostic value of a concomitant diagnosis of symptomatic or asymptomatic multiple myeloma (MM), as defined by the current International Myeloma Working Group (IMWG) criteria, in patients with immunoglobulin light chain amyloidosis (AL), are unknown. We studied 46 consecutive patients with AL who underwent quantification of serum M-protein and clonal bone marrow plasma cells, as well as a comprehensive evaluation for end organ damage by MM. Using standard morphology and CD138 immunohistochemical staining, 57% and 80% of patients were found to have concomitant MM, respectively. Nine patients exhibited end organ damage consistent with a diagnosis of symptomatic MM. While overall survival was similar between AL patients with or without concurrent myeloma (1-year overall survival 68% vs. 87%; P = 0.27), a diagnosis of symptomatic myeloma was associated with inferior outcome (1-year overall survival 39% vs. 81%; P = 0.005). Quantification of bone marrow plasma cells by both standard morphology and CD138 immunohistochemistry identified a much higher prevalence of concurrent MM in patients with AL than previously reported. Evaluation of bone marrow plasma cell infiltration and presence of myeloma associated end organ damage could be clinically useful for prognostication of patients with AL. © 2013 Blackwell Publishing Ltd.

  18. [The role of the assessment of heavy/light chain pairs of immunoglobulin in monoclonal gammopathies].

    Science.gov (United States)

    Ščudla, Vlastimil; Pika, Tomáš; Minařík, Jiří

    2015-01-01

    The aim of the paper is to inform about the contribution of novel, highly sensitive analytic technique for the assessment of serum immunoglobulins (Hevylite), enabling separate quantitative assessment of heavy/light chain pairs of immunoglobulin (HLC), i. e. the monoclonal ("involved") and polyclonal ("noninvolved") isotype including their ratio (HLC-r) in monoclonal gammopathies. We particularly target the characteristics of this technique, the compari-son of its clinical contribution with standard methods used in the diagnostics, course and the detection of relapse and progression of the disease, as well as the stratification, assessment of therapeutic outcome and prognosis in monoclonal gammopathy of undetermined significance, multiple myeloma, Waldenström´s macroglobulinemia, systemic AL-amyloidosis and some non-Hodgkin lymphomas. Present results show that in comparison with existing routinely used techniques the Hevylite method enriches clinical practice with the assessment of serum levels of "uninvolved" Ig. It enables the evaluation of the depth of "immunoparesis", and the determination of HLC-r index that is needful for the stratification of MM into "risk cohorts". It also contributes to prognostic assessment and improvement of the evaluation of the depth of therapeutic response. In MGUS individuals the HLC-r index provides information about the risk of malignant transformation. We await the results of ongoing validation studies that are expected to provide specific indications for Hevylite technique for MG in routine practice.

  19. Immunophenotypic analysis of abnormal plasma cell clones in bone marrow of primary systemic light chain amyloidosis patients

    Institute of Scientific and Technical Information of China (English)

    Hu Yang; Wang Mangju; Chen Yan; Chen Xue; Fang Fang; Liu Shiqin; Zhang Ying

    2014-01-01

    Background Primary systemic light chain amyloidosis (AL) is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry (FCM) could be used as an important indicator of AL diagnosis.Methods Fresh bone marrow samples were collected from 51 AL,21 multiple myeloma (MM),and 5 Waldenstr(o)m's macroglobulinemia (WM) patients.The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45,CD38,CD138,CD117,CD56,and CD19.Results In AL,light chain restriction could be identified in 31 cases (60.9%),in which the λ light chain restriction was found in 24 cases (77.4%).In MM,κ light chain restriction was found in 13 cases (61.9%),and λ light chain restriction in eight cases.CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM,but was positive to strongly positive in WM.In the bone marrow plasma cells of the 51 AL,78.4% were CD56+,68.6% were CD117+,and 88.2% were CD19-.While in the 21 MM cases,66.7% were CD56+,38.1% were CD117+,and 90.4% were CD19-.The plasmacytoid lymphocytes in the five WM patients were CD19+ and CD56-,CD117-.Conclusion Detection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL.

  20. Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

    Science.gov (United States)

    Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

    2015-12-04

    In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

  1. Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient

    DEFF Research Database (Denmark)

    Zhou, J; Hertz, Jens Michael; Leinonen, A;

    1992-01-01

    alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin...

  2. Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations

    Directory of Open Access Journals (Sweden)

    Der-Shan Sun

    2016-12-01

    Full Text Available Conventional photocatalysts are primarily stimulated using ultraviolet (UV light to elicit reactive oxygen species and have wide applications in environmental and energy fields, including self-cleaning surfaces and sterilization. Because UV illumination is hazardous to humans, visible light-responsive photocatalysts (VLRPs were discovered and are now applied to increase photocatalysis. However, fundamental questions regarding the ability of VLRPs to trigger DNA mutations and the mutation types it elicits remain elusive. Here, through plasmid transformation and β-galactosidase α-complementation analyses, we observed that visible light-responsive platinum-containing titania (TiO2 nanoparticle (NP-mediated photocatalysis considerably reduces the number of Escherichia coli transformants. This suggests that such photocatalytic reactions cause DNA damage. DNA sequencing results demonstrated that the DNA damage comprises three mutation types, namely nucleotide insertion, deletion and substitution; this is the first study to report the types of mutations occurring after photocatalysis by TiO2-VLRPs. Our results may facilitate the development and appropriate use of new-generation TiO2 NPs for biomedical applications.

  3. Visible Light-Responsive Platinum-Containing Titania Nanoparticle-Mediated Photocatalysis Induces Nucleotide Insertion, Deletion and Substitution Mutations.

    Science.gov (United States)

    Sun, Der-Shan; Tseng, Yao-Hsuan; Wu, Wen-Shiang; Wong, Ming-Show; Chang, Hsin-Hou

    2016-12-28

    Conventional photocatalysts are primarily stimulated using ultraviolet (UV) light to elicit reactive oxygen species and have wide applications in environmental and energy fields, including self-cleaning surfaces and sterilization. Because UV illumination is hazardous to humans, visible light-responsive photocatalysts (VLRPs) were discovered and are now applied to increase photocatalysis. However, fundamental questions regarding the ability of VLRPs to trigger DNA mutations and the mutation types it elicits remain elusive. Here, through plasmid transformation and β-galactosidase α-complementation analyses, we observed that visible light-responsive platinum-containing titania (TiO₂) nanoparticle (NP)-mediated photocatalysis considerably reduces the number of Escherichia coli transformants. This suggests that such photocatalytic reactions cause DNA damage. DNA sequencing results demonstrated that the DNA damage comprises three mutation types, namely nucleotide insertion, deletion and substitution; this is the first study to report the types of mutations occurring after photocatalysis by TiO₂-VLRPs. Our results may facilitate the development and appropriate use of new-generation TiO₂ NPs for biomedical applications.

  4. Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation.

    Science.gov (United States)

    Asselta, Rosanna; Robusto, Michela; Platé, Manuela; Santoro, Cristina; Peyvandi, Flora; Duga, Stefano

    2015-07-01

    Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes. In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene.

  5. Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy

    DEFF Research Database (Denmark)

    Toftmann Hansen, Charlotte; Pedersen, Per T.; Jensen, Bo Amdi;

    Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy.......Value of the free light chain analysis in the clinical evaluation of response in multiple myeloma patients receiving anti-myeloma therapy....

  6. Two unique mutations in the interleukin-2 receptor gamma chain gene (IL2RG) cause X-linked severe combined immunodeficiency arising in opposite parental germ lines

    Energy Technology Data Exchange (ETDEWEB)

    Puck, J.M.; Pepper, A.E. [National Institutes of Health, Bethesda, MD (United States)

    1994-09-01

    The gene encoding the gamma chain of the lymphocyte receptor for IL-2 lies in human X13.1 and is mutated in males with X-linked severe combined immunodeficiency (SCID). 27 X-linked SCID mutations have been found in our laboratory. Single strand conformation polymorphism (SSCP) analysis of genomic DNA using primers flanking each of the 8 exons was followed by direct sequencing of abnormally migrating fragments from SCID patients and family members. A 9 bp in-frame duplication insertion was found in IL2RG exon 5 of a patient from a large X-linked SCID pedigree; the resulting duplication of 3 extracellular amino acids, including the first tryptophan of the {open_quotes}WSXWS{close_quotes} cytokine binding motif, is predicted to disrupt interaction of the cytokine receptor chain with its ligand. Genetic linkage studies demonstrated that the grandmaternal X chromosome associated with SCID was contributed to 3 daughters, 2 obligate carriers and 1 woman of unknown status. However, this grandmother`s genomic DNA did not contain the insertion mutation, nor did she have skewed X-chromosome inactivation in her lymphocytes. That both obligate carrier daughters, but not the third daughter, had the insertion proved the grandmother to be a germline mosaic. A second proband had X-linked SCID with a branch point mutation due to substitution of T for A 15 bp 5{prime} of the start of IL2RG exon 3. This mutation resulted in undetectable IL2RG mRNA by Northern blot. Linkage analysis and sequencing of IL2RG DNA in this family proved the mutation to have originated in the germline of the proband`s grandfather, an immunocompetent individual who contributed an X chromosome with normal IL2RG to one daughter and a mutated X to the another.

  7. Immunoglobulin diversity in the phylogenetically primitive shark, Heterodontus francisci. Suggested lack of structural variation between light chains isolated from different animals.

    Science.gov (United States)

    Litman, G W; Scheffel, C; Gerber-Jenson, B

    1980-06-01

    A two-step procedure employing gel filtration and anion exchange chromatography has been utilized to isolate LMW immunoglobulin from the horned shark, Heterodontus francisci. Light chains obtained by complete reduction and alkylation of the parent protein have been compared by several analytical techniques. Amino acid composition data implies a limited degree of variation in the light chains isolated from individual animals. Polyacrylamide gel electrophoresis of the CNBr digests of the light chains reveal indistinguishable banding profiles of the major peptides. Isoelectric focusing indicates limited heterogeneity in the light chain spectrotype and identity in the pI of the majority of bands detectable by staining. The suggested degree of structural similarity in the light chains of this phylogenetically primitive shark is discussed in terms of the evolutionary position of the species and current theories concerning the origins of structural diversity in immunoglobulins.

  8. CSF Neurofilament Light Chain but not FLT3 Ligand Discriminates Parkinsonian Disorders

    Science.gov (United States)

    Herbert, Megan K.; Aerts, Marjolein B.; Beenes, Marijke; Norgren, Niklas; Esselink, Rianne A. J.; Bloem, Bastiaan R.; Kuiperij, H. Bea; Verbeek, Marcel M.

    2015-01-01

    The differentiation between multiple system atrophy (MSA) and Parkinson’s disease (PD) is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL), fms-like tyrosine kinase ligand (FLT3L), and total tau protein (t-tau) in cerebrospinal fluid (CSF) as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunosorbent assays, we measured CSF levels of NFL, FLT3L, and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients, and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85) in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94) in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. t-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA. PMID:25999911

  9. Serum neurofilament light chain levels are increased in patients with a clinically isolated syndrome.

    Science.gov (United States)

    Disanto, Giulio; Adiutori, Rocco; Dobson, Ruth; Martinelli, Vittorio; Dalla Costa, Gloria; Runia, Tessel; Evdoshenko, Evgeniy; Thouvenot, Eric; Trojano, Maria; Norgren, Niklas; Teunissen, Charlotte; Kappos, Ludwig; Giovannoni, Gavin; Kuhle, Jens

    2016-02-01

    Neurofilament light chain (NfL) represents a promising biomarker for axonal injury. We present the first exploratory study on serum NfL in patients with a clinically isolated syndrome (CIS) and healthy controls. We investigated serum NfL levels in 100 patients with CIS with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls. NfL levels were higher in FC (24.1 pg/mL (13.5-51.8)) and NC (19.3 pg/mL (13.6-35.2)) than in healthy controls (7.9 pg/mL (5.6-17.2)) (OR=5.85; 95% CI 2.63 to 13.02; p = 1.5 × 10(-5) and OR = 7.03; 95% CI 2.85 to 17.34; p = 2.3 × 10(-5), respectively). When grouping FC and NC, increased serum NfL concentration was also associated with increasing numbers of T2 hyperintense MRI lesions (OR = 2.36; 95% CI 1.21 to 4.59; p = 0.011), gadolinium-enhancing lesions (OR = 2.69; 95% CI 1.13 to 6.41; p=0.026) and higher disability scores (OR = 2.54; 95% CI 1.21 to 5.31; p = 0.013) at CIS diagnosis. If replicated in future studies, serum NfL may represent a reliable and easily accessible biomarker of early axonal damage in CIS and MS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  10. Neurofilament light chain level is a weak risk factor for the development of MS.

    Science.gov (United States)

    Arrambide, Georgina; Espejo, Carmen; Eixarch, Herena; Villar, Luisa M; Alvarez-Cermeño, José C; Picón, Carmen; Kuhle, Jens; Disanto, Giulio; Kappos, Ludwig; Sastre-Garriga, Jaume; Pareto, Deborah; Simon, Eva; Comabella, Manuel; Río, Jordi; Nos, Carlos; Tur, Carmen; Castilló, Joaquín; Vidal-Jordana, Angela; Galán, Ingrid; Arévalo, Maria J; Auger, Cristina; Rovira, Alex; Montalban, Xavier; Tintore, Mar

    2016-09-13

    To determine the prognostic value of selected biomarkers in clinically isolated syndromes (CIS) for conversion to multiple sclerosis (MS) and disability accrual. Data were acquired from 2 CIS cohorts. The screening phase evaluated patients developing clinically definite MS (CIS-CDMS) and patients who remained as CIS during a 2-year minimum follow-up (CIS-CIS). We determined levels of neurofascin, semaphorin 3A, fetuin A, glial fibrillary acidic protein, and neurofilament light (NfL) and heavy chains in CSF (estimated mean [95% confidence interval; CI]). We evaluated associations between biomarker levels, conversion, disability, and magnetic resonance parameters. In the replication phase, we determined NfL levels (n = 155) using a 900 ng/L cutoff. Primary endpoints in uni- and multivariate analyses were CDMS and 2010 McDonald MS. The only biomarker showing significant differences in the screening was NfL (CIS-CDMS 1,553.1 [1,208.7-1,897.5] ng/L and CIS-CIS 499.0 [168.8-829.2] ng/L, p NfL did not correlate with disability. In the replication phase, more NfL-positive patients, according to the cutoff, evolved to MS. Every 100-ng/L increase in NfL predicted CDMS (hazard ratio [HR] = 1.009, 95% CI 1.005-1.014) and McDonald MS (HR = 1.009, 95% CI 1.005-1.013), remaining significant for CDMS in the multivariate analysis (adjusted HR = 1.005, 95% CI 1.000-1.011). This risk was lower than the presence of oligoclonal bands or T2 lesions. NfL is a weak independent risk factor for MS. Its role as an axonal damage biomarker may be more relevant as suggested by its association with medium-term brain volume changes. © 2016 American Academy of Neurology.

  11. Myosin light chain kinase mediates intestinal barrier disruption following burn injury.

    Directory of Open Access Journals (Sweden)

    Chuanli Chen

    Full Text Available BACKGROUND: Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC phosphorylation mediated by MLC kinase (MLCK is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. METHODOLOGY/PRINCIPAL FINDINGS: Male balb/c mice were assigned randomly to either sham burn (control or 30% total body surface area (TBSA full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg, an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. CONCLUSIONS/SIGNIFICANCE: The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.

  12. Cardiac Light Chain Amyloidosis: The Role of Metal Ions in Oxidative Stress and Mitochondrial Damage.

    Science.gov (United States)

    Diomede, Luisa; Romeo, Margherita; Rognoni, Paola; Beeg, Marten; Foray, Claudia; Ghibaudi, Elena; Palladini, Giovanni; Cherny, Robert A; Verga, Laura; Capello, Gian Luca; Perfetti, Vittorio; Fiordaliso, Fabio; Merlini, Giampaolo; Salmona, Mario

    2017-09-20

    The knowledge of the mechanism underlying the cardiac damage in immunoglobulin light chain (LC) amyloidosis (AL) is essential to develop novel therapies and improve patients' outcome. Although an active role of reactive oxygen species (ROS) in LC-induced cardiotoxicity has already been envisaged, the actual mechanisms behind their generation remain elusive. This study was aimed at further dissecting the action of ROS generated by cardiotoxic LC in vivo and investigating whether transition metal ions are involved in this process. In the absence of reliable vertebrate model of AL, we used the nematode Caenorhabditis elegans, whose pharynx is an "ancestral heart." LC purified from patients with severe cardiac involvement intrinsically generated high levels of ROS and when administered to C. elegans induced ROS production, activation of the DAF-16/forkhead transcription factor (FOXO) pathway, and expression of proteins involved in stress resistance and survival. Profound functional and structural ROS-mediated mitochondrial damage, similar to that observed in amyloid-affected hearts from AL patients, was observed. All these effects were entirely dependent on the presence of metal ions since addition of metal chelator or metal-binding 8-hydroxyquinoline compounds (chelex, PBT2, and clioquinol) permanently blocked the ROS production and prevented the cardiotoxic effects of amyloid LC. Innovation and Conclusion: Our findings identify the key role of metal ions in driving the ROS-mediated toxic effects of LC. This is a novel conceptual advance that paves the way for new pharmacological strategies aimed at not only counteracting but also totally inhibiting the vicious cycle of redox damage. Antioxid. Redox Signal. 27, 567-582.

  13. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

  14. Light chain amyloidosis: Experience in a tertiary hospital: 2005-2013.

    Science.gov (United States)

    Krsnik, I; Cabero, M; Morillo, D; Segovia, J; García-Pavía, P; Gómez-Bueno, M; Salas, C

    2015-01-01

    AL amyloidosis is a rare condition whose management is undergoing changes due to recent advances in diagnosis and treatment. We describe a contemporary series of patients with AL amyloidosis to analyze the features that enable early diagnosis and optimal management. We recruited for analysis 32 patients (19 women; mean age, 63 years) treated consecutively at our center. Eighty-four percent of the patients presented with asthenia, dyspnea or edema, with a previous duration of symptoms of 8 months (median). Cardiac (21/32) and renal impairment were the most common type (11/32). All of the patients, except one, had a monoclonal component in serum/urine or abnormal values for free light chains (78%, λ). The bone marrow (BM) showed clonal plasmacytosis in 29 cases. All of the cardiac biopsies and 50% of the BM biopsies showed amyloid deposits. The results of the echocardiogram and/or cardiac resonance were abnormal in 27/30 cases. The median NT-proBNP value at diagnosis was 5200 ng/ml. Thirteen patients died due to heart failure, 2 due to rejection after heart transplantation, 2 due to pneumonia and 1 after a stroke. Ten patients did not undergo treatment, 12 were treated with bortezomib and 5 were treated with alkylating agents. Five patients underwent heart transplantation and 4 underwent autologous bone marrow transplantation. Fourteen patients achieved a complete hematologic response and 10 achieved organ response. The median survival was 17 months. Cardiac involvement is the major determinant of prognosis. Yield of involved organ biopsy is high (100% heart biopsies). Antineoplastic treatment with bortezomib and/or autologous bone marrow transplantation achieves hematological responses with improvements in organ impairment. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  15. Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and idiopathic pulmonary fibrosis.

    Directory of Open Access Journals (Sweden)

    Tom Groot Kormelink

    Full Text Available BACKGROUND: Idiopathic pulmonary fibrosis (IPF, a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP, a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF. METHODS: In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry. RESULTS: FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung. CONCLUSION: These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.

  16. Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia.

    Science.gov (United States)

    Rashid, Sajid; Grzmil, Pawel; Drenckhahn, Joerg-Detlef; Meinhardt, Andreas; Adham, Ibrahim; Engel, Wolfgang; Neesen, Juergen

    2010-01-01

    To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

  17. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

    Science.gov (United States)

    Rao, P Vasantha; Deng, Peifeng; Sasaki, Yasuharu; Epstein, David L

    2005-02-01

    Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM.

  18. Prognostic significance of serum free light chains in chronic lymphocytic leukemia.

    Science.gov (United States)

    Sarris, Katerina; Maltezas, Dimitrios; Koulieris, Efstathios; Bartzis, Vassiliki; Tzenou, Tatiana; Sachanas, Sotirios; Nikolaou, Eftychia; Efthymiou, Anna; Bitsani, Katerina; Dimou, Maria; Vassilakopoulos, Theodoros P; Siakantaris, Marina; Angelopoulou, Maria K; Kontopidou, Flora; Tsaftaridis, Panagiotis; Kafasi, Nikolitsa; Pangalis, Gerasimos A; Panayiotidis, Panayiotis P; Harding, Stephen; Kyrtsonis, Marie-Christine

    2013-01-01

    Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients' followup was 32 months (range 4-228). Results. Increased involved (restricted) sFLC (iFLC) was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment (P = 0.0005 and P = 0.000003, resp.) and overall survival (P = 0.05 and P = 0.003, resp.). They also correlated with β 2-microglobulin (P = 0.009 and P = 0.03, resp.), serum albumin (P = 0.009 for summated sFLC), hemoglobin (P < 0.001), abnormal LDH (P = 0.037 and P = 0.001, resp.), Binet stage (P < 0.05) and with the presence of beta symptoms (P = 0.004 for summated sFLC). Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters.

  19. Prognostic Significance of Serum Free Light Chains in Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Katerina Sarris

    2013-01-01

    Full Text Available Background. Serum free light chains (sFLC, the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients’ series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients’ followup was 32 months (range 4–228. Results. Increased involved (restricted sFLC (iFLC was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment (P=0.0005 and P=0.000003, resp. and overall survival (P=0.05 and P=0.003, resp.. They also correlated with β2-microglobulin (P=0.009 and P=0.03, resp., serum albumin (P=0.009 for summated sFLC, hemoglobin (P<0.001, abnormal LDH (P=0.037 and P=0.001, resp., Binet stage (P<0.05 and with the presence of beta symptoms (P=0.004 for summated sFLC. Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters.

  20. Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite.

    Directory of Open Access Journals (Sweden)

    Zhang-Fan Chen

    Full Text Available Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM, encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+/CaM-dependent kinase II (CaMKII inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.

  1. CSF neurofilament light chain but not FLT3 ligand discriminates Parkinsonian disorders

    Directory of Open Access Journals (Sweden)

    Megan Kristy Herbert

    2015-05-01

    Full Text Available The differentiation between multiple system atrophy (MSA and Parkinson’s disease (PD is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL, fms-like tyrosine kinase ligand (FLT3L and total tau protein (t-tau in cerebrospinal fluid (CSF as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunoassays (ELISAs, we measured CSF levels of NFL, FLT3L and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85 in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94 in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. T-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA.

  2. Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.

    Science.gov (United States)

    Yu, Shaorong; Xie, Li; Hou, Zhibo; Qian, Xiaoping; Yu, Lixia; Wei, Jia; Ding, Yitao; Liu, Baorui

    2011-09-01

    Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.

  3. Another way to view the chain conformation broadening of the line-width distribution measured in dynamic light scattering

    Institute of Scientific and Technical Information of China (English)

    吴奇; 牛爱珍

    1999-01-01

    In dynamic laser light scattering (LLS), for a given polydisperse sample, a line-width distribution G(Γ) or the translational diffusion coefficient distribution G(D) can be obtained from the measured time correlation function. For rigid colloid particles, G(Γ) can be directly related to the hydrodynamic size distribution. However, for flexible polymer chains, G(Γ) depends not only on the chain length distribution, but also on the relaxation of the chain conformation; that is, even for a monodisperse polymer sample there still exists a chain conformation distribution. If the time scale of the chain conformation relaxation is comparable to that of the translational diffusion, such as in the case of a very long polymer chain, the conformation relaxation might lead to an additional broadening in G (Γ). This "conformation broadening" has been directly observed for the first time by comparing two G(Γ) s obtained from a poly(N-isopropyl-acrylamide) solution at~25℃ and~32℃ at which the solution is ther

  4. A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Auewarakul Chirayu U

    2011-02-01

    Full Text Available Abstract Background BCR-ABL kinase domain (KD mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI in BCR-ABL-positive chronic myeloid leukemia (CML patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage. Methods A single-tube allele specific-polymerase chain reaction (AS-PCR method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC and direct sequencing were performed as a comparison to AS-PCR. Results T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution. Conclusion A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.

  5. Sensitive detection of the c-KIT c.1430G>T mutation by mutant-specific polymerase chain reaction in feline mast cell tumours.

    Science.gov (United States)

    Takanosu, M; Sato, M; Kagawa, Y

    2014-06-01

    Here, we describe the establishment of mutant-specific polymerase chain reaction (PCR) for detection of a c-KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c-KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c-KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant-specific PCR but in only 7.1% (5 of 70) by PCR-restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin-fixed paraffin-embedded sections or cells collected by fine needle aspiration. Mutant-specific PCR showed remarkably higher detection rate than did PCR-RFLP. DNA sequence analysis did not always yield identical results to those of mutant-specific PCR, suggesting heterogeneity of tumour cells. Mutant-specific PCR is a valid and efficient screening tool for detection of the c-KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR-RFLP and sequencing analysis.

  6. Relationship between EGFR and KRAS mutations and prognosis in Chinese patients with non-small cell lung cancer:a mutation analysis with real-time polymerase chain reaction using scorpion amplification refractory mutation system

    Institute of Scientific and Technical Information of China (English)

    高洁

    2012-01-01

    Objective To investigate the gene mutation of EGFR and KRAS in Chinese patients with non-small cell lung cancer (NSCLC) ,and to analyze the relationship between the gene mutations and the clinicopathological features and EGFR-TKI efficiency. Methods EGFR mutation was detected in 120 patients and KRAS mutation in 104 patients

  7. Branched-Chain in situ Hybridization for κ and λ Light Chains: A Powerful Ancillary Technique for Determining B-Cell Clonality in Cytology Samples

    Science.gov (United States)

    Arora, Kshitij; Chebib, Ivan; Zukerberg, Lawrence; Gandhi, Manoj; Rivera, Miguel; Ting, David; Deshpande, Vikram

    2017-01-01

    BACKGROUND Current immunohistochemical and in situ hybridization (ISH) assays are generally inconclusive for clonality unless plasmacytic differentiation is present. This study examined a series of cytology specimens and explored the ability of a branched-chain RNA (bRNA) ISH assay for immunoglobulin κ constant (IGKC) and immunoglobulin λ constant (IGLC) to detect a clonal population of B lymphocytes. METHODS Pathology databases were used to identify fine-needle aspiration biopsies (n = 28) and exfoliative cytology samples (n = 20). Demographic, flow cytometry, and excision biopsy results were recorded. bRNA ISH was performed on the Leica Bond platform with the following probes: IGKC, IGLC, immunoglobulin λ-like polypeptide 5 (IGLL5), and a housekeeping gene (HKG). RESULTS The bRNA ISH assay was validated with 30 surgical biopsies. On bRNA ISH, a clonal B-cell population (light-chain ratio > 10:1) was detected in 22 of 28 cases with a final diagnosis of lymphoma. In 2 cases, a κ predominance was present, although the ratio was <10:1. Eleven of the 17 κ-clonal lymphomas also expressed IGLL5, the latter recognized by the presence of an intranuclear signal. Two B-cell lymphomas lacked IGKC and IGLC, whereas 2 cases were negative for the HKG. In 12 of the 20 cases with reactive lymphoid tissue, bRNA ISH identified a polyclonal lymphoid population. No light-chain messenger RNA was detected in 6 cases (typically those associated with very few B cells). CONCLUSIONS The automated bRNA ISH platform is a robust technique for detecting a clonal B-cell population in cytology material. PMID:26524200

  8. Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis.

    Science.gov (United States)

    Wu, Qian; Wei, Zhiguo; Yang, Zhi; Wang, Tao; Ren, Liming; Hu, Xiaoxiang; Meng, Qingyong; Guo, Ying; Zhu, Qinghong; Robert, Jacques; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

    2010-05-01

    The reptiles are the last major taxon of jawed vertebrates in which immunoglobulin light chain isotypes have not been well characterized. Using the recently released genome sequencing data, we show in this study that the reptile Anolis carolinensis expresses both lambda and kappa light chain genes. The genomic organization of both gene loci is structurally similar to their respective counterparts in mammals. The identified lambda locus contains three constant region genes each preceded by a joining gene segment, and a total of 37 variable gene segments. In contrast, the kappa locus contains only a single constant region gene, and two joining gene segments with a single family of 14 variable gene segments located upstream. Analysis of junctions of the recombined VJ transcripts reveals a paucity of N and P nucleotides in both expressed lambda and kappa sequences. These results help us to understand the generation of the immunoglobulin repertoire in reptiles and immunoglobulin evolution in vertebrates.

  9. Light conditions alter accumulation of long chain polyprenols in leaves of trees and shrubs throughout the vegetation season.

    Science.gov (United States)

    Bajda, Agnieszka; Chojnacki, Tadeusz; Hertel, Józefina; Swiezewska, Ewa; Wójcik, Jacek; Kaczkowska, Alicja; Marczewski, Andrzej; Bojarczuk, Tomasz; Karolewski, Piotr; Oleksyn, Jacek

    2005-01-01

    In many plants belonging to angiosperms and gymnosperms the accumulation in leaves of long chain polyprenols and polyprenyl esters during growth in natural habitats depends on the light intensity. The amount of polyprenols in leaves is also positively correlated with the thickness of the leaf blade (SLA, specific leaf area). The polyprenol content of leaves shows seasonal changes with a maximum in autumn and a minimum in early summer with the difference between poorly and well illuminated plants persisting throughout the vegetation season.

  10. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    Science.gov (United States)

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Galtseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy.

  11. Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

    Directory of Open Access Journals (Sweden)

    James N. Cobley

    2016-04-01

    Full Text Available Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM. We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.

  12. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

    Science.gov (United States)

    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  13. Neurofilament light chain level is a weak risk factor for the development of MS

    Science.gov (United States)

    Arrambide, Georgina; Eixarch, Herena; Villar, Luisa M.; Alvarez-Cermeño, José C.; Picón, Carmen; Kuhle, Jens; Disanto, Giulio; Kappos, Ludwig; Sastre-Garriga, Jaume; Pareto, Deborah; Simon, Eva; Comabella, Manuel; Río, Jordi; Nos, Carlos; Tur, Carmen; Castilló, Joaquín; Vidal-Jordana, Angela; Galán, Ingrid; Arévalo, Maria J.; Auger, Cristina; Rovira, Alex; Montalban, Xavier

    2016-01-01

    Objective: To determine the prognostic value of selected biomarkers in clinically isolated syndromes (CIS) for conversion to multiple sclerosis (MS) and disability accrual. Methods: Data were acquired from 2 CIS cohorts. The screening phase evaluated patients developing clinically definite MS (CIS-CDMS) and patients who remained as CIS during a 2-year minimum follow-up (CIS-CIS). We determined levels of neurofascin, semaphorin 3A, fetuin A, glial fibrillary acidic protein, and neurofilament light (NfL) and heavy chains in CSF (estimated mean [95% confidence interval; CI]). We evaluated associations between biomarker levels, conversion, disability, and magnetic resonance parameters. In the replication phase, we determined NfL levels (n = 155) using a 900 ng/L cutoff. Primary endpoints in uni- and multivariate analyses were CDMS and 2010 McDonald MS. Results: The only biomarker showing significant differences in the screening was NfL (CIS-CDMS 1,553.1 [1,208.7–1,897.5] ng/L and CIS-CIS 499.0 [168.8–829.2] ng/L, p < 0.0001). The strongest associations were with brain parenchymal fraction change (rs = −0.892) and percentage brain volume change (rs = −0.842) at 5 years. NfL did not correlate with disability. In the replication phase, more NfL-positive patients, according to the cutoff, evolved to MS. Every 100-ng/L increase in NfL predicted CDMS (hazard ratio [HR] = 1.009, 95% CI 1.005–1.014) and McDonald MS (HR = 1.009, 95% CI 1.005–1.013), remaining significant for CDMS in the multivariate analysis (adjusted HR = 1.005, 95% CI 1.000–1.011). This risk was lower than the presence of oligoclonal bands or T2 lesions. Conclusions: NfL is a weak independent risk factor for MS. Its role as an axonal damage biomarker may be more relevant as suggested by its association with medium-term brain volume changes. PMID:27521440

  14. Heart failure induced by perinatal ablation of cardiac myosin light chain kinase

    Directory of Open Access Journals (Sweden)

    Yasmin F. K. Islam

    2016-10-01

    Full Text Available Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7 as opposed to 31.4 ± 1.0% (n = 11 in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12 relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11 and 5.81 ± 0.33 (wild/wild/Cre, n = 5, accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320 as opposed to 87.1 ± 1.6 μm (n = 360 in the controls. Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing

  15. Long term outcomes of cardiac transplant for immunoglobulin light chain amyloidosis: The Mayo Clinic experience

    Science.gov (United States)

    Grogan, Martha; Gertz, Morie; McCurdy, Arleigh; Roeker, Lindsey; Kyle, Robert; Kushwaha, Sudhir; Daly, Richard; Dearani, Joseph; Rodeheffer, Richard; Frantz, Robert; Lacy, Martha; Hayman, Suzanne; McGregor, Christopher; Edwards, Brooks; Dispenzieri, Angela

    2016-01-01

    AIM: To determine the outcome of orthotopic heart transplantation (OHT) in immunoglobulin light chain (AL) amyloidosis. METHODS: The medical records of patients with AL who underwent orthotopic heart transplantation at the Mayo Clinic in Rochester Minnesota from 1992 to 2011 were reviewed. Patients met at least one of the following at: New York Heart Association class IV heart failure, ventricular thickness > 15 mm, ejection fraction < 40%. Selection guidelines for heart transplant included age < 60 years, absence of multiple myeloma and significant extra-cardiac organ involvement. Baseline characteristics including age, gender, organ involvement, and New York Heart Association functional class were recorded. Laboratory data, waiting time until heart transplant, and type of treatment of the underlying plasma cell disorder were recorded. Survival from the time of OHT was calculated using Kaplan-Meier survival curves. Survival of patients undergoing OHT for AL was compared to that of non-amyloid patients undergoing OHT during the same time period. RESULTS: Twenty-three patients (median age 53 years) with AL received OHT. There were no deaths in the immediate perioperative period. Twenty patients have died post OHT. For the entire cohort, the median overall survival was 3.5 years (95%CI: 1.2, 8.2 years). The 1-year survival post OHT was 77%, the 2-year survival 65%, and the 5-year survival 43%. The 5-year survival for non-amyloid patients undergoing OHT during the same era was 85%. Progressive amyloidosis contributed to death in twelve patients. Of those without evidence of progressive amyloidosis, the cause of death included complications of autologous hematopoietic stem cell transplantation for 3 patients, post-transplant lymphoproliferative disorder for 2 patients; and for the remaining one death was related to each of the following causes: acute rejection; cardiac vasculopathy; metastatic melanoma; myelodysplastic syndrome; and unknown. Eight patients had

  16. Diagnostic value of clonality of surface immunoglobulin light and heavy chains in malignant lymphoproliferative disorders.

    Science.gov (United States)

    Batata, A; Shen, B

    1993-08-01

    Cell suspensions from the peripheral blood of B-chronic lymphoid leukemias (B-CLL, n = 274) and reactive lymphocytosis (RLC, n = 132) and from solid tissue samples of B-non-Hodgkin's lymphoma (B-NHL, n = 466) and reactive lymphadenopathy (RLA, n = 324) were analyzed to evaluate the diagnostic value of clonality of L- and H- chains in B-CLL and B-NHL. Cutoff levels for monoclonal L-chain (mono-L) and monoclonal H-chain (mono-H) were defined. In B-CLL, the association patterns of L- and H- chains were as follows: mono-L/mono-H, 245 cases (89.42%); mono-L/polyclonal H chain (poly-H), 4 (1.46%); polyclonal L chain (poly-L)/mono-H, 2 (0.73%); poly-L/poly-H, 2 (0.73%); undetected (und)-L/mono-H, 6 (2.19%); and und-L/und-H, 15 (5.47%). In B-NHL, the association patterns were mono-L/mono-H, 433 cases (92.92%); mono-L/poly-H, 4 (0.86%); poly-L/mono-H, 8 (1.72%); poly-L/poly-H, 2 (0.43%); und-L/mono-H, 4 (0.86%); and und-L/und-H, 15 (3.22%). Monoclonality of H chains are complementary to L-chain restriction, especially in the cases with poly-L or und-L, and should be considered as a positive criterion in determining surface immunoglobulin (SIg) clonality. Monoclonality of SIg assessed by both L and H chains is both sensitive and specific for the diagnosis of B-CLL and B-NHL, and their differentiation from RLC and RLA, since none of the cases of RLC and RLA showed monoclonal SIg.

  17. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain amp...... network. References 1 Cape, J., et al. (2011) Chem. Sci., 2 (4), 661-667. 2 Maurer, S. E., et al. (2009) Astrobiology, 9, 979-987. 3 Caschera, F., et al. Langmuir, In press....

  18. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

    Science.gov (United States)

    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  19. Evidence indicating independent assortment of framework and complementarity-determining segments of the variable regions of rabbit light chains. Delineation of a possible J minigene.

    Science.gov (United States)

    Kabat, E A; Wu, T T; Bilofsky, H

    1980-07-01

    Amino acid sequences of rabbit light chains show considerable evidence of independent assortment of framework (FR) and complementarity-determining (CDR) segments. This suggests that they are coded for by independent genetic units (minigaenes) and that individual light chains are assembled somatically by recombining these units. Identical FR sets with multiple members generally comprise chains with different specificities, whereas identical CDR sets tend to have chains of a single specificity. A J segment, which, by analogy with mouse light chains, is made up of the last two residues of CDR3 plus all of FR4, contained 18 different sets and could contribute to diversity generated by CDR3. The longest segment, FR3, had a very large number of sets. Evidence is presented showing that the number of sets could be substantially reduced by permitting FR3 to be formed by two independently assorting segments comprising residues 57-68 and 69-88.

  20. Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures.

    Science.gov (United States)

    Gao, Feng-shan; Bai, Jing; Zhang, Qiang; Xu, Chong-bo; Li, Yanmin

    2012-07-10

    Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    Energy Technology Data Exchange (ETDEWEB)

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.; (IIT); (Vermont); (Duke)

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  2. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    Science.gov (United States)

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  3. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene].

    Science.gov (United States)

    Oh, Hye Ryong; Lee, Mi Ja; Park, Geon; Moon, Dae Soo; Park, Young Jin; Jang, Sook Jin

    2009-06-01

    A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

  4. Dilated Cardiomyopathy Mutation (R134W) in Mouse Cardiac Troponin T Induces Greater Contractile Deficits against α-Myosin Heavy Chain than against β-Myosin Heavy Chain

    OpenAIRE

    Gollapudi, Sampath K.; Murali Chandra

    2016-01-01

    Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant — such as myofilament Ca2+ sensitivity — is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms...

  5. Rapid and efficient identification of the mouse leptin receptor mutation (C57BL/KsJ-db/db) by tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) analysis.

    Science.gov (United States)

    Jung, Harry; Nam, Hajin; Suh, Jun-Gyo

    2016-03-01

    The C57BLKS/J-Lepr(db) mouse has a point mutation in the leptin receptor gene and is one of the most useful animal model for non-insulin dependent diabetes mellitus in human. Since the homozygote of C57BLKS/J-Lepr(db) mouse is infertile, detection of point mutation in the leptin receptor gene is important for efficient maintaining strains as well as mass production of homozygotes. To develop a rapid and efficient genotyping method for C57BLKS/J-Lepr(db) mouse, the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) was used. The 407 and 199 bp PCR products were amplified from normal (+/+) mice; while the 407 and 268 bp PCR products were amplified from homozygotes (db/db) mice; and the 407, 268, and 199 bp PCR products were amplified from heterozygotes (db/+) mice. This result showed that the tetra-primer ARMS-PCR analysis by us is suitable to detect point mutation of the leptin receptor gene. Taken together, our method will dramatically reduce animal use for maintenance of strains as well as production of homozygote in the C57BLKS/J-Lepr(db) strains.

  6. Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

    Science.gov (United States)

    Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

    1994-03-01

    Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Multiplex polymerase chain reaction analysis of UV-A- and UV-B-induced delayed and early mutations in V79 Chinese hamster cells.

    Science.gov (United States)

    Dahle, Jostein; Noordhuis, Paul; Stokke, Trond; Svendsrud, Debbie Hege; Kvam, Egil

    2005-01-01

    We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.

  8. Three-dimensional Speckle Tracking Echocardiography in Light Chain Cardiac Amyloidosis: Examination of Left and Right Ventricular Myocardial Mechanics Parameters.

    Science.gov (United States)

    Urbano-Moral, Jose Angel; Gangadharamurthy, Dakshin; Comenzo, Raymond L; Pandian, Natesa G; Patel, Ayan R

    2015-08-01

    The study of myocardial mechanics has a potential role in the detection of cardiac involvement in patients with amyloidosis. This study aimed to characterize 3-dimensional-speckle tracking echocardiography-derived left and right ventricular myocardial mechanics in light chain amyloidosis and examine their relationship with brain natriuretic peptide. In patients with light chain amyloidosis, left ventricular longitudinal and circumferential strain (n=40), and right ventricular longitudinal strain and radial displacement (n=26) were obtained by 3-dimensional-speckle tracking echocardiography. Brain natriuretic peptide levels were determined. All myocardial mechanics measurements showed differences when compared by brain natriuretic peptide level tertiles. Left and right ventricular longitudinal strain were highly correlated (r=0.95, P<.001). Left ventricular longitudinal and circumferential strain were reduced in patients with cardiac involvement (-9±4 vs -16±2; P<.001, and -24±6 vs -29±4; P=.01, respectively), with the most prominent impairment at the basal segments. Right ventricular longitudinal strain and radial displacement were diminished in patients with cardiac involvement (-9±3 vs -17±3; P<.001, and 2.7±0.8 vs 3.8±0.3; P=.002). On multivariate analysis, left ventricular longitudinal strain was associated with the presence of cardiac involvement (odds ratio = 1.6; 95% confidence interval, 1.04 to 2.37; P=.03) independent of the presence of brain natriuretic peptide and troponin I criteria for cardiac amyloidosis. Three-dimensional-speckle tracking echocardiography-derived left and right ventricular myocardial mechanics are increasingly altered as brain natriuretic peptide increases in light chain amyloidosis. There appears to be a strong association between left ventricular longitudinal strain and cardiac involvement, beyond biomarkers such as brain natriuretic peptide and troponin I. Copyright © 2015 Sociedad Española de Cardiología. Published by

  9. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

    Science.gov (United States)

    Schlapschy, Martin; Fogarasi, Marton; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Aguib, Yasmine; Skerra, Arne

    2009-03-01

    An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

  10. Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.

    Science.gov (United States)

    Kirkham, Justin K; Park, Sung Hee; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur

    2016-01-01

    Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

  11. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  12. Evaluation of the serum free light chain (sFLC) analysis in prediction of response in symptomatic multiple myeloma patients

    DEFF Research Database (Denmark)

    Toftmann Hansen, Charlotte; Pedersen, Per T; Nielsen, Lars C;

    2014-01-01

    BACKGROUND: Observational data from clinical studies indicate that the goal of first-line therapy in newly diagnosed patients with symptomatic multiple myeloma (MM) should be very good partial response (VGPR) or better, preferably before high-dose treatment. We evaluated the value of early...... measurements of involved free light chains (iFLC) in prediction of high-quality responses. Measuring iFLC has a potential advantage due to a short half-life compared to the half-life of the M-protein. METHODS: In 36 multiple myeloma (MM) patients, we measured serial changes in iFLC and M-protein after start...

  13. Renal Light Chain Deposition Associated with the Formation of Intracellular Crystalline Inclusion Bodies in Podocytes: A Rare Case Report.

    Science.gov (United States)

    Wang, Yuan-da; Dong, Zhe-yi; Zhang, Xue-guang; Zhang, Wei; Yin, Zhong; Qiu, Qiang; Chen, Xiang-mei

    2016-01-01

    We herein report the case of an elderly woman with bone pain and proteinuria as the main clinical manifestations. The patient was diagnosed with the IgG κ type of multiple myeloma. Her renal pathology consisted of widespread κ light chain protein deposition associated with the formation of large quantities of rod-like crystals in podocytes. This phenomenon is very rare. We explored the significance of this crystal formation via a detailed and descriptive analysis and also performed a literature review, thus providing data to increase the available information about this type of disease.

  14. Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

    Directory of Open Access Journals (Sweden)

    Yong-Zhong Wang

    2011-12-01

    Full Text Available OBJECTIVES: Detection of mutations associated to nucleos(tide analogs and hepatitis B virus (HBV genotyping are essential for monitoring treatment of HBV infection. We developed a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR assay for the rapid detection of HBV genotypes and mutations associated with lamivudine, adefovir, and telbivudine resistance in HBV-infected patients. METHODS: HBV templates were amplified by PCR, followed by LDR and electrophoresis on a sequencer. The assay was evaluated using plasmids that contained wild-type or mutant HBV sequences and 216 clinical samples. RESULTS: The PCR-LDR assay and sequencing gave comparable results for 158 of the 216 samples (73.1% with respect to mutation detection and genotyping. Complete agreement between the two methods was observed for all the samples (100% at codon 180 and codon 204. Concordant results were observed for 99.4% of the 158 samples at codon 181 and 98.7% at codon 236. The genotyping results were completely concordant between the PCR-LDR assay and sequencing. The PCR-LDR assay could detect a proportion of 1% mutant plasmid in a background of wild-type plasmid. CONCLUSION: The PCR-LDR assay is sensitive and specific for detection of HBV genotypes and drug resistance mutations, and could be helpful for decision making in the treatment of HBV infection.

  15. Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.

    Science.gov (United States)

    Muth, J; Williams, P M; Williams, S J; Brown, M D; Wallace, D C; Karger, B L

    1996-12-01

    High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.

  16. Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

    Science.gov (United States)

    Iolascon, A; Parrella, T; Perrotta, S; Guardamagna, O; Coates, P M; Sartore, M; Surrey, S; Fortina, P

    1994-07-01

    Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.

  17. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

    Science.gov (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail

    2015-10-01

    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  18. A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.

    Science.gov (United States)

    Rodríguez-Rodríguez, Everardo Remi; Ledezma-Candanoza, Luis M; Contreras-Ferrat, Luis Gabriel; Olamendi-Portugal, Timoteo; Possani, Lourival D; Becerril, Baltazar; Riaño-Umbarila, Lidia

    2012-10-26

    Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

  19. Homozygosity for a novel mutation in the C1q C chain gene in a Turkish family with hereditary C1q deficiency

    DEFF Research Database (Denmark)

    Gulez, N; Genel, F; Atlihan, F;

    2010-01-01

    Hereditary complete deficiency of complement component C1q is associated with a high prevalence of systemic lupus erythematosus and increased susceptibility to severe recurrent infections. An 11-year-old girl was screened for immunodeficiency due to a history of recurrent meningitis and pneumonia....... Immunologic studies revealed absence of classic pathway hemolytic activity and undetectable levels of Clq. Exon-specific amplification of genomic DNA by polymerase chain reaction followed by direct sequence analysis revealed a novel homozygous missense mutation at codon 48 in the C1q C gene causing a glycine...

  20. Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

    Science.gov (United States)

    Deighan, William I; O'Kane, Maurice J; McNicholl, Feargal P; Keren, David F

    2016-11-01

    Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

  1. Molecular analysis of THH-induced mutations at HPRT locus in human promyelocytic leukemia cells with multiplex polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    LIU Sheng-xue; CAO Jia; AN Hui

    2002-01-01

    Objective: To study the genotoxicity and antitumor activity of a Chinese medicinal herb, Tripterygium Hypoglaucum (Level) Hutch (THH). Methods: The genotoxicity and antitumor activity of TH-H were investigated in human promyelocytic leukemia cells on the mutation of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene by using single cell clone culture, two-way screening counting, multiplex PCR amplification and gel electrophoresis. Results: The results showed that different mutant spectra existed between the spontaneous mutation and induced mutation by THH. Only 7. 7% (1/13) of spontaneous mutants showed deletion mutations, whereas the induced mutants included 46.6% (27/58) deletions. Mapping of all intragenic deletion breakpoints showed a random distribution in all 9 exons, but toward the 3'-end of the HPRT gene. Deletion of exon 1 only appeared when whole gene was deleted. Deletions of exon 7/8 and 9 often showed linkage deletions (71.4%). Conclusion: THH can induce the mutation, mainly deletions, of HPRT gene in human promyelocytic leukemia cells.

  2. Myosin light chain phosphorylation is required for peak power output of mouse fast skeletal muscle in vitro.

    Science.gov (United States)

    Bowslaugh, Joshua; Gittings, William; Vandenboom, Rene

    2016-11-01

    The skeletal myosin light chain kinase (skMLCK) catalyzed phosphorylation of the myosin regulatory light chain (RLC) is associated with potentiation of force, work, and power in rodent fast twitch muscle. The purpose of this study was to compare concentric responses of EDL from wild-type (WT) and skMLCK devoid (skMLCK(-/-)) muscles at a range of shortening speeds (0.05 to 0.70 V max) around that expected to produce maximal power (in vitro, 25 °C) both before (unpotentiated) and after (potentiated) a potentiating stimulus (PS). When collapsed across all speeds tested, neither unpotentiated force, work, or power differed between genotypes (all data n = 10, P muscles. For example, when collapsed across the six fastest speeds we tested, both concentric force and power were increased 30-34 % in WT but only 15-17 % in skMLCK(-/-) muscles. In contrast, at the two slowest speeds, these parameters were increased in WT but decreased in skMLCK(-/-) muscles (8-10 and 7-9 %, respectively). Intriguingly, potentiation of concentric force and power was optimal near speeds producing maximal power in both genotypes. Because the PS elevated RLC phosphorylation above resting levels in WT but not in skMLCK(-/-) muscles, our data suggest that skMLCK-catalyzed phosphorylation of the RLC is required for maximal concentric power output of mouse EDL muscle stimulated at high frequency in vitro.

  3. Assessment of free light chains in the cerebrospinal fluid of patients with lymphomatous meningitis – a pilot study

    Directory of Open Access Journals (Sweden)

    Scholz C

    2007-10-01

    Full Text Available Abstract Background Lymphomatous meningitis (LM represents a severe complication of malignant lymphomas. While clinical suspicion is raised by symptoms ranging from mild disturbances of sensation to severe pain or impaired consciousness, the definite diagnosis of LM is often difficult to obtain. Since B-cell lymphomas are clonally restricted to express either kappa or lambda immunoglobulin light chain, we hypothesised that analysis of free light chain (FLC ratios might facilitate the diagnosis of LM. Methods Kappa and lambda FLC were measured using a novel nephelometric assay in cerebrospinal fluid (CSF and serum from 17 patients. 5/17 suffered from LM as demonstrated by cytology, immunocytology, and/or imaging procedures. Results Measurement of FLC concentrations in CSF was achieved for all 17 patients. FLC levels in CSF were lower than serum FLC levels in samples for the same patient obtained at the same time (p Conclusion This is the first report demonstrating that a significant proportion of LM patients display an abnormal kappa/lambda FLC ratio in the CSF.

  4. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    Science.gov (United States)

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  5. Quantification of β region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

    Science.gov (United States)

    Paolini, Lucia

    2016-06-01

    Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

  6. Effective market influence. An effect chain analysis of NUTEK`s high-frequency lighting campaign

    Energy Technology Data Exchange (ETDEWEB)

    Goeransson, C.; Faugert, S. [SIPU Utvaerdering, Stockholm (Sweden); Baeckman, B.; Arndt, J. [B2B AB, Stockholm (Sweden)

    1994-12-31

    This report is an evaluation of NUTEK`s `Better lighting and more energy-efficient high-frequency fluorescent tube luminaries` campaign. It is concerned with the ways in which NUTEK can influence the spread and use of high-frequency (HF) lighting devices. It also shows that NUTEK actually has affected (contributed to) development in the area. The report analyses and quantifies (as far as possible) how much NUTEK has influenced the various parties concerned in the sector, their attitudes and their actions. 14 figs, 5 tabs

  7. Mutations in NDUFB11, encoding a complex I component of the mitochondrial respiratory chain, cause microphthalmia with linear skin defects syndrome.

    Science.gov (United States)

    van Rahden, Vanessa A; Fernandez-Vizarra, Erika; Alawi, Malik; Brand, Kristina; Fellmann, Florence; Horn, Denise; Zeviani, Massimo; Kutsche, Kerstin

    2015-04-01

    Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

  8. Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Wu, Ming-Shan; Cheng, Chao-An; Lin, Chih-Hung; Lee, Chiou-Yueh; Tseng, Shih-Jou; Tzeng, Chyng-Shyan; Chang, Chi-Yao

    2013-03-01

    To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

  9. Serum immunoglobulin free light chain measurements and heavy chain isotype usage provide insight into disease biology in patients with POEMS syndrome.

    Science.gov (United States)

    Stankowski-Drengler, Trista; Gertz, Morie A; Katzmann, Jerry A; Lacy, Martha Q; Kumar, Shaji; Leung, Nelson; Hayman, Suzanne R; Buadi, Francis; Kyle, Robert A; Rajkumar, S Vincent; Dispenzieri, Angela

    2010-06-01

    POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare paraneoplastic syndrome in which nearly all patients have a monoclonal lambda restricted plasma cell disorder. We investigated whether patients with POEMS have abnormal serum immunoglobulin free light chain (FLC) ratios. Fifty patients with newly diagnosed POEMS syndrome were assessed. Cystatin C levels were measured to discern whether subclinical renal insufficiency could account for FLC elevations in the presence of a normal FLC ratio. Forty-five patients (90%) had elevated lambda FLC; however, only nine (18%) had abnormal FLC ratios. The rise in serum FLC of POEMS patients appeared to be multifactorial-both a function of subclinical renal insufficiency and polyclonal activation of medullary and extramedullary plasma cells. Those patients expressing a clonal IgA were more likely to have clonal plasmacytosis observed on iliac crest biopsy than those with IgG. In summary, serum immunoglobulin profiles are unique in POEMS syndrome as compared with other plasma cell disorders.

  10. Genotype phenotype correlations of cardiac beta-myosin heavy chain mutations in Indian patients with hypertrophic and dilated cardiomyopathy

    DEFF Research Database (Denmark)

    Rai, Taranjit Singh; Ahmad, Shamim; Bahl, Ajay;

    2009-01-01

    consecutive patients diagnosed with HCM or DCM (69 with HCM and 61 with DCM) attending the cardiology clinic of Post Graduate Institute of Medical Education and Research were screened for mutations in the MYH7 gene. The control group for genetic studies consisted of 100 healthy subjects. We report 14...

  11. The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene

    DEFF Research Database (Denmark)

    Kølvraa, S; Gregersen, N; Blakemore, A I;

    1991-01-01

    RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII. PstI and TaqI and with an MCAD cDNA-clone as a probe....... Of 32 disease-causing alleles studied, 31 possessed the previously published A----G point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present....... In contrast, the same haplotype was present in only 23% of normal alleles (P less than or equal to 3.4 x 10(-18)). These findings are consistent with the existence of a pronounced founder effect, possibly combined with biological and/or sampling selection....

  12. A new era for homolytic aromatic substitution: replacing Bu3SnH with efficient light-induced chain reactions.

    Science.gov (United States)

    Gurry, Michael; Aldabbagh, Fawaz

    2016-04-28

    Herein is a pertinent review of recent photochemical homolytic aromatic substitution (HAS) literature. Issues with using the reductant Bu3SnH in an oxidative process where the net loss of a hydrogen atom occurs is discussed. Nowadays more efficient light-induced chain reactions are used resulting in HAS becoming a synthetic mechanism of choice rivaling organometallic, transition-metal and electrophilic aromatic substitution protocols. The review includes aromatic substitution as part of a tandem or cascade reaction, Pschorr reaction, as well as HAS facilitated by ipso-substitution, and Smiles rearrangement. Recently visible-light photoredox catalysis, which is carried out at room temperature has become one of the most important means of aromatic substitution. The main photoredox catalysts used are polypyridine complexes of Ru(ii) and Ir(iii), although eosin Y is an alternative allowing metal-free HAS. Other radical initiator-free aromatic substitutions have used 9-mesityl-10-methylacridinium ion and N,N-bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarboximide) as the photoredox catalyst, UV-light, photoinduced electron-transfer, zwitterionic semiquinone radical anions, and Barton ester intermediates.

  13. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  14. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    Energy Technology Data Exchange (ETDEWEB)

    Lima, V.V. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil); Lobato, N.S.; Filgueira, F.P. [Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO (Brazil); Webb, R.C. [Department of Physiology, Georgia Regents University, Augusta, GA (United States); Tostes, R.C. [Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Giachini, F.R. [Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT (Brazil)

    2014-08-15

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca{sup 2+}/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  15. Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

    OpenAIRE

    Balastik, M.; Ferraguti, F.; A. Pires da Silva; Lee, T; Alvarez-Bolado, G.; Lu, K.; Gruss, P

    2008-01-01

    TRIM RING finger proteins have been shown to play an important role in cancerogenesis, in the pathogenesis of some human hereditary disorders, and in the defense against viral infection, but the function of the majority of TRIM proteins remains unknown. Here, we show that TRIM RING finger protein TRIM2, highly expressed in the nervous system, is an UbcH5a-dependent ubiquitin ligase. We further demonstrate that TRIM2 binds to neurofilament light subunit (NF-L) and regulates NF-L ubiquitination...

  16. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    Science.gov (United States)

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-01-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  17. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    KAUST Repository

    Sharkhuu, Altanbadralt

    2014-06-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

  18. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate.

    Science.gov (United States)

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-06-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide.

  19. Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.

    Science.gov (United States)

    Van Calcar, Sandra C; Baker, Mei W; Williams, Phillip; Jones, Susan A; Xiong, Blia; Thao, Mai Choua; Lee, Sheng; Yang, Mai Khou; Rice, Greg M; Rhead, William; Vockley, Jerry; Hoffman, Gary; Durkin, Maureen S

    2013-01-01

    Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (≥0.44μmol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ≥0.44μmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to

  20. Identification of four new mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene in two patients

    DEFF Research Database (Denmark)

    Gregersen, N; Winter, V S; Corydon, M J

    1998-01-01

    We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disea...

  1. Respiratory chain complex I deficiency due to NDUFA12 mutations as a new cause of Leigh syndrome

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Rodenburg, Richard J; van den Brand, Mariël;

    2011-01-01

    This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed...

  2. Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus)

    DEFF Research Database (Denmark)

    Kim, Young Kyu; Lee, Jung Seok; Jung, Jae Wook

    2017-01-01

    abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.......Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used...... successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11...

  3. Structure–function–folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

    Indian Academy of Sciences (India)

    P M Krishna Mohan; Ramakrishna V Hosur

    2009-09-01

    The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure–function–folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

  4. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation

    DEFF Research Database (Denmark)

    Hansen, Charlotte Toftmann

    2016-01-01

    , data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information......Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally...... of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed....

  5. The 1909 Darwin celebration. Reexamining evolution in the light of Mendel, mutation, and meiosis.

    Science.gov (United States)

    Richmond, Marsha L

    2006-09-01

    In June 1909, scientists and dignitaries from 167 different countries gathered in Cambridge to celebrate the hundredth anniversary of Charles Darwin's birth and the fiftieth anniversary of the publication of Origin of Species. The event was one of the most magnificent commemorations in the annals of science. Delegates gathered within the cloisters of Cambridge University not only to honor the "hero" of evolution but also to reassess the underpinnings of Darwinism at a critical juncture. With the mechanism of natural selection increasingly under attack, evolutionary theory was in disarray. Against this backdrop, biologists weighed the impact of several new developments--the rediscovery of Mendel's laws of heredity, de Vriesian mutation theory, and the linkage of sex-cell division (recently named "meiosis") to the mechanism of heredity. The 1909 Darwin celebration thus represents a significant watershed in the history of modem biology that allows historians to assess the status of evolution prior to the advent of the chromosome theory of genetics.

  6. Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori.

    Science.gov (United States)

    Hong, Sun Mee; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2014-04-01

    The silkworm genome encodes three iron storage proteins or ferritins, Fer1HCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day-old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses during embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCH mRNA contains an iron-responsive element, suggesting this ferritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection of Bombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory pathway, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed of mannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms.

  7. Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays.

    Science.gov (United States)

    Reiman, Anne; Pandey, Sarojini; Lloyd, Kate L; Dyer, Nigel; Khan, Mike; Crockard, Martin; Latten, Mark J; Watson, Tracey L; Cree, Ian A; Grammatopoulos, Dimitris K

    2016-11-01

    Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories. Methods Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. Results In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method. Conclusions Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

  8. A Rare Case of Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma with Light Chain Discrepancy between B Lymphocyte Population and Serum Paraprotein.

    Science.gov (United States)

    Kim, Hyun Jeong; Hur, Mina; Kim, Hanah; Moon, Hee-Won; Yun, Yeo-Min; Kim, Sung Yong; Lee, Mark Hong

    2015-01-01

    Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma with bone marrow involvement, monotypic immunoglobulin (Ig) M and a light chain of neoplastic cells. A 68-year-old woman presented with fever, nausea, vomiting, and pancytopenia. Her serum albumin/globulin ratio was reversed, and monoclonal gammopathy of IgM, lambda type (23.20%, 1.58 g/dL) was detected. In her bone marrow, increased small lymphocytes were admixed with plasmacytoid lymphocytes and plasma cells. She was diagnosed as having lymphoplasmacytic variant of WM. Immunohistochemical stains and flow cytometic analysis revealed two distinct populations; monoclonal B cells (kappa+) and abnormal plasma cells (CD19-/CD56+/lambda+). She expired 19 days after admission due to septic shock. This is a rare case of WM exhibiting a light chain discrepancy between monoclonal B lymphocytes and paraprotein-secreting plasma cells. Light chain restriction may occur distinctly between lymphocyte and plasma cell populations in WM.

  9. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  10. Plasma vitamin D-binding protein (GC) factors, immunoglobulin G heavy chain (GM) allotypes and immunoglobulin kappa light chain (KM1) allotype in patients with sarcoidosis and in healthy control subjects

    DEFF Research Database (Denmark)

    Milman, Nils; Thymann, Mariann; Graudal, Niels

    2002-01-01

    and immunoglobulin kappa light chain (KM) allotype differed significantly from the distribution in healthy subjects. METHODS: GC 1S, 1F, 2 allotypes, GM 1, 2, 5 allotypes, and KM1 allotype were assessed in 44 patients with sarcoidosis and in healthy control subjects. RESULTS: There were no significant differences...

  11. Long-term survival in a child with severe encephalopathy, multiple respiratory chain deficiency and GFM1 mutations

    Science.gov (United States)

    Brito, Sara; Thompson, Kyle; Campistol, Jaume; Colomer, Jaime; Hardy, Steven A.; He, Langping; Fernández-Marmiesse, Ana; Palacios, Lourdes; Jou, Cristina; Jiménez-Mallebrera, Cecilia; Armstrong, Judith; Montero, Raquel; Artuch, Rafael; Tischner, Christin; Wenz, Tina; McFarland, Robert; Taylor, Robert W.

    2015-01-01

    Background: Mitochondrial diseases due to deficiencies in the mitochondrial oxidative phosphorylation system (OXPHOS) can be associated with nuclear genes involved in mitochondrial translation, causing heterogeneous early onset and often fatal phenotypes. Case report: The authors describe the clinical features and diagnostic workup of an infant who presented with an early onset severe encephalopathy, spastic-dystonic tetraparesis, failure to thrive, seizures and persistent lactic acidemia. Brain imaging revealed thinning of the corpus callosum and diffuse alteration of white matter signal. Genetic investigation confirmed two novel mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1 (mtEFG1), resulting in combined deficiencies of OXPHOS. Discussion: The patient shares multiple clinical, laboratory and radiological similarities with the 11 reported patients with mutations involving this gene, but presents with a stable clinical course without metabolic decompensations, rather than a rapidly progressive fatal course. Defects in GFM1 gene confer high susceptibility to neurologic or hepatic dysfunction and this is, to the best of our knowledge, the first described patient who has survived beyond early childhood. Reporting of such cases is essential so as to delineate the key clinical and neuroradiological features of this disease and provide a more comprehensive view of its prognosis. PMID:25852744

  12. Long-term survival in a child with severe encephalopathy, multiple respiratory chain deficiency and GFM1 mutations

    Directory of Open Access Journals (Sweden)

    Sara eBrito

    2015-03-01

    Full Text Available Background: Mitochondrial diseases due to deficiencies in the mitochondrial oxidative phosphorylation system (OXPHOS can be associated with nuclear genes involved in mitochondrial translation, causing heterogeneous early onset and often fatal phenotypes. Case report: The authors describe the clinical features and diagnostic workup of an infant who presented with an early onset severe encephalopathy, spastic-dystonic tetraparesis, failure to thrive, seizures and persistent lactic acidemia. Brain imaging revealed thinning of the corpus callosum and diffuse alteration of white matter signal. Genetic investigation confirmed two novel mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1 (mtEFG1, resulting in combined deficiencies of OXPHOS. Discussion: The patient shares multiple clinical, laboratory and radiological similarities with the eleven reported patients with mutations involving this gene, but presents with a stable clinical course without metabolic decompensations, rather than a rapidly progressive fatal course. Defects in GFM1 gene confer high susceptibility to neurologic or hepatic dysfunction and this is, to the best of our knowledge, the first described patient who has survived beyond early childhood. Reporting of such cases is essential so as to delineate the key clinical and neuroradiological features of this disease and provide a more comprehensive view of its prognosis.

  13. Abnormal kappa:lambda light chain ratio in circulating immune complexes as a marker for B cell activity in juvenile idiopathic arthritis.

    Science.gov (United States)

    Low, J M; Chauhan, A K; Moore, T L

    2007-01-01

    Patients with juvenile idiopathic arthritis (JIA) have been shown to have elevated levels of circulating immune complexes (CICs) which correlated with disease activity. Our aim was to assess B cell activity by measuring the amount of and the kappa:lambda chain immunoglobulin light (L) chain ratio in CICs from JIA patients and to determine potential evidence for either an antigen-driven response or B-cell receptor editing. We used an enzyme-linked immunosorbent assay to measure kappa and lambda chains present in the CICs from the sera of patients with JIA. Statistical analysis was performed using Pearson's correlation, one-way ANOVA and Bonferroni post hoc analysis. Sera from 44 JIA patients were examined for the concentration of L chains in CICs. Healthy controls had a kappa:lambda chain ratio of 1.2:1, whereas this ratio was reversed among JIA subgroups with RF-positive polyarthritis (1:1.2), RF-negative polyarthritis (1:1.3), oligoarthritis (1:2.3) and systemic-onset arthritis (1:2.5). In addition, overall lambda chain selection was not significantly associated with a particular immunoglobulin heavy (H) chain and occurred with all immunoglobulin isotypes. We showed preferential selection of lambda chains contributing to the formation of potentially pathogenic CICs from JIA patients, of all onset types compared to healthy controls, in an H chain-independent manner. The reversal of kappa:lambda chain ratio within the JIA CICs and association with all immunoglobulin isotypes demonstrated the potential for L chain editing. Furthermore, we conclude that a reversal of the normal kappa:lambda chain ratio in JIA CICs may be used as a marker for increased B-cell activity.

  14. Mutation of histidine residues in CP47 leads to destabilization of the photosystem II complex and to impairment of light energy transfer.

    Science.gov (United States)

    Shen, G; Eaton-Rye, J J; Vermaas, W F

    1993-05-18

    Site-directed mutagenesis has been used to change conserved histidine residues in hydrophobic regions of the photosystem II chlorophyll-binding protein CP47 in the cyanobacterium Synechocystis sp. PCC 6803. Nine mutants with one, four mutants with two, and four mutants with three His mutations in CP47 have been generated and characterized. Mutation of any one of seven different His residues to Tyr leads to slower photoautotrophic growth and apparent destabilization of the PS II complex. Mutations introduced into multiple His residues in one mutant exhibited a cumulative effect. Replacing His by Asn leads to a much smaller effect than observed upon mutation to Tyr. This is consistent with the hypothesis that the mutated His residues are chlorophyll ligands: Asn can substitute as chlorophyll ligand, whereas Tyr cannot. Further evidence supporting a role of the mutated His residues in chlorophyll binding comes from measurements of the light intensity needed to half-saturate oxygen evolution. All His mutants with impaired PS II function needed higher light intensities for half-saturation than wild type. A possible explanation for this decrease in antenna efficiency in the mutants is a loss of the Mg in the chlorophyll due to a loss of the fifth ligand, and thus the formation of a pheophytin molecule in the antenna. We conclude that conserved His residues in hydrophobic regions of CP47 indeed are chlorophyll ligands and that these ligands are important for PS II stability as well as efficient antenna function.

  15. Short-chain acyl-CoA dehydrogenase gene mutation (c.319C>T) presents with clinical heterogeneity and is candidate founder mutation in individuals of Ashkenazi Jewish origin.

    Science.gov (United States)

    Tein, Ingrid; Elpeleg, Orly; Ben-Zeev, Bruria; Korman, Stanley H; Lossos, Alexander; Lev, Dorit; Lerman-Sagie, Tally; Leshinsky-Silver, Esther; Vockley, Jerry; Berry, Gerard T; Lamhonwah, Anne-Marie; Matern, Dietrich; Roe, Charles R; Gregersen, Niels

    2008-02-01

    We report 10 children (7 male, 3 female), 3 homozygous for c.319C>T mutation and 7 heterozygous for c.319C>T on one allele and c.625G>A variant on the other in the short-chain acyl-CoA dehydrogenase (SCAD) gene (ACADS). All were of Ashkenazi Jewish origin in which group we found a c.319C>T heterozygote frequency of 1:15 suggesting the presence of a founder mutation or selective advantage. Phenotype was variable with onset from birth to early childhood. Features included hypotonia (8/10), developmental delay (8/10), myopathy (4/10) with multicore changes in two and lipid storage in one, facial weakness (3/10), lethargy (5/10), feeding difficulties (4/10) and congenital abnormalities (3/7). One female with multiminicore myopathy had progressive external ophthalmoplegia, ptosis and cardiomyopathy with pneumonia and respiratory failure. Two brothers presented with psychosis, pyramidal signs, and multifocal white matter abnormalities on MRI brain suggesting additional genetic factors. Two other infants also had white matter changes. Elevated butyrylcarnitine (4/8), ethylmalonic aciduria (9/9), methylsuccinic aciduria (6/7), decreased butyrate oxidation in lymphoblasts (2/4) and decreased SCAD activity in fibroblasts or muscle (3/3) were shown. Expression studies of c.319C>T in mouse liver mitochondria showed it to be inactivating. c.625G>A is a common variant in ACADS that may confer disease susceptibility. Five healthy parents were heterozygous for c.319C>T and c.625G>A, suggesting reduced penetrance or broad clinical spectrum. We conclude that the c.319C>T mutation can lead to wide clinical and biochemical phenotypic variability, suggesting a complex multifactorial/polygenic condition. This should be screened for in individuals with multicore myopathy, particularly among the Ashkenazim.

  16. Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

    Science.gov (United States)

    Gomez, Diana I; Fisher-Hoch, Susan P; Bordt, Andrea S; Quitugua, Teresa N; Robledo, Jaime; Alvarez, Nataly; Correa, Nidia; McCormick, Joseph B; Restrepo, Blanca I

    2010-05-01

    Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.

  17. Fast side chain replacement in proteins using a coarse-grained approach for evaluating the effects of mutation during evolution.

    Science.gov (United States)

    Grahnen, Johan A; Kubelka, Jan; Liberles, David A

    2011-08-01

    For high-throughput structural genomic and evolutionary bioinformatics approaches, there is a clear need for fast methods to evaluate substitutions structurally. Coarse-grained methods are both powerful and fast, and a coarse-grained approach to position the substituted side chains is presented. Through the application of a coarse-grained method, a speed-up on the single- residue replacement, of at least sevenfold is achieved compared with modern all-atom approaches. At the same time, this approach maintains a small median RMSD from the leading all-atom approach (as measured in coarse-grained space), and predicts the conformation of point mutants with similar accuracy and generates biologically realistic side chain angles. This method is also substantially more predictable in its run time, making it useful for high-throughput studies of protein structural evolution. To demonstrate the utility of this method, it has been implemented in a forward simulation of sequences threaded through the SH2 domains, with selective pressures to fold and bind specifically. The relative substitution rates across the protein structure and at the binding interface are reflective of those observed in SH2 domain evolution. The algorithm has been implemented in C++, with the source code and binaries (currently supported for Linux systems) freely available as SARA at http://www.wyomingbioinformatics.org/LiberlesGroup/SARA .

  18. The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

    Science.gov (United States)

    DiBella, Linda M.; Sakato, Miho; Patel-King, Ramila S.; Pazour, Gregory J.; King, Stephen M.

    2004-01-01

    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins. PMID:15304520

  19. Clinical usefulness of serum free light chains measurement in patients with multiple myeloma: comparative analysis of two different tests

    Directory of Open Access Journals (Sweden)

    Tadeusz Kubicki

    2017-01-01

    Full Text Available Introduction: There are two commercially available tests for measurement of serum free light chains (sFLC in multiple myeloma (MM patients – Freelite and N Latex FLC. The aim of this study was to perform an assessment and direct comparison of the usefulness of the methods in routine clinical practice.Methods: 40 refractory/relapsed MM patients underwent routine disease activity assessment studies, along with sFLC analysis using both assays. Correlation and concordance between the tests and sensitivity of studied methods of sFLC assessment were established. Special attention was focused on sFLC results in patients finally evaluated after completing the treatment. Results: A weak correlation for the measurement of both κ [Passing–Bablok slope (PB = 0.7681] and λ chains [(PB = 1.542] was found. Using Bland–Altman plots, a bias of 0.0467 (κ and -0.2133 (λ between the measurements was documented. The concordance coefficient equaled 0.87 for κ, 0.62 for λ and 0.52 for κ/λ ratio. Ten patients had an abnormal Freelite assay κ/λ ratio and normal N Latex FLC κ/λ ratio. Three of these patients had negative serum protein electrophoresis results and fulfilled diagnostic criteria of stringent complete remission (sCR according to N Latex FLC (but not according to Freelite. When the κ/λ ratio obtained by both methods was compared to patients’ serum/urine protein electrophoresis and immunofixation results, sensitivity of Freelite and N Latex FLC was established to be 62.5% and 41%, respectively. Conclusions: There was no strong correlation or concordance between the two assays, and the sensitivity in terms of sFLC detection was different. This may cause problems when diagnosis of sCR is considered.

  20. Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

    This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

  1. Trends in Survival and Renal Recovery in Patients with Multiple Myeloma or Light-Chain Amyloidosis on Chronic Dialysis.

    Science.gov (United States)

    Decourt, Alexandre; Gondouin, Bertrand; Delaroziere, Jean Christophe; Brunet, Philippe; Sallée, Marion; Burtey, Stephane; Dussol, Bertrand; Ivanov, Vadim; Costello, Regis; Couchoud, Cecile; Jourde-Chiche, Noemie

    2016-03-07

    Monoclonal gammopathies (MGs) with renal involvement can lead to ESRD caused by myeloma cast nephropathy (MCN), immunoglobulin light chain amyloidosis (ALA), or light-chain deposition disease (LCDD). Few studies have focused on the prognosis of patients with MG on chronic dialysis. We evaluated the outcomes of patients with MG incident on chronic dialysis in France. All incident patients registered in the Renal Epidemiology and Information Network Registry between 2002 and 2011 with ESRD caused by ALA, LCDD, or MCN were included. Patient's survival, censored for renal transplantation, renal recovery, and loss to follow-up, as well as renal outcomes were analyzed and compared with a control group. Risk factors and causes of death were analyzed. We included 1459 patients, comprising 265 (18%) patients with ALA, 334 (23%) patients with LCDD, and 861 (59%) patients with MCN. Median age was 72 years, and 56% were men. Median follow-up was 13.1 months. Renal recovery was observed in 9.1% of patients and more frequent after 2006. Kidney transplantation was rare in this population (2.3%). Among 1272 patients who remained on dialysis, 67% died. Median survival on dialysis was 18.3 months. Main causes of death were malignancies (34.4%), cardiovascular diseases (18%), infections (13.3%), and cachexia (5.2%). Independent risk factors of death were age (hazard ratio [HR], 1.03 per year increase; 95% confidence interval [95% CI], 1.02 to 1.03), frailty (HR, 1.93; 95% CI, 1.58 to 2.36), congestive heart failure (HR, 1.54; 95% CI, 1.23 to 1.93), and dialysis initiation on a central catheter (HR, 1.40; 95% CI, 1.11 to 1.75). Factors associated with a lower risk of death were year of dialysis initiation (HR, 0.95 per year increase; 95% CI, 0.91 to 0.99) and high BP (HR, 0.80; 95% CI, 0.67 to 0.97). Survival of patients with ALA, LCDD, or MCN on chronic dialysis is poor but has improved over time. Progressive malignancy is the main cause of death in this population. Renal recovery

  2. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    Energy Technology Data Exchange (ETDEWEB)

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L. (IIT); (UW-MED)

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  3. Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Shelly Woody

    Full Text Available Myosin II (MyoII is required for insulin-responsive glucose transporter 4 (GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC of MyoIIA via myosin light chain kinase (MLCK. The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy ethane-N,N,N',N'-tetra acetic acid, (BAPTA (in the presence of insulin impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

  4. Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris.

    Science.gov (United States)

    A R, Sudheer Reddy; Satheeshkumar, Padikara Kutty; Vijayalakshmi, Mookambeswaran A

    2013-09-01

    Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1-A2) and light chains (A3-C1-C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.

  5. Successful management of refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis by vincristine adriamycin dexamethasone chemotherapy: a case report

    Directory of Open Access Journals (Sweden)

    Mima Akira

    2010-10-01

    Full Text Available Abstract Introduction Refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation is rarely reported and has a poor prognosis in general (a median survival of 1.6 months. Moreover, the optimum treatment for this condition is still undecided. This is the first report on the successful use of vincristine, adriamycin and dexamethasone chemotherapy for refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis without cardiac decompensation. Case presentation We report the case of a 68-year old Japanese male with systemic immunoglobulin light chain amyloidosis presenting with bilateral pleural effusion (more severe on the right side in the absence of cardiac decompensation that was refractory to diuretic therapy. The patient was admitted for fatigue, exertional dyspnea, and bilateral lower extremity edema. He had been receiving intermittent melphalan and prednisone chemotherapy for seven years. One month before admission, his dyspnea had got worse, and his chest radiograph showed bilateral pleural effusion; the pleural effusion was ascertained to be a transudate. The conventionally used therapeutic measures, including diuretics and thoracocentesis, failed to control pleural effusion. Administration of vincristine, adriamycin, and dexamethasone chemotherapy led to successful resolution of the effusion. Conclusion Treatment with vincristine, adriamycin, and dexamethasone chemotherapy was effective for the refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation and appears to be associated with improvement in our patient's prognosis.

  6. ASSESSMENT OF THE RESIDUAL TUMOR IN PATIENTS WITH MULTIPLE MYELOMA BASED ON THE ANALYSIS OF THE FREE LIGHT CHAINS OF IMMUNOGLOBULINS IN BLOOD SERUM

    Directory of Open Access Journals (Sweden)

    T. A. Мitina

    2013-01-01

    Full Text Available Efficiency of the multiple myeloma treatment with chemotherapy including bortezomib was assessed based on determination of the level of immunoglobulin free light chains in blood serum. The method enables estimation of changes in kinetic parameters of the residual tumor, detection of the disease course prognosis, and the choice of the optimal approach to the disease therapy.

  7. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen

  8. Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway

    NARCIS (Netherlands)

    Wang, Siyuan; Chen, Cheng; Su, Ke; Zha, Dongqing; Liang, Wei; Hillebrands, J L; van Goor, Harry; Ding, Guohua

    2016-01-01

    Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen m

  9. Cardiomyopathy-related mutation (A30V) in mouse cardiac troponin T divergently alters the magnitude of stretch activation in α- and β-myosin heavy chain fibers.

    Science.gov (United States)

    Mickelson, Alexis V; Gollapudi, Sampath K; Chandra, Murali

    2017-01-01

    The present study investigated the functional consequences of the human hypertrophic cardiomyopathy (HCM) mutation A28V in cardiac troponin T (TnT). The A28V mutation is located within the NH2 terminus of TnT, a region known to be important for full activation of cardiac thin filaments. The functional consequences of the A28V mutation in TnT remain unknown. Given how α- and β-myosin heavy chain (MHC) isoforms differently alter the functional effect of the NH2 terminus of TnT, we hypothesized that the A28V-induced effects would be differently modulated by α- and β-MHC isoforms. Recombinant wild-type mouse TnT (TnTWT) and the mouse equivalent of the human A28V mutation (TnTA30V) were reconstituted into detergent-skinned cardiac muscle fibers extracted from normal (α-MHC) and transgenic (β-MHC) mice. Dynamic and steady-state contractile parameters were measured in reconstituted muscle fibers. Step-like length perturbation experiments demonstrated that TnTA30V decreased the magnitude of the muscle length-mediated recruitment of new force-bearing cross bridges (ER) by 30% in α-MHC fibers. In sharp contrast, TnTA30V increased ER by 55% in β-MHC fibers. Inferences drawn from other dynamic contractile parameters suggest that directional changes in ER in TnTA30V + α-MHC and TnTA30V + β-MHC fibers result from a divergent impact on cross bridge-regulatory unit (troponin-tropomyosin complex) cooperativity. TnTA30V-mediated effects on Ca(2+)-activated maximal tension and instantaneous muscle fiber stiffness (ED) were also divergently affected by α- and β-MHC. Our study demonstrates that TnTA30V + α-MHC and TnTA30V + β-MHC fibers show contrasting contractile phenotypes; however, only the observations from β-MHC fibers are consistent with the clinical data for A28V in humans.

  10. Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain

    KAUST Repository

    Coluccio, M. L.

    2015-09-04

    Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10−12 M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components.

  11. Detection of single amino acid mutation in human breast cancer by disordered plasmonic self-similar chain.

    Science.gov (United States)

    Coluccio, Maria Laura; Gentile, Francesco; Das, Gobind; Nicastri, Annalisa; Perri, Angela Mena; Candeloro, Patrizio; Perozziello, Gerardo; Proietti Zaccaria, Remo; Gongora, Juan Sebastian Totero; Alrasheed, Salma; Fratalocchi, Andrea; Limongi, Tania; Cuda, Giovanni; Di Fabrizio, Enzo

    2015-09-01

    Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10(-12) M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components.

  12. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    Science.gov (United States)

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  13. A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

    Science.gov (United States)

    Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

    2014-01-01

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

  14. Cherenkov light production from the α-emitting decay chains of (223)Ra, (212)Pb, and (149)Tb for Cherenkov Luminescence Imaging.

    Science.gov (United States)

    Wood, V; Ackerman, N L

    2016-12-01

    Cherenkov Luminescence Imaging (CLI) is a new method to image radioactive therapeutic and diagnostic agents, primarily in preclinical studies. This study used Geant4 and Python to generate the predicted Cherenkov light production as a function of time for a set of isotopic chains of interest for targeted alpha therapy: (223)Ra, (212)Pb, and (149)Tb. All are shown to produce substantial Cherenkov light, though time delays between initial decays and the production of Cherenkov light requires caution in interpreting CLI. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;

    1992-01-01

    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...

  16. Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa.

    Science.gov (United States)

    Kuhle, Jens; Barro, Christian; Andreasson, Ulf; Derfuss, Tobias; Lindberg, Raija; Sandelius, Åsa; Liman, Victor; Norgren, Niklas; Blennow, Kaj; Zetterberg, Henrik

    2016-10-01

    Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, pNfL measurements between the platforms were highly correlated (r=1.0, pNfL levels were highly related between ECL assay and Simoa (r=0.86, pNfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases.

  17. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  18. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    Science.gov (United States)

    Bacioglu, Mehtap; Maia, Luis F; Preische, Oliver; Schelle, Juliane; Apel, Anja; Kaeser, Stephan A; Schweighauser, Manuel; Eninger, Timo; Lambert, Marius; Pilotto, Andrea; Shimshek, Derya R; Neumann, Ulf; Kahle, Philipp J; Staufenbiel, Matthias; Neumann, Manuela; Maetzler, Walter; Kuhle, Jens; Jucker, Mathias

    2016-07-01

    A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

  19. Molecular and Functional Analyses of the Fast Skeletal Myosin Light Chain2 Gene of the Korean Oily Bitterling, Acheilognathus koreensis

    Directory of Open Access Journals (Sweden)

    Hyun Kook Cho

    2013-08-01

    Full Text Available We identified and characterized the primary structure of the Korean oily bitterling Acheilognathus koreensis fast skeletal myosin light chain 2 (Akmlc2f, gene. Encoded by seven exons spanning 3955 bp, the deduced 168-amino acid AkMLC2f polypeptide contained an EF-hand calcium-binding motif and showed strong homology (80%–98% with the MLC2 proteins of Ictalurus punctatus and other species, including mammals. Akmlc2f mRNA was highly enriched in skeletal muscles, and was detectable in other tissues. The upstream regions of Akmlc2f included a TATA box, one copy of a putative MEF-2 binding site and several putative C/EBPβ binding sites. The functional activity of the promoter region of Akmlc2f was examined using luciferase and red fluorescent protein reporters. The Akmlc2f promoter-driven reporter expressions were detected and increased by the C/EBPβ transcription factor in HEK293T cells. The activity of the promoter of Akmlc2f was also confirmed in the developing zebrafish embryo. Although the detailed mechanism underlying the expression of Akmlc2f remains unknown, these results suggest the muscle-specific expression of Akmlc2f transcript and the functional activation of Akmlc2f promoter by C/EBPβ.

  20. Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

    Science.gov (United States)

    Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

    2017-09-01

    The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

  1. A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

    Science.gov (United States)

    Patel-King, Ramila S.; Benashski, Sharon E.; Harrison, Alistair; King, Stephen M.

    1997-01-01

    Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex–encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm. PMID:9166408

  2. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    Science.gov (United States)

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  3. Identification and characterization of the Bombyx mori myosin II essential light chain and its effect in BmNPV infection

    Directory of Open Access Journals (Sweden)

    L Hao

    2015-02-01

    Full Text Available Myosin, as a type of molecular motor, is mainly involved in muscle contraction. Recently, myosin research has made considerable progress. However, the function of Bombyx mori myosin remains unclear. In this study, we cloned the BmMyosin II essential light chain (BmMyosin II ELC gene from a cDNA library of silkworm, which had an open reading frame (ORF of 444 bp encoding 147 amino acids (about 16 kDa. After analyzing their sequences, BmMyosin II ELC was similar to the ELCs of 27 other Myosin II types, which contained EFh domain that bound Ca2+. In addition, 28 sequences had five motifs, motifs 1 and 3 were relatively conserved. We constructed two vectors with BmMyosin to transfect MGC803 or BmN, monolayer wound healing of cells indicated they can promote cell migration successfully. For three fifth instar silkworms, Bm306, BmNB, BmBC8, we mainly analyzed the change of BmMyosin II ELC from transcription and translation after infecting with nucleopolyhedrovirus (BmNPV. We found that gene expression of resistant strains were higher than susceptible strains at 12 h, while the result of the translation level was opposite that of the transcription level. Through in vitro protein interactions, we found BmMyosin II ELC can interact with BmNPV ubiquitin.

  4. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

  5. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    Science.gov (United States)

    Hansen, Charlotte Toftmann

    2016-06-01

    Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed.

  6. Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production

    Directory of Open Access Journals (Sweden)

    Akihiro Watari

    2017-04-01

    Full Text Available Background/Aims: Although proinflammatory cytokine–induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. Methods: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER and paracellular permeability were measured. Production levels and localization of tight junction (TJ proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC and MLC kinase (MLCK mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Results: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Conclusion: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.

  7. Rebeccamycin Attenuates TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Inhibiting Myosin Light Chain Kinase Production.

    Science.gov (United States)

    Watari, Akihiro; Sakamoto, Yuta; Hisaie, Kota; Iwamoto, Kazuki; Fueta, Miho; Yagi, Kiyohito; Kondoh, Masuo

    2017-01-01

    Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation. © 2017 The Author(s)Published by S. Karger AG, Basel.

  8. Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.

    Science.gov (United States)

    Okada, Chisa; Nakamura, Akio; Tomioka, Shigeo; Kohama, Kazuhiro; Kaneko, Takako S

    2010-08-01

    A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 microM, and the V(max) was 1.17 nkat/mg of protein. Ca(2+) (10 microM) inhibited the activity of the enzyme, and Mg(2+) (8.5 microM) activated the dephosphorylation of PMLC. Mn(2+) (1.6 microM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.

  9. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    Sagar P Mahale

    Full Text Available The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC. However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division.

  10. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  11. [Effect of vitamin E on myosin light chain kinase activity and endothelial permeability of the artery in atherosclerotic rabbit].

    Science.gov (United States)

    Zhu, Hua-qing; Ren, Bin; Xiong, Jiang-xia; Hu, Ruo-lei; Liu, Xiao-ying; Wang, Xue; Zhang, Su-mei; Liu, Hui-lan; Jiang, Zhi-kui; Zhou, Qing; Gui, Shu-yu; Wang, Yuan

    2003-08-01

    To study the effect of vitamin E (Vit E) on the myosin light chain kinase(MLCK) activity and the endothelial permeability of the artery in atherosclerotic rabbits. The MLCK activity of rabbit artery was measured by incorporation of gamma-(32)P. The endothelial permeability was accessed by immunofluorescence. The model of atherosclerosis was established after rabbits were fed with cholesterol for four weeks. The activity of MLCK increased markedly, and there was significantly statistical difference compared with the normal control (Prabbits were fed with cholesterol for twelve weeks or with cholesterol and Vit E for twelve weeks, the activity of MLCK did not change markedly, and there was no statistical difference compared with the normal control, respectively (P>0.05). The permeability of arterial wall was increased after the rabbits were fed with cholesterol for four weeks, and the permeability increased even more obviously after the rabbits were fed with cholesterol for twelve weeks. The permeability appeared to be decreased when Vit E was added into the cholesterol feeding. The change in integrity of arterial wall may be associated with the increase of the activity of MLCK. Vit E may decrease the MLCK activity. Vit E may decrease the endothelial permeability of atherosclerotic rabbits.

  12. Auto-SCT improves survival in systemic light chain amyloidosis: a retrospective analysis with 14-year follow-up.

    Science.gov (United States)

    Parmar, S; Kongtim, P; Champlin, R; Dinh, Y; Elgharably, Y; Wang, M; Bashir, Q; Shah, J J; Shah, N; Popat, U; Giralt, S A; Orlowski, R Z; Qazilbash, M H

    2014-08-01

    Optimal treatment approach continues to remain a challenge for systemic light chain amyloidosis (AL). So far, Auto-SCT is the only modality associated with long-term survival. However, failure to show survival benefit in randomized study raises questions regarding its efficacy. We present a comparative outcome analysis of Auto-SCT to conventional therapies (CTR) in AL patients treated over a 14-year period at our institution. Out of the 145 AL amyloidosis patients, Auto-SCT was performed in 80 patients with 1-year non-relapse mortality rate of 12.5%. Novel agents were used as part of induction therapy in 56% of transplant recipients vs 46% of CTR patients. Hematological and organ responses were seen in 74.6% and 39% in the Auto-SCT arm vs 53% and 12% in the CTR arm, respectively. The projected 5-year survival for Auto-SCT vs CTR was 63% vs 38%, respectively. Landmark analysis of patients alive at 1-year after diagnosis showed improved 5-year OS of 72% with Auto-SCT vs 65% in the CTR arm. In the multivariate analysis, age Auto-SCT were associated with improved survival. In conclusion, Auto-SCT is associated with long-term survival for patients with AL amyloidosis.

  13. Melphalan, lenalidomide and dexamethasone for the treatment of immunoglobulin light chain amyloidosis: results of a phase II trial.

    Science.gov (United States)

    Sanchorawala, Vaishali; Patel, Jaymin M; Sloan, J Mark; Shelton, Anthony C; Zeldis, Jerome B; Seldin, David C

    2013-05-01

    We report results of a phase II trial of combination of melphalan, lenalidomide, and dexamethasone for the treatment of immunoglobulin light chain (AL) amyloidosis. The primary objectives were tolerability and hematologic response rate; secondary objectives were organ responses and survival. Treatment protocol consisted of melphalan 5 mg/m(2)/day for four days, lenalidomide 10 mg/day for 21 days and dexamethasone 20-40 mg once a week every 28 days for a total of 12 cycles. Sixteen subjects were enrolled of whom 14 completed at least 3 cycles and were evaluable for response. Grade 3/4 toxicities were experienced by 88% (n=14), the most common being myelosuppression (n=7). Dose reductions occurred in 85% (n=12 of 14) of subjects. Hematologic partial and complete responses were achieved by 43% (n=6 of 14) and 7% (n=1 of 14), respectively. The median overall survival has not been reached and median progression-free survival is 24 months. In conclusion, this combination is associated with significant myelosuppression leading to dose modifications and producing minor hematologic responses in AL amyloidosis. http://clinicaltrials.gov/ct2/show/NCT00679367.

  14. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    Science.gov (United States)

    Passerini, Gabriella; Sangalli, Francesca; Moiola, Lucia; Colombo, Bruno; Locatelli, Massimo; Comi, Giancarlo

    2016-01-01

    Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p < 0.001 for all comparisons). KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p < 0.001 and p < 0.001, resp.) and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients. PMID:28116160

  15. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Gabriella Passerini

    2016-01-01

    Full Text Available Background. The presence of CSF oligoclonal bands (OBs is an independent prognostic factor for multiple sclerosis (MS, but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS, other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p<0.001 for all comparisons. KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p<0.001 and p<0.001, resp. and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index. Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients.

  16. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    Science.gov (United States)

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

  17. Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells.

    Science.gov (United States)

    Wang, Xiaobo; Olson, Jenessa R; Rasoloson, Dominique; Ellenbecker, Mary; Bailey, Jessica; Voronina, Ekaterina

    2016-12-15

    PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression.

  18. Diagnostic reference range of κ/λ free light chain ratio to screen for Bence Jones proteinuria is not significantly influenced by GFR.

    Science.gov (United States)

    Schmidt-Hieltjes, Yvonne; Elshof, Clemens; Roovers, Lian; Ruinemans-Koerts, Janneke

    2016-05-01

    The aim of our study was to analyse whether the κ/λ free light chain ratio reference range for screening for Bence Jones proteinuria should be dependent on the estimated glomerular filtration rate (eGFR). The serum κ/λ free light chain ratio, eGFR, serum M-protein and Bence Jones protein were measured in 544 patients for whom Bence Jones protein analysis was ordered. In the population of patients without Bence Jones proteinuria or a M-protein (n = 402), there is no gradual increase in κ/λ free light chain ratio with diminishing eGFR. The κ/λ free light chain ratio in this group was 0.56-1.86 (95% interval). With this diagnostic reference range of the κ/λ ratio, 105 of the 110 patients with Bence Jones protein could be identified correctly. Only five patients with Bence Jones proteinuria (proteinuria. A κ/λ free light chain ratio in serum can be used safely and efficiently to select urine samples which should be analysed for Bence Jones proteinuria with an electrophoresis/immunofixation technique. Using this diagnostic reference range, the number of urine samples which should be analysed by electrophoresis/immunofixation could be reduced by 74%. The diagnostic reference interval can be determined best in a group of patients for whom Bence Jones analysis is indicated. For calculation of this reference range, the eGFR value does not need to be taken into account. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Detection of the value of consecutive serum total light chain (sTLC) in patients diagnosed with diffuse large B cell lymphoma.

    Science.gov (United States)

    Zhai, Linzhu; Zhao, Yuanyuan; Peng, Songguo; Zhu, Ke; Yu, Rongjian; Chen, Hailong; Lin, Tongyu; Lin, Lizhu

    2016-12-01

    There are limited data on serum total light chain (sTLC) in lymphoma and its relative role on the outcome of diffuse large B cell lymphoma (DLBCL) patients. Blood samples from 46 cases newly diagnosed with DLBCL were collected consecutively during chemotherapy to detect sTLC, IgG, IgA, and IgM levels. Clinical data and survival outcomes were analyzed according to the results of sTLC measurements. In summary, 22 patients (47.8 %) had abnormal k or λ light chain, respectively, and 6 patients (13.0 %) had both abnormal k and λ light chains before chemotherapy. Patients with elevated k light chain more frequently displayed multiple extra-nodal organ involvement (P = 0.01) and had an inferior overall survival (OS) (P = 0.041) and progression-free survival (PFS) (P = 0.044) compared to patients with normal level of k light chain. Furthermore, patients with elevated level of both k and λ also exhibited significant association with shorter OS (P = 0.002) and PFS (P = 0.009). Both elevated k alone and concurrent elevated k and λ had independent adverse effects on PFS (P = 0.031 and P = 0.019, respectively). sTLC level was reduced gradually by treatment in this study and reached the lowest point after the fourth cycle of chemotherapy, which was consistent with the disease behavior during chemotherapy. Considering the small sample size of this study, these results should be confirmed in a larger prospective study.

  20. Long-term reversibility of renal dysfunction associated to light chain deposition disease with bortezomib and dexamethasone and high dose therapy and autologous stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Tomás J. González-López

    2011-11-01

    Full Text Available A 63-year-old woman presented with progressive renal insufficiency, until a glomerular filtration rate (GFR of 12 mL/min. A renal biopsy demonstrated glomerular deposition of immunoglobulin k light chain. The presence of a small population of monoclonal plasmacytes producing an only light k monoclonal component was demonstrated and Bortezomib and Dexamethasone (BD was provided as initial therapy. After seven courses of therapy, renal function improved without dialysis requirements up to a GFR 31 mL/min. Under hematological complete response (HCR the patient underwent high dose of melphalan (HDM and autologous peripheral blood stem cell transplant. Fifty-four months later the patient remains in HCR and the GFR has progressively improved up to 48 mL/min. This report describes a notably renal function improvement in a patient with Light Chain Deposition Disease after therapy with BD followed by HDM, which can support this treatment as a future option for these patients.

  1. Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR for diagnosis of natural infection with canine distemper virus

    Directory of Open Access Journals (Sweden)

    Wong Min-Liang

    2010-06-01

    Full Text Available Abstract Background Canine distemper virus (CDV is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR of the matrix (M gene to the fusion (F gene (designated M-F UTR in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Results Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. Conclusions The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

  2. Single base mutation in the pro. alpha. 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro. alpha. 2(I) chain

    Energy Technology Data Exchange (ETDEWEB)

    Tromp, G.; Prockop, D.J. (Thomas Jefferson Univ., Philadelphia, PA (USA))

    1988-07-01

    Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro{alpha}2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro{alpha}2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro{alpha}2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro{alpha}2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro{alpha}2(I) gene except that four subclones had a single base mutation at the 3{prime} end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3{prime} splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro{alpha}2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro{alpha}2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.

  3. Lighting

    Data.gov (United States)

    Federal Laboratory Consortium — Lighting Systems Test Facilities aid research that improves the energy efficiency of lighting systems. • Gonio-Photometer: Measures illuminance from each portion of...

  4. Dual role of G-runs and hnRNP F in the regulation of a mutation-activated pseudoexon in the fibrinogen gamma-chain transcript.

    Directory of Open Access Journals (Sweden)

    Valeria Rimoldi

    Full Text Available Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T. This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.

  5. Dual role of G-runs and hnRNP F in the regulation of a mutation-activated pseudoexon in the fibrinogen gamma-chain transcript.

    Science.gov (United States)

    Rimoldi, Valeria; Soldà, Giulia; Asselta, Rosanna; Spena, Silvia; Stuani, Cristiana; Buratti, Emanuele; Duga, Stefano

    2013-01-01

    Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.

  6. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four vλ6 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Wall, J.S.; Gupta, V.; Wilkerson, M.; Schell, M.; Loris, R.; Adams, P.; Solomon, A.; Stevens, F.; Dealwis, C.

    2004-04-01

    Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (V{sub L}) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the V{sub L} domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable V{sub {lambda}}6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic V{sub {lambda}}6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of

  7. Light

    DEFF Research Database (Denmark)

    Prescott, N.B.; Kristensen, Helle Halkjær; Wathes, C.M.

    2004-01-01

    This chapter presents the effect of artificial light environments (light levels, colour, photoperiod and flicker) on the welfare of broilers in terms of vision, behaviour, lameness and mortality......This chapter presents the effect of artificial light environments (light levels, colour, photoperiod and flicker) on the welfare of broilers in terms of vision, behaviour, lameness and mortality...

  8. Optical anisotropy of polyimide and polymethacrylate containing photocrosslinkable chalcone group in the side chain under irradiation of a linearly polarized UV light

    CERN Document Server

    Choi, D H

    2002-01-01

    Photocrosslinkable soluble polyimide and polymethacrylate compound were synthesized for studying the optically induced anisotropy of the thin films. Chalcone group was introduced into the side chain unit of two polymers. We observed a photodimerization behavior between the double bonds in the chalcone group and an optical anisotropy of these materials by irradiation of a linearly polarized UV light (LPL). Optical anisotropy of the thin film was also investigated by using polarized UV absorption spectroscopy.The dynamic property of optical anisotropy in photoreactive polyimide was compared to that in polymethacrylate containing chalcone group in the side chain.

  9. Serum Free Light Chain Assay and κ/λ Ratio: Performance in Patients With Monoclonal Gammopathy-High False Negative Rate for κ/λ Ratio

    Science.gov (United States)

    Singh, Gurmukh

    2017-01-01

    Background Serum free light chain assay (SFLCA) and κ/λ ratio, and protein electrophoretic methods are used in the diagnosis and monitoring of monoclonal gammopathies. Methods Results for serum free light chains, serum and urine protein electrophoreses and immunofixation electrophoreses in 468 patients with a diagnosis of monoclonal gammopathy were compared. The results of the two methods were graded as concordant, non-concordant or discordant with the established diagnoses to assess the relative performance of the methods. Results of κ/λ ratio in samples with monoclonal protein detectable by electrophoretic methods were also analyzed. Results Protein electrophoreses results were concordant with the established diagnoses significantly more often than κ/λ ratio. The false negative rate for κ/λ ratio was higher than that for electrophoretic methods. κ/λ ratio was falsely negative in about 27% of the 1,860 samples with detectable monoclonal immunoglobulin. The false negative rate was higher in lesions with lambda chains (32%) than those with kappa chains (24%). The false negative rate for κ/λ ratio was over 55% in samples with monoclonal gammopathy of undetermined significance. Even at first encounter, the false negative rates for κ/λ ratios for monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma were 66.98%, 23.08%, and 30.15%, respectively, with false negative rate for lambda chain lesions being higher. Conclusions Electrophoretic studies of serum and urine are superior to SFLCA and κ/λ ratio. Abnormal κ/λ ratio, per se, is not diagnostic of monoclonal gammopathy. A normal κ/λ ratio does not exclude monoclonal gammopathy. False negative rates for lesions with lambda chain are higher than those for lesions with kappa chains. Electrophoretic studies of urine are underutilized. Clinical usefulness and medical necessity of SFLCA and κ/λ ratio is of questionable value in routine clinical testing. PMID:27924175

  10. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Directory of Open Access Journals (Sweden)

    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  11. Acute heart failure with cardiomyocyte atrophy induced in adult mice by ablation of cardiac myosin light chain kinase.

    Science.gov (United States)

    Massengill, Michael T; Ashraf, Hassan M; Chowdhury, Rajib R; Chrzanowski, Stephen M; Kar, Jeena; Warren, Sonisha A; Walter, Glenn A; Zeng, Huadong; Kang, Byung-Ho; Anderson, Robert H; Moss, Richard L; Kasahara, Hideko

    2016-07-01

    Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. To mimic acute cMLCK reduction in adult hearts, the floxed-Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3(flox/flox)/merCremer mice (Mylk3-KO), and compared with two control mice (Mylk3(flox/flox) and Mylk3(+/+)/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  12. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    Science.gov (United States)

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  13. Kinesin light chain 1 suppression impairs human embryonic stem cell neural differentiation and amyloid precursor protein metabolism.

    Directory of Open Access Journals (Sweden)

    Rhiannon L Killian

    Full Text Available The etiology of sporadic Alzheimer disease (AD is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC expressing reduced levels of the kinesin light chain 1 (KLC1 Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs, isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases.

  14. Kinetics of myosin light chain kinase activation of smooth muscle myosin in an in vitro model system.

    Science.gov (United States)

    Hong, Feng; Facemyer, Kevin C; Carter, Michael S; Jackson, Del R; Haldeman, Brian D; Ruana, Nick; Sutherland, Cindy; Walsh, Michael P; Cremo, Christine R; Baker, Josh E

    2013-11-26

    During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

  15. Inhibition of Myosin light-chain kinase attenuates cerebral edema after traumatic brain injury in postnatal mice.

    Science.gov (United States)

    Rossi, Janet L; Todd, Tracey; Bazan, Nicolas G; Belayev, Ludmila

    2013-10-01

    Traumatic brain injury (TBI) in children less than 8 years of age leads to decline in intelligence and executive functioning. Neurological outcomes after TBI correlate to development of cerebral edema, which affect survival rates after TBI. It has been shown that myosin light-chain kinase (MLCK) increases cerebral edema and that pretreatment with an MLCK inhibitor (ML-7) reduces cerebral edema. The aim of this study was to determine whether inhibition of MLCK after TBI in postnatal day 24 (PND-24) mice would prevent breakdown of the blood-brain barrier (BBB) and development of cerebral edema and improve neurological outcome. We used a closed head injury model of TBI. ML-7 or saline treatment was administered at 4 h and every 24 h until sacrifice or 5 days after TBI. Mice were sacrificed at 24 h, 48 h, and 72 h and 7 days after impact. Mice treated with ML-7 after TBI had decreased levels of MLCK-expressing cells (20.7±4.8 vs. 149.3±40.6), less albumin extravasation (28.3±11.2 vs. 116.2±60.7 mm(2)) into surrounding parenchymal tissue, less Evans Blue extravasation (339±314 vs. 4017±560 ng/g), and showed a significant difference in wet/dry weight ratio (1.9±0.07 vs. 2.2±0.05 g), compared to saline-treated groups. Treatment with ML-7 also resulted in preserved neurological function measured by the wire hang test (57 vs. 21 sec) and two-object novel recognition test (old vs. new, 10.5 touches). We concluded that inhibition of MLCK reduces cerebral edema and preserves neurological function in PND-24 mice.

  16. Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

    Science.gov (United States)

    Prochniewicz, Ewa; Guhathakurta, Piyali; Thomas, David D.

    2013-01-01

    We have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, in order to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final anisotropy cooperatively and to a similar extent, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of saturating ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin. PMID:23339370

  17. Urinary free light chains may help to identify infection in patients with elevated systemic inflammation due to rheumatic disease.

    Science.gov (United States)

    Bramlage, Carsten P; Froelich, Britta; Wallbach, Manuel; Minguet, Joan; Grupp, Clemens; Deutsch, Cornelia; Bramlage, Peter; Müller, Gerhard A; Koziolek, Michael

    2017-04-01

    The risk of infection in patients with rheumatic diseases is elevated, but a clear marker to differentiate the cause of the systemic inflammation is missing. We assessed the ability urinary immunoglobulin free light chains (FLCs) to indicate the presence of infection in patients with rheumatic disease. We performed a retrospective analysis of patients with rheumatic disease attending the Georg-August University Hospital in Goettingen, Germany, from January 2011 to December 2013. Subjects were included if they had urine levels of κ and λ FLCs available. A reference group of patients without autoimmune disease, but with documented infection, was constructed. A total of 1500 patients had their urinary FLCs quantified during the study period. Of the 382 patients with rheumatic disease, 172 (45%) displayed no systemic inflammation, 162 (42%) had inflammation due to the underlying disease activity, and 48 (13%) had inflammation due to a confirmed infection. Urinary FLC concentrations were much higher in patients with rheumatic diseases and infection (κ 68.8 ± 81.8 mg/L, λ 31.4 ± 53.5 mg/L) compared to those with inflammation due to rheumatic disease activity (κ 22.7 ± 26.3 mg/L, λ 8.1 ± 9.1 mg/L, κ p inflammation due to rheumatic disease activity from that due to the additional presence of infection. The ability to quantify these proteins in urine provides a simple alternative to the use of blood.

  18. Receptor-associated protein blocks internalization and cytotoxicity of myeloma light chain in cultured human proximal tubular cells.

    Directory of Open Access Journals (Sweden)

    Sule Sengul

    Full Text Available BACKGROUND: Free light chains (LCs are among the many ligands that bind to cubilin/megalin for endocytosis via the clathrin-dependent endosomal/lysosomal pathway. Receptor associated protein (RAP, is a 39 kDA high-affinity, chaperone-like ligand for megalin that assists in the proper folding and functioning of megalin/cubilin. Although RAP is known to inhibit ligand binding to megalin/cubilin, its effect on LC endocytosis has not been shown directly. METHODS AND PRINCIPAL FINDINGS: We investigated whether RAP can block the endocytosis of LC in cultured human proximal tubule cells and whether this can prevent LC cytotoxicity. Immunofluorescence microscopy and flow cytometry showed that fluorescently labeled LC endocytosis was markedly inhibited in HK-2 cells pretreated with human RAP. The effect of RAP was dose-dependent, and was predominantly on endocytosis as it had no effect on the small acid-washable fraction of LC bound to cell membrane. RAP significantly inhibited LC induced cytokine production and phosphorylation of ERK1/2 and p38 MAPK. Prolonged exposure to LC for 48 h resulted in epithelial-to-mesenchymal transformation in HK-2 cells as evidenced by marked reduction in the expression of the epithelial cell marker E-cadherin, and increased the expression of the mesenchymal marker α-SMA, which was also prevented by RAP in the endocytosis medium. CONCLUSIONS: RAP inhibited LC endocytosis by ∼88% and ameliorated LC-induced cytokine responses and EMT in human PTCs. The results not only provide additional evidence that LCs endocytosis occurs via the megalin/cubilin endocytic receptor system, but also show that blocking LC endocytosis by RAP can protect proximal tubule cells from LC cytotoxicity.

  19. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    Science.gov (United States)

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene.

  20. The Association between Polyclonal Combined Serum Free Light Chain Concentration and Mortality in Individuals with Early Chronic Kidney Disease.

    Directory of Open Access Journals (Sweden)

    Lakhvir K Assi

    Full Text Available A major component of increased mortality risk in people with chronic kidney disease (CKD is associated with non-traditional cardiovascular risk factors including markers of inflammation. We studied whether a novel marker of systemic inflammation, elevated serum combined polyclonal immunoglobulin free light chains (cFLC, was an independent risk factor for increased all-cause mortality in people with CKD stage 3. In a prospective community based cohort study, 1695 participants with stage 3 CKD and no cases of monoclonal gammopathy had cFLC concentrations measured. cFLC levels were determined using the summation of Freelite kappa and lambda assays. All other bioclinical variables were collected at the time of sample collection. Kaplan-Meier plots and Cox proportional hazards analysis was used to assess the relationship between high cFLC levels (>43.3 mg/L and mortality. There were 167 deaths (10% after a median of 1375 days. cFLC levels at recruitment were higher in participants who died compared with those who were alive at the end of the study; median: 46.5 mg/L (IQR: 36.1-65.4 mg/L and 35.4 mg/L (28.1-46.6 mg/L respectively, P 43.3 mg/L levels had an increased risk of mortality compared to people with normal cFLC levels (P <0.001. Elevated cFLC levels were independently associated with worse survival (Hazard ratio: 1.50; 95% confidence interval: 1.04-2.16; P=0.03. Other independent risk factors for worse survival were: older age, male gender, previous cardiovascular event, lower eGFR and higher high sensitivity C-reactive protein (hsCRP. To conclude, high cFLC levels predict increased mortality in people with stage 3 CKD, independent of established risk factors and other markers of inflammation.

  1. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

    Science.gov (United States)

    Ashokkumar, Balasubramaniem; Nabokina, Svetlana M; Ma, Thomas Y; Said, Hamid M

    2009-09-01

    The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

  2. Prognostic value of left atrial function in systemic light-chain amyloidosis: a cardiac magnetic resonance study.

    Science.gov (United States)

    Mohty, Dania; Boulogne, Cyrille; Magne, Julien; Varroud-Vial, Nicolas; Martin, Sylvain; Ettaif, Hind; Fadel, Bahaa M; Bridoux, Frank; Aboyans, Victor; Damy, Thibaud; Jaccard, Arnaud

    2016-09-01

    Cardiac involvement in systemic light-chain amyloidosis (AL) imparts an adverse impact on outcome. The left atrium (LA), by virtue of its anatomical location and muscular wall, is commonly affected by the amyloid process. Although LA infiltration by amyloid fibrils leads to a reduction in its pump function, the infiltration of the left ventricular (LV) myocardium results in diastolic dysfunction with subsequent increase in filling pressures and LA enlargement. Even though left atrial volume (LAV) is an independent prognostic marker in many cardiomyopathies, its value in amyloid heart disease remains to be determined. In addition, few data are available as to the prognostic value of LA function in systemic AL. Using cardiac magnetic resonance (CMR), the current study aims to assess the prognostic significance of the maximal LAV and total LA emptying fraction (LAEF) in patients with AL. Fifty-four consecutive patients (age 66 ± 10 years, 59% males) with confirmed systemic AL and mean LV ejection fraction of 60 ± 12% underwent CMR. As compared with patients with no or minimal cardiac involvement (Mayo Clinic [MC] stage I), those at moderate and high risk (MC stages II and III) had significantly larger indexed maximal LAV (36 ± 15 vs. 46 ± 13 vs. 52 ± 19 mL/m(2), P = 0.03) and indexed minimal LAV (20 ± 6 vs. 34 ± 11 vs. 44 ± 17 mL/m(2), P 16% (37 ± 11 vs. 94 ± 4%, P = 0.001). In multivariate analysis, lower LAEF remained independently associated with a higher risk of 2-year mortality (HR = 1.08 per 1% decrease, 95% CI: 1.02-1.15, P = 0.003). In patients with systemic AL, LAEF as assessed by CMR is associated with NYHA functional class, MC stage, myocardial LGE and 2-year mortality. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  3. The value of serum immunoglobulin free light chain assessment in patients with monoclonal gammopathies and acute renal failure.

    Science.gov (United States)

    Cirit, Mustafa; Uzüm, Atilla; Ozen, Pınar; Sentürk, Banu A; Bozkaya, Giray; Payzin, Bahriye; Ural, Orçun

    2012-12-01

    Immunoglobulin free light chain (FLC) abnormalities are common in patients with monoclonal gammopathies and the kidneys are the most affected organs. Immunoassays that provide quantitative measurement of FLC in serum indicate monoclonal FLC production based on the presence of an abnormal FLC kappa:lambda (κ:λ) ratio. The aim of this study was to assess the utility of serum FLC measurement as a diagnostic tool for detecting plasma cell dyscrasias in comparison to standard assays, and to ascertain its sensitivity and specificity in patients with acute renal failure (ARF). Sera from 82 patients with ARF were assessed using serum protein electrophoresis (SPE), serum immunofixation electrophoresis (SIFE), and FLC measurement. The sensitivity and specificity of the FLC ratio in identifying which ARF patients had multiple myeloma (MM) was compared to those of SPE and SIFE. Among the 82 patients with ARF, 7 were diagnosed as MM using SPE, SIFE, and bone marrow biopsy techniques. In total, 8 patients did not have a FLC κ:λ ratio that was within the published reference range (0:26-1:65); the FLC κ:λ ratio based on FLC measurement had a specificity of 96% and sensitivity of 71%, and positive and negative predictive values of 62.9% and 97.3%, respectively, for the diagnosis of MM. The sensitivity and specificity of the FLC κ:λ ratio for diagnosing MM in patients that presented with ARF were lower than those of SPE and SIFE. To further delineate the utility of the FLC κ:λ ratio additional prospective, well-designed large-scale studies are needed. None declared.

  4. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    Science.gov (United States)

    Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Chomwong, Sudarat; Senapin, Saengchan; Tassanakajon, Anchalee; Amparyup, Piti

    2016-01-01

    Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

  5. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

    Science.gov (United States)

    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  6. A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Jensen, T G

    1993-01-01

    157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells...

  7. Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains.

    Science.gov (United States)

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Kim, Sang-Jin; Urm, Sang-Hwa; Seog, Dae-Hyun

    2014-01-01

    Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

  8. A First Report of Endoscopic Ultrasound for the Diagnosis of Pancreatic Amyloid Deposition in Immunoglobulin Light Chain (AL Amyloidosis (Primary Amyloidosis

    Directory of Open Access Journals (Sweden)

    Somashekar G Krishna

    2013-05-01

    Full Text Available Context Pancreatic involvement in systemic light chain (AL-amyloidosis is exceedingly rare. Prior reports of endoscopicultrasound (EUS for the diagnosis of amyloidosis are also limited. Case report We report the first description of EUS-guided fine needle aspiration (FNA for the diagnosis of primary AL-amyloidosis involving the pancreas. Conclusion EUS-FNA can be effectively utilized for the characterization and cytologic diagnosis of pancreatic amyloidosis and potentially other accessible extraluminal amyloid deposits.

  9. De novo DNA Methyltransferases Dnmt3a and Dnmt3b regulate the onset of Igκ light chain rearrangement during early B-cell development.

    Science.gov (United States)

    Manoharan, Anand; Du Roure, Camille; Rolink, Antonius G; Matthias, Patrick

    2015-08-01

    Immunoglobulin genes V(D)J rearrangement during early lymphopoiesis is a critical process involving sequential recombination of the heavy and light chain loci. A number of transcription factors act together with temporally activated recombinases and chromatin accessibility changes to regulate this complex process. Here, we deleted the de novo DNA methyltransferases Dnmt3a and Dnmt3b in early B cells of conditionally targeted mice, and monitored the process of V(D)J recombination. Dnmt3a and Dnmt3b deletion resulted in precocious recombination of the immunoglobulin κ light chain without impairing the differentiation of mature B cells or overall B-cell development. Ex vivo culture of IL-7 restricted early B-cell progenitors lacking Dnmt3a and Dnmt3b showed precocious Vκ-Jκ rearrangements that are limited to the proximal Vκ genes. Furthermore, B-cell progenitors deficient in Dnmt3a and Dnmt3b showed elevated levels of germline transcripts at the proximal Vκ genes, alterations in methylation patterns at Igκ enhancer sites and increased expression of the transcription factor E2A. Our data suggest that Dnmt3a and Dnmt3b are critical to regulate the onset of Igκ light chain rearrangement during early B-cell development.

  10. Substitution of arginine for glycine at position 154 of the {alpha}1 chain of type I collagen in a variant of osteogenesis imperfecta: Comparison to previous cases with the same mutation

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, J.; Tromp, G.; Kuivaniemi, H.; Prockop, D.J. [Thomas Jefferson Univ., Philadelphia, PA (United States); Castells, S. [Univ. Hospital of Brooklyn, NY (United States)

    1996-01-11

    A substitution of arginine for glycine at amino acid position 154 of the {alpha}1(I) collagen chain was found in a father and his three children. The phenotype of the patients includes manifestations of types I and III/IV osteogenesis imperfecta, but appears to be milder than that of the previously described two unrelated patients that had the identical mutation in the {alpha}1(I) collagen chain. The variability in the phenotype raises the possibility of epistatic loci or environmental effects on expression of the disorder. 35 refs., 3 figs., 2 tabs.

  11. Somatic mutations in the Ig variable region genes and expression of novel Cmu-germline transcripts in a B-lymphoma cell line ("Farage") not producing Ig polypeptide chains.

    Science.gov (United States)

    Hochberg, M; Gabay, C; Laskov, R

    1998-08-01

    Non-Hodgkin's B-lymphomas (B-NHL) are a very heterogeneous group of B-cell neoplasias originating from the germinal centers of lymphatic follicles. Thus, they represent a suitable experimental model to study the molecular basis of certain key events which take place in the lymphatic follicles, including somatic hypermutation and heavy chain isotypic switch. An unusual B-NHL cell line ("Farage") not producing Ig polypeptide chains was previously shown to rearrange its IgH and Igkappa genes and transcribe seemingly normal size mu and kappa mRNAs. In an attempt to characterize the phenotype of Farage cells better and to elucidate the molecular basis of the failure of Farage cells to synthesize Ig chains, we sequenced its VH and Vkappa rearranged gene segments by PCR and RT-PCR. It was found that both V genes are somatically, heavily mutated compared to their germline counterparts. In addition, this rearranged VDJ gene of the heavy chain is not transcribed. Instead, the Farage cells express a low level of a new family of germline transcripts starting with a VH like sequence, continuing with a small segment of the 3'VH germline flanking region, and ending within the Cmu region. These transcripts lack D and J segments and do not contain the open reading frame of the full-length Cmu protein. Thus, Farage cells fail to produce mu heavy chains due to silencing of the expression of the conventional VDJCmu transcript and expression of unusual Cmu-germline transcripts. In contrast to the IgH genes, the rearranged VJ gene of Farage is transcribed and gives rise to a full-size kappa-mRNA. This transcript, however, is not translated to a full-length kappa-chain, as it contains a stop codon in its coding region. All the above show that Farage cells are unable to produce Ig polypeptide chains, due to somatic mutations altering the kappa-chain gene, and mutations and/or regulatory events that shutoff the transcription of the IgH gene. The heavily mutated Vkappa and Vkappa genes

  12. Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion.

    Science.gov (United States)

    Gao, Ling; Zheng, Yan-Jun; Gu, Shan-Shan; Tan, Ji-Liang; Paul, Christian; Wang, Yi-Gang; Yang, Huang-Tian

    2014-12-01

    Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca(2+) sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin-myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca(2+) transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca(2+). These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca(2+)-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca(2+), while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca(2+)-stimulated ATPase activity, which subsequently suppresses

  13. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    Science.gov (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  14. Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

    Science.gov (United States)

    Olsen, Rikke K J; Koňaříková, Eliška; Giancaspero, Teresa A; Mosegaard, Signe; Boczonadi, Veronika; Mataković, Lavinija; Veauville-Merllié, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F; Konstantopoulou, Vassiliki; Möslinger, Dorothea; Feichtinger, René G; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazzà, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M; Gregersen, Niels; Mayr, Johannes A; Horvath, Rita; Barile, Maria; Prokisch, Holger

    2016-06-02

    Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

  15. Molecular identification of the light brown apple moth (Lepidoptera: Tortricidae) in California using a polymerase chain reaction assay of the internal transcribed spacer 2 locus.

    Science.gov (United States)

    Barr, N B; Ledezma, L A; Vasquez, J D; Epstein, M; Kerr, P H; Kinnee, S; Sage, O; Gilligan, T M

    2009-12-01

    A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

  16. A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects

    Science.gov (United States)

    Augière, Céline; Mégy, Simon; El Malti, Rajae; Boland, Anne; El Zein, Loubna; Verrier, Bernard; Mégarbané, André; Deleuze, Jean-François; Bouvagnet, Patrice

    2015-01-01

    Background A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5. Methods and Results A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface. Conclusions Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin. PMID:26061005

  17. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

    Science.gov (United States)

    Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

    2016-06-23

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

  18. The 4C5 cell-impermeable anti-HSP90 antibody with anti-cancer activity, is composed of a single light chain dimer.

    Science.gov (United States)

    Sidera, Katerina; El Hamidieh, Avraam; Mamalaki, Avgi; Patsavoudi, Evangelia

    2011-01-01

    MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics.

  19. Light

    CERN Document Server

    Robertson, William C

    2003-01-01

    Why is left right and right left in the mirror? Baffled by the basics of reflection and refraction? Wondering just how the eye works? If you have trouble teaching concepts about light that you don t fully grasp yourself, get help from a book that s both scientifically accurate and entertaining with Light. By combining clear explanations, clever drawings, and activities that use easy-to-find materials, this book covers what science teachers and parents need to know to teach about light with confidence. It uses ray, wave, and particle models of light to explain the basics of reflection and refraction, optical instruments, polarization of light, and interference and diffraction. There s also an entire chapter on how the eye works. Each chapter ends with a Summary and Applications section that reinforces concepts with everyday examples. Whether you need a deeper understanding of how light bends or a good explanation of why the sky is blue, you ll find Light more illuminating and accessible than a college textbook...

  20. Light-Induced ESR Studies of Quadrimolecular Recombination Kinetics of Photogenerated Charge Carriers in Regioregular Poly(3-alkylthiophene)/C60 Composites: Alkyl Chain Dependence

    Science.gov (United States)

    Tanaka, Hisaaki; Hasegawa, Naoki; Sakamoto, Tomotaka; Marumoto, Kazuhiro; Kuroda, Shin-ichi

    2007-08-01

    Light-induced ESR (LESR) measurements have been performed on the composites of regioregular poly(3-alkylthiophene) (RR-P3AT) and C60 by using polymers having different alkyl chains (CmH2m+1 with m=6, 8, 10, 12). The quadrimolecular recombination (QR) kinetics of photogenerated charge carriers, previously reported, have been confirmed for all the composites from the excitation power (Iex) dependence of the LESR intensity showing an ˜Iex0.25 dependence. The time decay of LESR intensity is also consistent with the QR model. Considering that only bimolecular recombination is observed in regiorandom polymer composites, the occurrence of QR strongly suggests the formation of doubly charged states, either bipolarons or polaron pairs on the regioregular polymer chains. On the other hand, the QR rate constant γ has been found to exhibit weak alkyl chain dependence, contrary to the case of the field-effect mobility of pure regioregular polymers with systematic alkyl chain dependence. This implies the significant contribution of the polymer and fullerene interface in determining γ.

  1. Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

    Science.gov (United States)

    Reddy, P; Sparvoli, A; Fagioli, C; Fassina, G; Sitia, R

    1996-01-01

    Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi. Images PMID:8641273

  2. A Markov Chain-based quantitative study of angular distribution of photons through turbid slabs via isotropic light scattering

    Science.gov (United States)

    Li, Xuesong; Northrop, William F.

    2016-04-01

    This paper describes a quantitative approach to approximate multiple scattering through an isotropic turbid slab based on Markov Chain theorem. There is an increasing need to utilize multiple scattering for optical diagnostic purposes; however, existing methods are either inaccurate or computationally expensive. Here, we develop a novel Markov Chain approximation approach to solve multiple scattering angular distribution (AD) that can accurately calculate AD while significantly reducing computational cost compared to Monte Carlo simulation. We expect this work to stimulate ongoing multiple scattering research and deterministic reconstruction algorithm development with AD measurements.

  3. Repair and actio spectrum of oxygen-independent lethality of near uv light on Haemophilus influenzae and lack of mutation

    Energy Technology Data Exchange (ETDEWEB)

    Cabrera-Juarez, E.; Setlow, J.K.

    1980-01-01

    Haemophilus influenzae has been inactivated anaerobically at 313, 334, 365, and 405 nm, and exhibits the greatest sensitivity at 334 nm. The rec1 and uvr1 mutants show the greatest increase in sensitivity over the wild-type at 313 nm, but differences could be seen also at the other wavelengths. Anaerobic irradiation is less effective for killing at all the wavelengths than irradiation under aerobic conditions, but the greatest difference was observed at 365 nm. No induced mutation was seen as a result of anaerobic irradiation at 334 nm, although purified transforming DNA can be mutated at this wavelength.

  4. On the role of long-chain aldehydes in the light reaction in Photobacterium phosphoreum enzyme preparations

    NARCIS (Netherlands)

    Terpstra, Willemke

    1960-01-01

    1. (1) Active luciferase-DPNH-oxidase preparations from Photobacterium phosphoreum generally contain some aldehyde-attacking enzyme, probably ADH. Under the experimental conditions applied this enzyme appears to attack decanal, but not palmital. 2. (2) The presence of long-chain aldehydes in the en

  5. Effect of the mutation of carotenoids on the dynamics of energy transfer in light- harvesting complexes (LH2) from Rhodobacter sphaeroides 601 at room temperature

    Institute of Scientific and Technical Information of China (English)

    Liu Wei-Min; Liu Yuan; Liu Rang-Jun; Yan Yong-Li; Guo Li-Jun; Xu Chun-He; Qian Shi-Xiong

    2006-01-01

    Energy transfers in two kinds of peripheral light-harvesting complexes (LH2) of Rhodobacter sphaeroides (RS) 601 are studied by using femtosecond pump-probe spectroscopy with tunable laser wavelength at room temperature. These two complexes are native LH2 (RS601) and green carotenoid mutated LH2 (GM309). The obtained results demonstrate that, compared with spheroidenes with ten conjugated double bonds in native RS601, carotenoid in GM309 containing neurosporenes with nine conjugated double bonds can lead to a reduction in energy transfer rate in the B800-to-B850 band and the disturbance in the energy relaxation processes within the excitonic B850 band.

  6. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    Science.gov (United States)

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  7. Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries.

    Science.gov (United States)

    Injeti, Elisha R; Sandoval, Renan J; Williams, James M; Smolensky, Alexander V; Ford, Lincoln E; Pearce, William J

    2008-12-01

    Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

  8. P53 GENE MUTATIONS IN NON-SMALL CELL LUNG CANCER DETECTED BY POLYMERASE CHAIN REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    赵永良; 吴德昌; 项晓琼; 张宝仁; 周乃康; 胡迎春

    1999-01-01

    Mutations of the p53 tumor suppressor gone are the most frequent genetic akerations detected in human lung cancer. To assess the pathogenic significance of p53 gone alterations in Chlnege non-small cell lung cancer (NSCLC), 74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5-8) using exon-specific PCR, single-gtrand conformation polymorphimax (PCR-SSCP). p53 mutations were observed in 55.4% (41/74) of the samples.No linkaiges were detected between the incidence of p53 mutations and histological type, lymph node metastasis, age or sex. Significant association between p53 mutations and degree of differentiation in edenotmremmnas, not in squamous cell carcinomas, was observed, The frequency of p53 mutations in(65. 3%) was higher than in nonsmokers (33. 3%) and reached stafisrical significance. We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis. These results suggest that smcking could be an important factor in lung carcinogenesis, p53 mutation is a worse prognosis indicator in ade and nocarcinomas and related to high aggressive behavior of human lung cancer.

  9. P53 GENE MUTATIONS IN NON-SMALL CELL LUNG CANCER DETECTED BY POLYMERASE CHAIN REACTION SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ Mutations of the p53 tumor suppressor gene are the most frequent genetic alterations detected in human lung cancer. To assess the pathogenic significance of p53 gene alterations in Chinese non-small cell lung cancer(NSCLC),74 paired samples of primary lung cancer and normal lung tissue far away from the cancer were analyzed for mutations of the p53 gene(exons 5-8) using exon-specific PCR, single-strand conformation polymorphism (PCR-SSCP). p53 mutations were observed in 55.4%(41/74) of the samples. No linkages were detected between the incidence of p53 mutations and histological type, lymph node metastasis,age or sex. Significant association between p53 mutations and degree of differentiation in adenocarcinomas, not in squamous cell carcinomas, was observed. The frequency of p53 mutations in smokers(65.3%) was higher than in nonsmokers(33.3%) and reached statistical significance.We also found p53 mutations in 6/7 samples which had tissue invasion and distant metastasis.These results suggest that smoking could be an important factor in lung carcinogenesis,p53 mutation is a worse prognosis indicator in adenocarcinomas and related to high aggressive behavior of human lung cancer.

  10. Two CpG mutational hot spots in the X-linked interleukin-2 receptor gamma chain gene (IL2RG) may cause 15% of all human severe combined immunodeficiency

    Energy Technology Data Exchange (ETDEWEB)

    Pepper, A.E.; Puck, J.M. [National Center for Human Genome Research, Bethseda, MD (United States); Liu, X. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)

    1994-09-01

    Severe combined immunodeficiency (SCID), a syndrome of profoundly imp