Role of residual kidney function and convective volume on change in beta2-microglobulin levels in hemodiafiltration patients

NARCIS (Netherlands)

Penne, E. Lars; van der Weerd, Neelke C.; Blankestijn, Peter J.; van den Dorpel, Marinus A.; Grooteman, Muriel P. C.; Nubé, Menso J.; ter Wee, Piet M.; Lévesque, Renée; Bots, Michiel L.

2010-01-01

Removal of beta2-microglobulin (beta2M) can be increased by adding convective transport to hemodialysis (HD). The aim of this study was to investigate the change in beta2M levels after 6-mo treatment with hemodiafiltration (HDF) and to evaluate the role of residual kidney function (RKF) and the

Levels of Beta-2 Microglobulin and Cystatin C in Beta Thalassemia Major Patients

Directory of Open Access Journals (Sweden)

Ayse Kacar

2014-03-01

Full Text Available Aim: Thalassemia is accepted to be the most common genetic disease in the world. This study was performed to establish whether there was a glomerular renal damage, which was usually a less mentioned subject in patients with Beta Thalassemia Major, and to compare urea, creatinine and creatinine clearance with early indicators of kidney damage as Cystatin-C and %u03B2-2 microglobulin as on determining the glomerular damage. Material and Method: This study was prospectively performed in patients, who were regularly followed in the children hematology outpatient clinic with a diagnosis of Beta Thalassemia Major. Results: There was no statistically significant difference between urea and levels of creatinine clearance and Cystatin-C. There was a statistically negative relationship between creatinine and creatinine clearance at an advanced level as 53.7% (p: 0.002, p

Kinetically controlled thermal response of beta2-microglobulin amyloid fibrils.

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Sasahara, Kenji; Naiki, Hironobu; Goto, Yuji

2005-09-23

Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 degrees C for characterizing the thermal response of beta2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity-temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid beta peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern.

Urinary albumin and beta 2-microglobulin excretion rates in patients with systemic lupus erythematosus

DEFF Research Database (Denmark)

Parving, H H; Sørensen, S F; Mogensen, C E

1980-01-01

The daily urinary albumin and beta 2-microglobulin excretion rates were measured with sensitive radioimmunoassays in 14 patients with systemic lupus erythematosus (SLE). The duration of SLE ranged from 0.5 to 18 years, mean 10 years. The mean age was 37 years. All patients except 5 received...

Fractional excretion of beta-2-microglobulin in the urine of patients with normal or reduced renal function and hepatic coma

DEFF Research Database (Denmark)

Hansen, P B; Dalhoff, K; Joffe, P

1991-01-01

The purpose of this prospective study was to evaluate beta-2-microglobulin (beta 2m) as a differential diagnostic indicator between hepatic nephropathy (HN) and acute tubulointerstitial nephropathy (ATIN) in patients with reduced renal function and hepatic coma, and to determine whether beta 2m e...

Formation of a stable oligomer of beta-2 microglobulin requires only transient encounter with Cu(II).

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Calabrese, Matthew F; Miranker, Andrew D

2007-03-16

Beta-2 Microglobulin (beta2m) is a small, globular protein, with high solubility under conditions comparable to human serum. A complication of hemodialysis in renal failure patients is the deposition of unmodified beta2m as amyloid fibers. In vitro, exposure of beta2m to equimolar Cu(2+) under near-physiological conditions can result in self-association leading to amyloid fiber formation. Previously, we have shown that the early steps in this process involve a catalyzed structural rearrangement followed by formation of discrete oligomers. These oligomers, however, have a continued requirement for Cu(2+) while mature fibers are resistant to addition of metal chelate. Here, we report that the transition from Cu(2+) dependent to chelate resistant states occurs in the context of small oligomers, dimeric to hexameric in size. These species require Cu(2+) to form, but once generated, do not need metal cation for stability. Importantly, this transition occurs gradually over several days and the resulting oligomers are isolatable and kinetically stable on timescales exceeding weeks. In addition, formation is enhanced by levels of urea similar to those found in hemodialysis patients. Our results are consistent with our hypothesis that transient encounter of full-length wild-type beta2m with transition metal cation at the dialysis membrane interface is causal to dialysis related amyloidosis.

Serum beta-2 microglobulin levels for predicting acute kidney injury complicating aortic valve replacement.

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Zaleska-Kociecka, Marta; Skrobisz, Anna; Wojtkowska, Izabela; Grabowski, Maciej; Dabrowski, Maciej; Kusmierski, Krzysztof; Piotrowska, Katarzyna; Imiela, Jacek; Stepinska, Janina

2017-10-01

Acute kidney injury complicating both transcatheter and surgical aortic valve replacement is associated with high rates of morbidity and mortality. The aim of this study was to investigate the role of serum beta 2 (β2) microglobulin, cystatin C and neutrophil gelatinase-associated lipocalin levels in detecting periprocedural acute kidney injury. Eighty consecutive patients who were 70 years of age or older and who were having surgical (n = 40) or transcatheter (n = 40) aortic valve replacement were recruited in a prospective study. The biomarkers were tested before the procedure, 6 times afterwards, at discharge and at a 6-month follow-up visit. The baseline β2-microglobulin level was the strongest predictor of acute kidney injury as a complication of transcatheter aortic valve replacement [odds ratio (OR) 5.277, P = 0.009]. Its level 24 h after the procedure reached the largest area under the curve (AUC) of 0.880 (P regression analysis, the levels of β2-microglobulin and cystatin C 24 h after the procedure were significantly associated with acute kidney injury after transcatheter valve replacement (OR 38.15, P = 0.044; OR 1782, P = 0.019, respectively). In the surgical aortic valve replacement group, the highest AUCs belonged to β2-microglobulin and cystatin C at 24 h (AUC = 0.808, P = 0.003 and AUC = 0.854, P = 0.001, respectively). Their higher values were also associated with acute kidney injury (OR 17.2, P = 0.018; OR 965.6, P = 0.02, respectively). A persistent increase in the postoperative levels of β2-microglobulin following acute kidney injury was associated with the progression of chronic kidney disease for 6 months after both transcatheter (OR 6.56, P = 0.030) and surgical (OR 7.67, P = 0.03) aortic valve replacements. Serum β2-microglobulin had the potential to predict acute kidney injury complicating transcatheter valve replacement and to diagnose it as early as 24 h after both the

Creatine kinase BB and beta-2-microglobulin as markers of CNS metastases in patients with small-cell lung cancer

DEFF Research Database (Denmark)

Pedersen, A G; Bach, F W; Nissen, Mogens Holst

1985-01-01

Creatine kinase (CK) and its BB isoenzyme (CK-BB) were measured in CSF in 65 evaluable patients suspected of CNS metastases secondary to small-cell lung cancer (SCLC). In addition, CSF and plasma levels of beta-2-microglobulin (beta-2-m) were measured in a group of 73 evaluable patients. Of the 65...

Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Claësson, M H

1987-01-01

the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...

T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

DEFF Research Database (Denmark)

Dunon, D; Kaufman, J; Salomonsen, J

1990-01-01

beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

[Effect of sulfonation of polyethersulfone sheets on the adsorption of beta2-microglobulin].

Science.gov (United States)

Cheng, Liping; Sun, Shudong; Yue, Yilun; Huang, Jia; Mao, Huayi; Liang, Bo

2005-06-01

This study was performed to evaluate the adsorption of beta2-microglobulin(beta2 M) by blood dialysis membrane materials which are polyethersulfone (PES), sulfonated polyethersulfones, (PES-SO3Na-I and PES-SO3Na-I ) in vitro incubated in human serum and radiolabeled beta2M (125I-beta2 M) solution respectively. In these experiments, the materials were incubated in 125I-beta2 M solution and human serum at the appointed time ranging from 15 minutes to four hours at 37 degrees C, and then the amounts of 125I-beta2M and serum beta2M adsorbed by materials were measured by radioimmunoassay (RIA). In the 125I-beta2 M system, amounts of 125I-beta2M adsorbed by the materials decreased in sequence of PES-SO3 Na-II > PES-SO3Na-I > PES. In the serum system, amounts of beta2M adsorbed reached maximums at 30 minutes and the final adsorptions decreased in sequence of PES-SO3Na-II > PES-SO3Na-I > PES. Sulfonated PES removes beta2M more than PES does and the adsorption of beta2M increases with the increase in the degree of sulfonation. Its ability to remove significant amount of beta2M may result in less beta2M available for incorporation into amyloid. The use of PES-SO3Na membranes lessens the likelihood of dialysis-related amyloidosis (DRA) development, which remains a major source of morbidity for patients treated with long-term hemodialysis.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter

DEFF Research Database (Denmark)

Riegert, P; Andersen, R; Bumstead, N

1996-01-01

a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II...... but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show...

Mammalian amyloidogenic proteins promote prion nucleation in yeast.

Science.gov (United States)

Chandramowlishwaran, Pavithra; Sun, Meng; Casey, Kristin L; Romanyuk, Andrey V; Grizel, Anastasiya V; Sopova, Julia V; Rubel, Aleksandr A; Nussbaum-Krammer, Carmen; Vorberg, Ina M; Chernoff, Yury O

2018-03-02

Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

Science.gov (United States)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

Science.gov (United States)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Urinary alpha 1-microglobulin as an indicator protein of renal tubular dysfunction caused by environmental cadmium exposure

Energy Technology Data Exchange (ETDEWEB)

Tohyama, C.; Kobayashi, E.; Saito, H.; Sugihara, N.; Nakano, A.; Mitane, Y.

1986-06-01

An epidemiologic investigation was carried out to clarify the significance of the urinary excretion of alpha 1-microglobulin (alpha 1-MG) in people aged 50 years and over living in a Cd-polluted area in Japan. Approximately 80% of the population participated in the health examination. The urinary and serum levels and the relative clearance of alpha 1-MG to creatinine clearance were compared with various parameters (age, urinary beta 2-microglobulin (beta 2-MG), total protein, Cd, Cu and Zn, serum beta 2-MG, creatinine and blood urea nitrogen and relative clearances of alpha 1-MG, beta 2-MG, inorganic phosphate and uric acid). It was found that the urinary excretion of alpha 1-MG is closely associated with the urinary Cd and Cu and with the indices of renal dysfunction listed above. These results suggest that the urinary alpha 1-MG level markedly reflects a degree of proximal tubular dysfunction and that it may be useful as one of the screening measures for proximal tubular dysfunction caused by environmental Cd exposure.

Beta-2-mikroglobulin ved medicinske sygdomme

DEFF Research Database (Denmark)

Hansen, P B; Olsen, Niels Vidiendal

1989-01-01

Beta-2-microglobulin (beta 2M) is a low-molecular protein which is filtered freely over the glomeruli. Under normal circumstances, more than 99.9% is resorbed in the proximal tubuli of the kidneys and is metabolized there. In renal disease with damage to this segment of the nephron, eg acute tubulo...

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

DEFF Research Database (Denmark)

Zhao, Heng; Stet, René J M; Skjødt, Karsten

2008-01-01

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

Association of Beta-2 Microglobulin with Inflammation and Dislipidemia in High-Flux Membrane Hemodialysis Patients.

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Topçiu-Shufta, Valdete; Miftari, Ramë; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend

2016-10-01

Higher than expected cardiovascular mortality in hemodialysis patients, has been attributed to dyslipidemia as well as inflammation. Beta2-Microglobulin (β2M) is an independent predictor of outcome for hemodialysis patients and a representative substance of middle molecules. In 40 patients in high-flux membrane hemodialysis, we found negative correlation of β2M with high density lipoprotein (r=-0.73, p<0.001) and albumin (r= -0.53, p<0.001) and positive correlation with triglycerides (r=0.69, p<0.001), parathyroid hormone (r=0.58, p < 0.05) and phosphorus (r= 0.53, p<0.001). There was no correlation of β2M with C- reactive protein (CRP) and interleukin-6 (IL-6). During the follow-up period of three years, 6 out of 40 patients have died from cardiovascular events. In high-flux membrane hemodialysis patients, we observed a significant relationship of β2M with dyslipidemia and mineral bone disorders, but there was no correlation with inflammation.

Cerebrospinal fluid beta-2-microglobulin in adult patients with acute leukemia or lymphoma

DEFF Research Database (Denmark)

Hansen, P B; Kjeldsen, L; Dalhoff, K

1992-01-01

Beta-2-microglobulin (B2m) was measured in the cerebrospinal fluid (CSF) and serum from 18 adults with acute lymphoblastic leukemia, acute myeloblastic leukemia or lymphoma in order to detect early central nervous system (CNS) involvement or relapse. Six had CNS-involvement documented by neurologic...... determination of CSF-B2m alone may be a useful and sensitive marker of CNS-dissemination in acute leukemia and malignant lymphoma. Using the criteria of CSF-B2m greater than 160 nmol/l as a positive diagnostic test the sensitivity of the test was 100%, the specificity was 76%. The same values for the CSF...

Kinetics of beta2-microglobulin and phosphate during hemodialysis: effects of treatment frequency and duration.

Science.gov (United States)

Leypoldt, John K

2005-01-01

Current understanding of beta2-microglobulin (beta2M) and phosphate (or inorganic phosphorus) kinetics during hemodialysis is reviewed. The postdialysis:predialysis concentration ratio for beta2M is determined by dialyzer clearance for beta2M, treatment time, patient body size (specifically, extracellular fluid volume), and total ultrafiltration volume during the treatment. Evaluation of these treatment parameters can be used to calculate dialyzer clearance for beta2M; however, such calculated values are only approximations, since they neglect intradialytic generation, nonrenal (nondialyzer) clearance, and postdialysis rebound of beta2M. The detailed kinetics of beta2M during hemodialysis are best described using a two-compartment model. Theoretical predictions from such two-compartment models suggest that the product of dialyzer clearance for beta2M and weekly treatment duration, independent of treatment frequency, is the main determinant of plasma beta2M concentrations. The kinetics of phosphate removal during hemodialysis are incompletely understood. Phosphate is removed from both extracellular and intracellular compartments during hemodialysis; the plasma phosphate concentration levels off after the first 1 or 2 hours of treatment and plasma concentrations can rebound even before therapy is complete. Increases in dialyzer clearance of phosphate have been previously achieved only by increasing dialysis membrane surface area or by the use of hemodiafiltration. A four-compartment model of phosphate kinetics proposed recently by Spalding et al. suggests that the major barrier to phosphate removal is limited transfer of phosphate between the intracellular and extracellular compartments, although other complex factors also play important roles. Theoretical predictions using the model of Spalding et al. suggest that increasing either treatment frequency or treatment duration can increase phosphate removal. The kinetics of beta2M are representative of middle molecules

A β2-microglobulin cleavage variant fibrillates at near-physiological pH

International Nuclear Information System (INIS)

Corlin, Dorthe B.; Johnsen, Christina K.; Nissen, Mogens H.; Heegaard, Niels H.H.

2009-01-01

β 2 -microglobulin (β 2 m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β 2 m are still largely unknown. In vitro, acidic conditions, pH 2 m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β 2 m-found in the circulation of many dialysis patients-is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β 2 m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β 2 m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β 2 m. This may explain the apparent lack of detectable amounts of the variant β 2 m in extracts of amyloid from DRA patients.

Association of Beta-2 Microglobulin with Inflammation and Dislipidemia in High-Flux Membrane Hemodialysis Patients

Science.gov (United States)

Topçiu–Shufta, Valdete; Miftari, Ramë; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend

2016-01-01

Background: Higher than expected cardiovascular mortality in hemodialysis patients, has been attributed to dyslipidemia as well as inflammation. Beta2-Microglobulin (β2M) is an independent predictor of outcome for hemodialysis patients and a representative substance of middle molecules. Results: In 40 patients in high-flux membrane hemodialysis, we found negative correlation of β2M with high density lipoprotein (r=-0.73, p<0.001) and albumin (r= -0.53, p<0.001) and positive correlation with triglycerides (r=0.69, p<0.001), parathyroid hormone (r=0.58, p < 0.05) and phosphorus (r= 0.53, p<0.001). There was no correlation of β2M with C- reactive protein (CRP) and interleukin-6 (IL-6). During the follow-up period of three years, 6 out of 40 patients have died from cardiovascular events. Conclusion: In high-flux membrane hemodialysis patients, we observed a significant relationship of β2M with dyslipidemia and mineral bone disorders, but there was no correlation with inflammation. PMID:27994294

Urinary excretion of beta 2-glycoprotein-1 (apolipoprotein H) and other markers of tubular malfunction in "non-tubular" renal disease.

Science.gov (United States)

Flynn, F V; Lapsley, M; Sansom, P A; Cohen, S L

1992-07-01

To determine whether urinary beta 2-glycoprotein-1 assays can provide improved discrimination between chronic renal diseases which are primarily of tubular or glomerular origin. Urinary beta 2-glycoprotein-1, retinol-binding protein, alpha 1-microglobulin, beta 2-microglobulin, N-acetyl-beta-D-glucosa-minidase and albumin were measured in 51 patients with primary glomerular disease, 23 with obstructive nephropathy, and 15 with polycystic kidney disease, and expressed per mmol of creatinine. Plasma beta 2-glycoprotein-1 was assayed in 52 patients and plasma creatinine in all 89. The findings were compared between the diagnostic groups and with previously published data relating to primary tubular disorders. All 31 patients with plasma creatinine greater than 200 mumol/l excreted increased amounts of beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin, and 29 had increased N-acetyl-beta-D-glucosaminidase; the quantities were generally similar to those found in comparable patients with primary tubular pathology. Among 58 with plasma creatinine concentrations under 200 mumol/l, increases in beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin excretion were less common and much smaller, especially in those with obstructive nephropathy and polycystic disease. The ratios of the excretion of albumin to the other proteins provided the clearest discrimination between the patients with glomerular or tubular malfunction, but an area of overlap was present which embraced those with obstructive nephropathy and polycystic disease. Increased excretion of beta 2-glycoprotein-1 due to a raised plasma concentration or diminution of tubular reabsorption, or both, is common in all the forms of renal disease investigated, and both plasma creatinine and urinary albumin must be taken into account when interpreting results. Ratios of urinary albumin: beta 2-glycoprotein-1 greater than 1000 are highly suggestive of primary glomerular disease and

Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin

DEFF Research Database (Denmark)

Nybo, Mads; Nielsen, E H; Svehag, S E

1999-01-01

component nor heparan sulfate did significantly alter the A beta-induced CA. The results indicate that not only fibrillar A beta but also oligomers of, in particular, beta 2M from patients with dialysis-associated amyloidosis are capable of inducing CA at supra-physiological concentrations....

Neopterin and Beta-2 Microglobulin Relations to Immunity and Inflammatory Status in Nonischemic Dilated Cardiomyopathy Patients

Directory of Open Access Journals (Sweden)

Celina Wojciechowska

2014-01-01

Full Text Available Background. The aim of the study was to assess the relationships among serum neopterin (NPT, β2-microglobulin (β2-M levels, clinical status, and endomyocardial biopsy results of dilated cardiomyopathy patients (DCM. Methods. Serum NPT and β-2 M were determined in 172 nonischaemic DCM patients who underwent right ventricular endomyocardial biopsy and 30 healthy subjects (ELISA test. The cryostat biopsy specimens were assessed using histology, immunohistology, and immunochemistry methods (HLA ABC, HLA DR expression, CD3 + lymphocytes, and macrophages counts. Results. The strong increase of HLA ABC or HLA DR expression was detected in 27.2% patients—group A—being low in 72.8% patients—group B. Neopterin level was increased in patients in group A compared to healthy controls 8.11 (4.50–12.57 versus 4.99 (2.66–8.28 nmol/L (P<0.05. β-2 microglobulin level was higher in DCM groups A (2.60 (1.71–3.58 and B (2.52 (1.51–3.72 than in the control group 1.75 (1.28–1.96 mg/L, P<0.001. Neopterin correlated positively with the number of macrophages in biopsy specimens (P<0.05 acute phase proteins: C-reactive proteins (P<0.05; fibrinogen (P<0.01; and NYHA functional class (P<0.05 and negatively with left ventricular ejection fraction (P<0.05. Conclusions. Neopterin but not β-2 microglobulin concentration reflected immune response in biopsy specimens. Neopterin correlated with acute phase proteins and stage of heart failure and may indicate a general immune and inflammatory activation in heart failure.

Fractional excretion of beta-2-microglobulin in the urine of patients with normal or reduced renal function and hepatic coma

DEFF Research Database (Denmark)

Hansen, P B; Dalhoff, K; Joffe, P

1991-01-01

The purpose of this prospective study was to evaluate beta-2-microglobulin (beta 2m) as a differential diagnostic indicator between hepatic nephropathy (HN) and acute tubulointerstitial nephropathy (ATIN) in patients with reduced renal function and hepatic coma, and to determine whether beta 2m...... excretion could be used as a marker of renal impairment before increased serum creatinine (S-Cr) concentration or decreased creatinine clearance (Cr-Cl). Finally, the use of beta 2m as a prognostic indicator was investigated. Eighteen patients in hepatic coma grade III-IV were entered in the study and were...... to the small number of patients. FE-beta 2m could not predict the development of renal failure earlier than the increase in S-Cr or decrease in Cr-Cl. However, a few patients who survived paracetamol intoxication had increased FE-beta 2M in the beginning of the coma and normal S-Cr and Cr-Cl. Patients who died...

Serum levels of beta2-microglobulin and free light chains of immunoglobulins are associated with systemic disease activity in primary Sjögren's syndrome. Data at enrollment in the prospective ASSESS cohort.

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Jacques-Eric Gottenberg

Full Text Available To analyze the clinical and immunological characteristics at enrollment in a large prospective cohort of patients with primary Sjögren's syndrome (pSS and to investigate the association between serum BAFF, beta2-microglobulin and free light chains of immunoglobulins and systemic disease activity at enrollment.Three hundred and ninety five patients with pSS according to American-European Consensus Criteria were included from fifteen centers of Rheumatology and Internal Medicine in the "Assessment of Systemic Signs and Evolution of Sjögren's Syndrome" (ASSESS 5-year prospective cohort. At enrollment, serum markers were assessed as well as activity of the disease measured with the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI.Patient median age was 58 (25(th-75(th: 51-67 and median disease duration was 5 (2-9 years. Median ESSDAI at enrollment was 2 (0-7 with 30.9% of patients having features of systemic involvement. Patients with elevated BAFF, beta2-microglobulin and kappa, lambda FLCS had higher ESSDAI scores at enrollment (4 [2]-[11] vs 2 [0-7], P = 0.03; 4 [1]-[11] vs 2 [0-7], P< 0.0001; 4 [2]-[10] vs 2 [0-6.6], P< 0.0001 and 4 [2-8.2] vs 2 [0-7.0], P = 0.02, respectively. In multivariate analysis, increased beta2-microglobulin, kappa and lambda FLCs were associated with a higher ESSDAI score. Median BAFF and beta2-microglobulin were higher in the 16 patients with history of lymphoma (1173.3(873.1-3665.5 vs 898.9 (715.9-1187.2 pg/ml, P = 0.01 and 2.6 (2.2-2.9 vs 2.1 (1.8-2.6 mg/l, P = 0.04, respectively.In pSS, higher levels of beta2-microglobulin and free light chains of immunoglobulins are associated with increased systemic disease activity.

Isolation and characterization of chicken and turkey beta 2-microglobulin

DEFF Research Database (Denmark)

Skjødt, K; Welinder, K G; Crone, M

1986-01-01

Chicken and turkey beta 2-m were isolated from citrated plasma in sequential use of three chromatographic steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta 2-m by reaction with a beta 2-m specific monoclonal...... (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta 2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. EM280 was 15.9 for chicken beta 2-m and 16.4 for turkey beta 2-m......, and is incompatible with a previously published sequence also thought to be from turkey beta 2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given. Udgivelsesdato: 1986-Dec...

β2-microglobulin test in the diagnosis of chronic renal diseases

International Nuclear Information System (INIS)

Trusov, V.V.; Filimonov, M.A.

1985-01-01

A study was made of the content of low molecular protein B 2 -microglobulin in the blood and urine of 126 patients with chronic renal diseases and 95 healthy persons. As a result of the study it was shown that B 2 -microglobulin concentration in the blood grows with age. The maximum level of B 2 -microglobulin was marked in patients with chronic glomerulonephritis. A high level of the urinary eXcretion of B 2 -microglobUlin with a moderate rise of its concentration in the blood is typical of patients with chronic pyehlonephritis during exacerbation. Indices of the B 2 -microglobulin test are closely related to renal function. The B 2 -microglobulin test is of great diagnostic significance as it proVides an opportunity to establish the nature of protenuria, site and expression of renal pathologic processes

A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

DEFF Research Database (Denmark)

Sylvester-Hvid, C; Buus, S

1999-01-01

of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule...... electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained....

Usefulness of Beta2-Microglobulin as a Predictor of All-Cause and Nonculprit Lesion-Related Cardiovascular Events in Acute Coronary Syndromes (from the PROSPECT Study).

Science.gov (United States)

Möckel, Martin; Muller, Reinhold; Searle, Julia; Slagman, Anna; De Bruyne, Bernard; Serruys, Patrick; Weisz, Giora; Xu, Ke; Holert, Fabian; Müller, Christian; Maehara, Akiko; Stone, Gregg W

2015-10-01

In the Providing Regional Observations to Study Predictors of Events in the Coronary Tree (PROSPECT) study, plaque burden, plaque composition, and minimal luminal area were associated with an increased risk of adverse cardiovascular events arising from untreated atherosclerotic lesions (vulnerable plaques) in patients with acute coronary syndromes (ACS). We sought to evaluate the utility of biomarker profiling and clinical risk factors to predict 3-year all-cause and nonculprit lesion-related major adverse cardiac events (MACEs). Of 697 patients who underwent successful percutaneous coronary intervention (PCI) for ACS, an array of 28 baseline biomarkers was analyzed. Median follow-up was 3.4 years. Beta2-microglobulin displayed the strongest predictive power of all variables assessed for all-cause and nonculprit lesion-related MACE. In a classification and regression tree analysis, patients with beta2-microglobulin >1.92 mg/L had an estimated 28.7% 3-year incidence of all-cause MACE; C-peptide 1.92 mg/L identified a cohort with a 3-year rate of 18.5%, and C-peptide PROSPECT study, beta2-microglobulin strongly predicted all-cause and nonculprit lesion-related MACE within 3 years after PCI in ACS. C-peptide and HDL provided further risk stratification to identify angiographically mild nonculprit lesions prone to future MACE. Copyright © 2015 Elsevier Inc. All rights reserved.

Expected packing density allows prediction of both amyloidogenic and disordered regions in protein chains

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Galzitskaya, Oxana V; Garbuzynskiy, Sergiy O; Lobanov, Michail Yu [Institute of Protein Research, Russian Academy of Sciences, 142290, Pushchino, Moscow Region (Russian Federation)

2007-07-18

The determination of factors that influence conformational changes in proteins is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for 9 of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of formation of amyloid fibrils is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value for predicting both disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.

Rediscovering Beta-2 Microglobulin As a Biomarker across the Spectrum of Kidney Diseases

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Christos P. Argyropoulos

2017-06-01

Full Text Available There is currently an unmet need for better biomarkers across the spectrum of renal diseases. In this paper, we revisit the role of beta-2 microglobulin (β2M as a biomarker in patients with chronic kidney disease and end-stage renal disease. Prior to reviewing the numerous clinical studies in the area, we describe the basic biology of β2M, focusing in particular on its role in maintaining the serum albumin levels and reclaiming the albumin in tubular fluid through the actions of the neonatal Fc receptor. Disorders of abnormal β2M function arise as a result of altered binding of β2M to its protein cofactors and the clinical manifestations are exemplified by rare human genetic conditions and mice knockouts. We highlight the utility of β2M as a predictor of renal function and clinical outcomes in recent large database studies against predictions made by recently developed whole body population kinetic models. Furthermore, we discuss recent animal data suggesting that contrary to textbook dogma urinary β2M may be a marker for glomerular rather than tubular pathology. We review the existing literature about β2M as a biomarker in patients receiving renal replacement therapy, with particular emphasis on large outcome trials. We note emerging proteomic data suggesting that β2M is a promising marker of chronic allograft nephropathy. Finally, we present data about the role of β2M as a biomarker in a number of non-renal diseases. The goal of this comprehensive review is to direct attention to the multifaceted role of β2M as a biomarker, and its exciting biology in order to propose the next steps required to bring this recently rediscovered biomarker into the twenty-first century.

Urine β2 Microglobulin and other Biochemical Indices in β Thalassemia Major

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Yazdan Ghandi

2009-12-01

Full Text Available To find if some indices have predictive value for renal complications. We conducted a cross sectional and included 80 patients with the age ranged 5-17 years, all with the proven diagnosis of β-thalassemia major. A urine and 5 ml of blood sample were obtained from all of the cases. Biochemical indices such as serum levels of creatinine, Na, Mg, Hb, and ferritin and also urine levels of Na, Mg, creatinine and β2 microglobulin was measured. All data analysis was performed using SPSS 14.0. P-Spearman test was applied to assess correlation between urine beta-2-microglobulin and other variables. Patients GFR was in normal range. Abnormal level of urine β2 microglobulin was reported in 44 patients (55%. P Spearman test proved correlation only between urine β2 microglobulin and FE-Mg. We concluded that renal proximal tubular dysfunction may oocur in children with β thalassemia major without clinical manifestations of renal dysfunction or decrease in GFR. We warn not to rely only on GFR as a early indicator for renal complications among children with β thalassemia major.

Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

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Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

2009-04-01

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Amyloidogenic determinants are usually not buried

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Karletidi Carolina-Maria

2009-07-01

Full Text Available Abstract Background Amyloidoses are a group of usually fatal diseases, probably caused by protein misfolding and subsequent aggregation into amyloid fibrillar deposits. The mechanisms involved in amyloid fibril formation are largely unknown and are the subject of current, intensive research. In an attempt to identify possible amyloidogenic regions in proteins for further experimental investigation, we have developed and present here a publicly available online tool that utilizes five different and independently published methods, to form a consensus prediction of amyloidogenic regions in proteins, using only protein primary structure data. Results It appears that the consensus prediction tool is slightly more objective than individual prediction methods alone and suggests several previously not identified amino acid stretches as potential amyloidogenic determinants, which (although several of them may be overpredictions require further experimental studies. The tool is available at: http://biophysics.biol.uoa.gr/AMYLPRED. Utilizing molecular graphics programs, like O and PyMOL, as well as the algorithm DSSP, it was found that nearly all experimentally verified amyloidogenic determinants (short peptide stretches favouring aggregation and subsequent amyloid formation, and several predicted, with the aid of the tool AMYLPRED, but not experimentally verified amyloidogenic determinants, are located on the surface of the relevant amyloidogenic proteins. This finding may be important in efforts directed towards inhibiting amyloid fibril formation. Conclusion The most significant result of this work is the observation that virtually all, to date, experimentally determined amyloidogenic determinants and the majority of predicted, but not yet experimentally verified short amyloidogenic stretches, lie 'exposed' on the surface of the relevant amyloidogenic proteins, and also several of them have the ability to act as conformational 'switches'. Experiments

Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

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Sukhdeep Kumar

Full Text Available Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD varieties. We establish (a that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT, resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

Amniotic Fluid β2- Microglobulin Measurements

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Emine Aydın

2016-02-01

Full Text Available OBJECTIVE: To determine β2-microglobulin levels in amniotic fluid during the course of second trimester. STUDY DESIGN: One hundred patient’s amniotic fluid β2-microglobulin levels had been evaluated retrospectively (March-October 2009. The most common amniocentesis indication was advanced maternal age (33.3%. Others were; high risk result for triple test (18.5%, high risk result for double test (6.48%, ventriculomegaly (5.55%, obstetric history for fetus with down syndrome (4.62%, the presence of soft markers on ultrasound (13.8%, others (17.8%. Patients average gravida was 2.66 (range: 1-6, parity was 0.75 (range: 0-3, abortion was 0.65 (range: 0-3. RESULTS: All patients were at second trimester and the average gestational week was 17.7 (range 15- 22. Patients were divided into four groups (15th, 16th, 17-18th and 19-20th gestational weeks. We have demonstrated that amniotic fluid β2-microglobulin levels are increased progressively throughout the second trimester. We have specified normal β2-microglobulin values of each gestational week/period in order to be used in clinical practice. CONCLUSION: We have demonstrated that amnionic fluid β2-microglobulin levels are increased progressively throughout the second trimester.

Modified human beta 2-microglobulin (desLys(58)) displays decreased affinity for the heavy chain of MHC class I and induces nitric oxide production and apoptosis

DEFF Research Database (Denmark)

Wang, M; Harhaji, L; Lamberth, K

2009-01-01

Beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8+ T cells. beta2m can be modified in vivo and in vitro by proteolytic cleavage...... by complement C1 and subsequent carboxypeptidase B-like activity--processes that lead to the generation of desLys(58) beta2m (dbeta2m). This work aims to study the effect of dbeta2m on peptide binding to MHC-I, the influence of dbeta2m on the binding of beta2m to the MHC-I heavy chain and the biological...... activity of dbeta2m. Both beta2m and dbeta2m are able to support the generation of MHC-I/peptide complexes at 18 degrees C, but complexes formed in the presence of dbeta2m destabilize at 37 degrees C. Moreover, a 250 times higher concentration of dbeta2m than of beta2m is needed to displace MHC...

Systemic lupus erythematosus activity and beta two microglobulin levels

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Thelma Larocca Skare

Full Text Available CONTEXT AND OBJECTIVE: Systemic lupus erythematosus (SLE is an autoimmune disease with a cyclical clinical course. Evaluation of the clinical activity of this disease is important for choosing the correct treatment. The objective of this study was to analyze the value of beta-2 microglobulin (β2M serum levels in determining SLE clinical activity.DESIGN AND SETTING: Cross-sectional analytical study conducted at the rheumatology outpatient clinic of a private university hospital.METHODS: 129 SLE patients were studied regarding disease activity using SLEDAI (SLE Disease Activity Index and cumulative damage using SLICC ACR (SLE International Collaborating Clinics/American College of Rheumatology Damage Index for SLE. At the same time, the β2M serum level, ESR (erythrocyte sedimentation rate, anti-dsDNA (anti-double-stranded DNA and C3 and C4 complement fractions were determined.RESULTS: β2M levels correlated positively with SLEDAI (P = 0.02 and ESR (P = 0.0009 and negatively with C3 (P = 0.007. Patients who were positive for anti-dsDNA had higher β2M serum levels (P = 0.009.CONCLUSION: β2M levels are elevated in SLE patients with active disease.

The Significance of Serum beta2-Microglobulin Measurement in Various Renal Diseases

Energy Technology Data Exchange (ETDEWEB)

Koong, Sung Soo; Oh, Ha Yong; Han, Jin Suk; Lee, Jung Sang [Seoul National University College of Medicine, Seoul (Korea, Republic of)

1985-03-15

To evaluate change of serum beta{sub 2}-microglobulin concentration (sbeta{sub 2}-MG) and the usefulness of sbeta{sub 2}-MG and sbeta{sub 2}-MG/serum creatinine concentration (sCr) ratio in various renal diseases, sbeta{sub 2}-MG and sCr were measured in 25 normal controls and 90 patients of various renal diseases (16 cases of glomerulonephritis, 12 cases of acute renal failure, 8 cases of chronic renal failure, 24 cases of nephrotic syndrome, 15 cases of tubulointerstitial diseases and 15 cases of lupus nephritis) using Phadebas beta{sub 2}-Micro Test kits. The results were as follows; 1) In normal control, the mean value of sbeta{sub 2}-MG was 1.65+-0.41 mg/l and the mean value of sbeta{sub 2}-MG/sCr ratio was 0.14+-0.05. 2) In various renal diseases, the mean value of sbeta{sub 2}-MG was 6.74+-5.47 mg/l. The mean value of sbeta{sub 2}-MG/sCr ratio was 0.24+-0.11 and significantly elevated than that of normal contro1. (P<0.05). 3) The correlation between sbeta-2-MG and sCr in glomerular and tubulointerstitial disease was log sbeta{sub 2}-MG=0.90 log sCr-0.48 and its correlation coefficient was 0.78 (P<0.05). 4) In glomerular disease, the correlation between sbeta{sub 2}-MG and sCr was log sbeta{sub 2}-MG=0.89 log sCr-0.46 (r-0.76) and in tubulointerstitial disease, it was log sbeta{sub 2}-MG=0.95 1og sCr-0.59 (r-0.87). There was no significant difference between the two groups (p<0.05). 5) Among 32 cases of glomerular and tubulointerstitial disease patients, whose sCr was within normal range, 17 cases showed elevated sbeta{sub 2}-MG. The mean values of sbeta{sub 2}-MG/sCr ratio in these patients was 0.30+-0.14 and significantly elevated than that of normal control (p<0.05). 6) In 15 cases of lupus nephritis, 12 cases showed elevated sbeta{sub 2}-MG with normal sCr and 12 cases showed elevated sbeta{sub 2}-MG/sCr ratio. With above results, It was found that the sbeta{sub 2}-MG can be used as an index of glomerular filtration rate as in the case of sCr and thats

Beta-2 Microglobulin Levels in Hemodialysis Patients

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Mumtaz Asim

2010-01-01

Full Text Available The objective of the study was to determine the level of β2-microglobulin (β2-m in hemodialysis (HD patients and the factors affecting it. This cross sectional, hospital based study was conducted between September and December 2008 at the Hemodialysis unit of Shalamar Hospital, Lahore. All patients with end-stage renal disease (ESRD who were on main-tenance HD for more than three months were included in the study. Patients with acute renal failure and on dialysis for less than three months were excluded. Demographic data were collec-ted and details of dialysis (type of dialyzers, dialysate bath, membrane used were recorded. Blood samples of the patients were drawn for hematological (hemoglobin, hematocrit, bioche-mical (urea, creatinine, uric acid, albumin and β2-m level measurement. The total number of patients studied was 50. The major causes of ESRD included diabetes mellitus and hypertension seen in 37 (74% and 10 patients (20%, respectively. The β2-m levels were significantly elevated in the study patients; 92.6 ± 17.1 mg/L with a range of 54 to 130 mg/L as compared to 2.0 ± 1.29 mg/L in the control group. The patients′ age had a statistically significant relationship with the β2-m level. The major reason for increased β2-m level was use of low-flux dialyzers. Synthetic polysulphone membrane, bicarbonate, ultra pure dialysate and duration on HD were not asso-ciated with high β2-m levels. Also, we found an inverse relationship between β2-m levels and serum albumin of the study patients. Our study suggests that the β2-m levels are significantly high in dialysis patients. Use of low-flux dialyzer seems to be the major reason for the high β2-m levels. Age and albumin have statistically significant relationship with β2-m levels.

Urinary β2 Microglobulin in Workers Exposed to Arc Welding Fumes

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Khosro Sadeghniiat-Haghighi

2011-11-01

Full Text Available Welding is a process in which two or more metals are attached by the use of heat and, in some cases, pressure. Direct exposure and inhalation of welding fumes causes acute and chronic side effects in humans. Kidney damage is one of these important side effects. β2 microglobulin is an 11.8 kilodalton protein and levels increase in the case of some inflammatory and viral diseases, or kidney malfunction and autoimmune diseases. In this study measurements of β2 microglobulin were used as a criterion for assessing effects on the kidneys of workers exposed to welding fumes. The study population were electric arc welders in an industrial plant in Tehran, Iran. For control we selected workers who did not have any exposure to welding fumes. Both groups were selected on the basis of a questionnaire and the consideration of criteria for inclusion and exclusion. In the end 50 cases and 50 controls were chosen. A urine sample was collected from all participants and urinary pH was set to between 6-8 using NaOH (1M. Sample transportation to the laboratory complied with the related standards. The samples were assessed using the ORG 5BM kit. For quantitative assessment of β2 microglobulin we used the Enzyme-linked Immunosorbent Assay (ELISA method. The ages of the welders ranged from 21 to 48 years (mean=30.5±5.9 yrs and of controls from 23 to 56 years (mean=31.8±5.9 yrs. Mean employment duration was 7.86±5.01years (range 2 to 27 years for welders. Mean β2 microglobulin level was 0.10±0.096 μg/ml in welders and 0.11±0.06 in controls. This difference was not statistically significant (P=0.381. In conclusion we don't find that exposure to electric arc welding fumes cause a significant change in urinary β2 microglobulin compared to the control group.

An elevated serum beta-2-microglobulin level is an adverse prognostic factor for overall survival in patients with early-stage Hodgkin disease.

Science.gov (United States)

Chronowski, Gregory M; Wilder, Richard B; Tucker, Susan L; Ha, Chul S; Sarris, Andreas H; Hagemeister, Fredrick B; Barista, Ibrahim; Hess, Mark A; Cabanillas, Fernando; Cox, James D

2002-12-15

The relative importance of prognostic factors in patients with early-stage Hodgkin disease remains controversial. The purpose of this study was to evaluate prognostic factors among patients who received chemotherapy before radiotherapy. From 1987 to 1995, 217 consecutive patients ranging in age from 16 to 88 years (median, 28 years) with Ann Arbor Stage I (n = 55) or II (n = 162) Hodgkin disease underwent chemotherapy before radiotherapy at a single center. Most were treated on prospective studies. Patients received a median of three cycles of induction chemotherapy. Mitoxantrone, vincristine, vinblastine, and prednisone (NOVP), doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), cyclophosphamide, vinblastine, procarbazine, prednisone, doxorubicin, bleomycin, dacarbazine, and CCNU (CVPP/ABDIC), or other chemotherapeutic regimens were given to 160, 18, 15, 10, and 14 patients, respectively. The median radiotherapy dose was 40 Gy. Serum beta-2-microglobulin (beta-2M) levels ranged from 1.0 to 4.1 mg/L (median, 1.7 mg/L; upper limit of normal, 2.0 mg/L). We studied univariate and multivariate associations between survival and the following clinical features: serum beta-2M level above 1.25 times the upper limit of normal (n = 12), male gender (n = 113), hypoalbuminemia (n = 11), and bulky mediastinal disease (n = 94). Follow-up of surviving patients ranged from 0.9 to 13.4 years (median, 6.6 years) and 92% were observed for 3.0 or more years. Nineteen patients have died. Only elevation of the serum beta-2M level was an independent adverse prognostic factor for overall survival (P = 0.0009). The prognostic significance of a simple, widely available, and inexpensive blood test, beta-2M, has not been studied routinely in patients with Hodgkin disease and should be tested prospectively in large, cooperative group trials. Copyright 2002 American Cancer Society.DOI 10.1002/cncr.10998

Disrupting self-assembly and toxicity of amyloidogenic protein oligomers by "molecular tweezers" - from the test tube to animal models.

Science.gov (United States)

Attar, Aida; Bitan, Gal

2014-01-01

Despite decades of research, therapy for diseases caused by abnormal protein folding and aggregation (amyloidoses) is limited to treatment of symptoms and provides only temporary and moderate relief to sufferers. The failure in developing successful disease-modifying drugs for amyloidoses stems from the nature of the targets for such drugs - primarily oligomers of amyloidogenic proteins, which are distinct from traditional targets, such as enzymes or receptors. The oligomers are metastable, do not have well-defined structures, and exist in dynamically changing mixtures. Therefore, inhibiting the formation and toxicity of these oligomers likely will require out-of-the-box thinking and novel strategies. We review here the development of a strategy based on targeting the combination of hydrophobic and electrostatic interactions that are key to the assembly and toxicity of amyloidogenic proteins using lysine (K)-specific "molecular tweezers" (MTs). Our discussion includes a survey of the literature demonstrating the important role of K residues in the assembly and toxicity of amyloidogenic proteins and the development of a lead MT derivative called CLR01, from an inhibitor of protein aggregation in vitro to a drug candidate showing effective amelioration of disease symptoms in animal models of Alzheimer's and Parkinson's diseases.

Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta(2)-microglobulin

DEFF Research Database (Denmark)

Heegaard, N.H.H.; Jørgensen, T.J.D.; Rozlosnik, N.

2005-01-01

. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that Delta K58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is I order of magnitude faster in Delta K58-beta(2)M than in wt-beta(2)m...... in the circulation of dialysis patients. This beta(2)M variant, Delta K58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)M...

Sequential changes in luminal microflora and mucosal cytokine expression during developing of colitis in HLA-B27/beta2-microglobulin transgenic rats.

Science.gov (United States)

Hata, K; Andoh, A; Sato, H; Araki, Y; Tanaka, M; Tsujikawa, T; Fujiyama, Y; Bamba, T

2001-11-01

Transgenic rats expressing HLA-B27 and human beta2-microglobulin (HLA-B27 rats) spontaneously develop chronic colitis resembling human inflammatory bowel disease. We investigated the sequential changes in the luminal bacterial flora and mucosal cytokine mRNA expression in this model. HLA-B27 rats were maintained in a specific pathogen-free environment, and luminal microflora was evaluated by standard bacterial culture technique. The expression of mucosal cytokine mRNA was analysed by RT-PCR methods. Clinical symptoms of colitis appeared at 8 weeks of age. The total number of obligate anaerobes was higher than those of facultative anaerobes during the experimental period. At 6 weeks of age, the colonization of Bacteroides spp., Bifidobacterium spp. and Lactobacillus spp. was already detectable at high concentrations, whereas Clostridium spp. and Eubacterium spp. were not detected. The expression of proinflammatory cytokines (IL-Ibeta, IL-8 and TNF-alpha) appeared at 8 weeks of age, and these were detectable until 17 weeks. A similar pattern was observed in the expression of Th1 cytokines (IL-2, IL-12 and IFN-gamma). On the other hand, the expression of Th2 cytokines (IL-4, IL-10 and TGF-beta) was weak. IL-4 mRNA expression was weakly detectable only at 6 and 8 weeks of age. The expression of IL-10 and TGF-beta mRNA was scarcely detectable throughout the experimental period. The development of colitis may be mediated by both the predominant expression of Th1 cytokines and the weakness of Th2 cytokine expression in the mucosa. The colonization of anaerobic bacteria, especially Bacteroides spp., may be initiating and promoting these cytokine responses.

Proteomic profiling reveals α1-antitrypsin, α1-microglobulin, and clusterin as preeclampsia-related serum proteins in pregnant women.

Science.gov (United States)

Hsu, Te-Yao; Hsieh, T'sang-T'ang; Yang, Kuender D; Tsai, Ching-Chang; Ou, Chia-Yu; Cheng, Bi-Hua; Wong, Yi-Hsun; Hung, Hsuan-Ning; Chou, An-Kuo; Hsiao, Chang-Chun; Lin, Hao

2015-10-01

Preeclampsia is a major cause of mortality in pregnant women but the underlying mechanism remains unclear to date. In this study, we attempted to identify candidate proteins that might be associated with preeclampsia in pregnant women by means of proteomics tools. Differentially expressed proteins in serum samples obtained from pregnant women with severe preeclampsia (n = 8) and control participants (n = 8) were identified using two-dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Additional serum samples from 50 normal and 41 pregnant women with severe preeclampsia were analyzed by immunoassay for validation. Ten protein spots were found to be upregulated significantly in women with severe preeclampsia. These protein spots had the peptide mass fingerprints matched to α1-antitrypsin, α1-microglobulin, clusterin, and haptoglobin. Immunoassays in an independent series of serum samples showed that serum α1-antitrypsin, α1-microglobulin, and clusterin levels of severe preeclampsia patients (n = 41) were significantly higher than those in the normal participants (n = 50; α1-antitrypsin 295.95 ± 50.94 mg/dL vs. 259.31 ± 33.90 mg/dL, p = 0.02; α1-microglobulin 0.029 ± 0.004 mg/mL vs. 0.020 ± 0.004 mg/mL, p proteins by proteomics analysis enables further understanding of the pathophysiology of preeclampsia. Further studies are warranted to investigate the role of these biomarkers in prediction of this disease. Copyright © 2015. Published by Elsevier B.V.

Enhanced expression in vivo of HLA-ABC antigens and beta 2-microglobulin on human lymphoid cells induced by human interferon-alpha in patients with lung cancer. Enhanced expression of class I major histocompatibility antigens prior to treatment

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Plesner, T; Larsen, J K

1985-01-01

than 0.5, respectively) by day-to-day analysis of an untreated healthy control group. An increased expression of both HLA-ABC (mean 55%, P less than 0.0005) and beta 2m (mean 23%, P less than 0.01) was also observed prior to treatment in the lung cancer patients when compared to a group of age matched......The effect of cloned human interferon-alpha (IFN-alpha) on the expression of HLA-ABC antigens (HLA-ABC) and beta 2-microglobulin (beta 2m) on human peripheral lymphoid cells in vivo was studied by cytofluorometry using monoclonal antibodies and fluorescein-labelled rabbit anti-mouse immunoglobulin....... A significant increase in the mean fluorescence intensity of HLA-ABC (median 59%, P less than 0.001) and beta 2m (median 57%, P less than 0.001) on small lymphoid cells was observed 24 h after initiation of IFN-alpha treatment (50 X 10(6) units IFN-alpha/m2 three times a week). The enhanced expression...

Beyond the neurotransmitter-focused approach in treating Alzheimer's disease: drugs targeting beta-amyloid and tau protein.

Science.gov (United States)

Panza, Francesco; Solfrizzi, Vincenzo; Frisardi, Vincenza; Imbimbo, Bruno P; Capurso, Cristiano; D'Introno, Alessia; Colacicco, Anna M; Seripa, Davide; Vendemiale, Gianluigi; Capurso, Antonio; Pilotto, Alberto

2009-12-01

Drugs currently used to treat Alzheimer's Disease (AD) have limited therapeutic value and do not affect the main neuropathological hallmarks of the disease, i.e., senile plaques and neurofibrillar tangles. Senile plaques are mainly formed of beta-amyloid (Abeta), a 42-aminoacid peptide. Neurofibrillar tangles are composed of paired helical filaments of hyperphosphorylated tau protein. New, potentially disease-modifying, therapeutic approaches are targeting Abeta and tau protein. Drugs directed against Abeta include active and passive immunization, that have been found to accelerate Abeta clearance from the brain. The most developmentally advanced monoclonal antibody directly targeting Abeta is bapineuzumab, now being studied in a large Phase III clinical trial. Compounds that interfere with proteases regulating Abeta formation from amyloid precursor protein (APP) are also actively pursued. The discovery of inhibitors of beta-secretase, the enzyme that regulates the first step of the amyloidogenic metabolism of APP, has been revealed to be particularly difficult due to inherent medicinal chemistry problems, and only one compound (CTS-21166) has reached clinical testing. Conversely, several compounds that inhibit gamma-secretase, the pivotal enzyme that generates Abeta, have been identified, the most advanced being LY-450139 (semagacestat), now in Phase III clinical development. Compounds that stimulate alpha-secretase, the enzyme responsible for the non-amyloidogenic metabolism of APP, are also being developed, and one of them, EHT-0202, has recently entered Phase II testing. Potent inhibitors of Abeta aggregation have also been identified, and one of such compounds, PBT-2, has provided encouraging neuropsychological results in a recently completed Phase II study. Therapeutic approaches directed against tau protein include inhibitors of glycogen synthase kinase- 3 (GSK-3), the enzyme responsible for tau phosphorylation and tau protein aggregation inhibitors. NP-12

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

Science.gov (United States)

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

Directory of Open Access Journals (Sweden)

French Frank S

2004-08-01

Full Text Available Abstract Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed

The interaction between beta 2-microglobulin (beta 2m) and purified class-I major histocompatibility (MHC) antigen

DEFF Research Database (Denmark)

Pedersen, L O; Hansen, A S; Olsen, A C

1994-01-01

been generated recently and this paper reports on a similar assay for the interaction between beta 2m and class I. As a model system human beta 2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined...... by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nM. The rate of association at 37 degrees C is very fast (the ka is around 5 x 10(4)/M/s) whereas the dissociation is slow (the kd is around 8 x 10(-6)/s); the ratio of dissociation...

A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

Science.gov (United States)

2011-05-01

mineral density in men with genetic hemochromatosis and HFE gene mutation. Osteoporos Int 2005; 16:1809–14. b2-Microglobulin Confers EMT and Cancer...In this project, we evaluate the molecular mechanism of AR gene expression at the transcriptional level regulated by β2M during prostate cancer...SREBP-1 targeted gene and has been shown to be a metabolic oncogene, cytoplasm), NADH oxidase 5 (Nox5, a SREBP-1 targeted gene and is a key enzyme for

MHC class I phenotype and function of human beta 2-microglobulin transgenic murine lymphocytes

DEFF Research Database (Denmark)

Bjerager, L; Pedersen, L O; Bregenholt, S

1996-01-01

. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous h beta 2m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/h beta 2m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS......-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous h beta 2m to MHC-I, in particular, to the H-2Db molecule through an exchange with endogenous mouse beta 2m. In contrast to normal H-2Db molecules, hybrid H-2Db expressed on the surface of transgenic lymphocytes...... binds radiolabelled peptide in the absence of exogenous added h beta 2m suggesting that a stable fraction of hybrid H-2Db molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than...

A temporal study on #betta#2-microglobulin as marker for diagnosis and monitoring of multiple myeloma

International Nuclear Information System (INIS)

Neri, B.

1982-01-01

The most important objective carried on, in the field of oncology has been the finding of new means to improve diagnosis and monitoring of neoplastic diseases. So we have chronobiologically studied the behaviour of #betta# 2 -microglobulin, in three groups of patients: Group A: 11 healthy subjects; Group B: 11 untreated patients with multiple myeloma and Group C: 11 multiple myeloma patients, after treatment with polychemoterapy. #betta# 2 -microglobulin, was assayed in serum obtained at six consecutive 4-hourly intervals, by radioimmunoassay, the levels and the various rhythm parameters were analyzed by ''Group mean cosinor'' test. The data suggest, that a circadian rhythm for #betta# 2 -microglobulin in the three groups is not detectable; but ''mesor test'' establishes significant differences (p 2 -microglobulin, in diagnosis and monitoring of multiple myeloma, is suggested

The Drosophila melanogaster DmCK2beta transcription unit encodes for functionally non-redundant protein isoforms.

Science.gov (United States)

Jauch, Eike; Wecklein, Heike; Stark, Felix; Jauch, Mandy; Raabe, Thomas

2006-06-07

Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2alpha subunit and/or a regulatory CK2beta subunit may exist. The phosphotransferase activity of members of the protein kinase CK2alpha family is thought to be independent of second messengers but is modulated by interaction with CK2beta-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2alpha subunit and three genes encoding for CK2beta-like proteins are present. The X-linked DmCK2beta transcription unit encodes for several CK2beta protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2beta-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2beta isoforms encoded by the X-linked DmCK2beta transcription unit influence their functional properties. In addition, we find that CK2beta-like proteins encoded by the autosomal D. melanogaster genes CK2betates and CK2beta' cannot fully substitute for a loss of CK2beta isoforms encoded by DmCK2beta.

Data supporting beta-amyloid dimer structural transitions and protein–lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures

Directory of Open Access Journals (Sweden)

Sara Y. Cheng

2016-06-01

Full Text Available This data article supports the research article entitled “Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface” [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

Beta-2 Microglobulin Kidney Disease Test

Science.gov (United States)

... Diabetes Diarrhea Disseminated Intravascular Coagulation (DIC) Down Syndrome Ebola Virus Infection Endocrine System and Syndromes Epilepsy Excessive ... is then reabsorbed by the renal proximal tubules , structures that reclaim water, proteins, vitamins, minerals, and other ...

First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

Science.gov (United States)

Sankaranarayanan, Sethu; Holahan, Marie A; Colussi, Dennis; Crouthamel, Ming-Chih; Devanarayan, Viswanath; Ellis, Joan; Espeseth, Amy; Gates, Adam T; Graham, Samuel L; Gregro, Allison R; Hazuda, Daria; Hochman, Jerome H; Holloway, Katharine; Jin, Lixia; Kahana, Jason; Lai, Ming-tain; Lineberger, Janet; McGaughey, Georgia; Moore, Keith P; Nantermet, Philippe; Pietrak, Beth; Price, Eric A; Rajapakse, Hemaka; Stauffer, Shaun; Steinbeiser, Melissa A; Seabrook, Guy; Selnick, Harold G; Shi, Xiao-Ping; Stanton, Matthew G; Swestock, John; Tugusheva, Katherine; Tyler, Keala X; Vacca, Joseph P; Wong, Jacky; Wu, Guoxin; Xu, Min; Cook, Jacquelynn J; Simon, Adam J

2009-01-01

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Amyloid-β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect.

Science.gov (United States)

Honda, Ryo

2018-04-12

Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β-rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer's disease amyloid-β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP-amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological indicators of cadmium exposure and toxicity

Energy Technology Data Exchange (ETDEWEB)

Shaikh, Z A; Smith, L M

1986-01-01

The increasing environmental and occupational exposure of populations to cadmium creates the need for biological indicators of cadmium exposure and toxicity. The advantages and disadvantages of monitoring blood cadmium, urinary, fecal, hair, and tissue cadmium, serum creatine, beta 2-microglobulin, alpha 1-anti-trypsin and other proteins, and urinary amino acids, enzymes, total proteins, glucose, beta 2-microglobulin, retinol-binding protein, lysozyme, and metallothionein are discussed. It is concluded that urinary cadmium, metallothionein and beta 2-microglubulin may be used together to assess cadmium exposure and toxicity. 66 references.

Design, Engineering and Application of an Amyloidogenic Protein, SBAFP-m1, for use in Nanotechnological Applications

Science.gov (United States)

Peralta, Maria del Refugio

Nanotechnology relies on collaborations across scientific disciplines such as physics, engineering, chemistry and biology. In nanotechnology, researchers manipulate molecules on the nanometer scale for various applications, ranging from tissue engineering, nanowire synthesis, and alternative energy devices. By utilizing various biological scaffolds, namely amyloid fibrils, the work of nanometer molecular control can be achieved through the use of self-assembly systems. Here, a systematic design scheme was developed to engineer protein based amyloid fibrils and was successfully applied to the design of two, unique self-assembled monomers, SBAFP-m1 and RGAFP-m1, from naturally occurring ice binding proteins found in insects and plants. A highly idealized, in-register dimer interface was designed and experimentally synthesized and demonstrated to form micron long amyloid fibrils (Chapter 2). The strength and resistance of the designer amyloid fibrils formed by SBAFP-m1 were probed in Chapter 3. Most notably, the ultimate tensile strength of SBAFP-m1 fibrils was experimentally determined to be 2.1 +/- 1.7 GPa, on par with that of naturally occurring amyloid fibrils in literature and steel. The fibrils were found to maintain their beta-sheet structure over a wide range of temperatures, from - 80 °C to 90 °C. Fibrils were resistant to common protein denaturants like 8M urea, 2.5 M guanidine hydrochloride, 2.5 M NaCl, organic solvents (methanol, ethanol, isopropanol and acetone), and across the pH range two to 11. SBAFP-m1 was mutated to add a 5x cysteine tag to the N-terminus, allowing for gold nanoparticle conjugation along the fibril axis (Chapter 4). The gold-conjugated fibrils were then enhanced with silver to produce nanowires. Various attempts to selectively synthesize heterogeneous fibrils from SBAFP-m1 mutants were attempted in Chapter 5. An attempt to de-stabilize the homogeneous fibril assembly through unfavorable homogeneous protein interactions was not

Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

Science.gov (United States)

Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

2012-11-01

The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

DEFF Research Database (Denmark)

Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

2002-01-01

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

Crystal structure of the human beta2 adrenergic G-protein-coupled receptor

DEFF Research Database (Denmark)

Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Rosenbaum, Daniel M

2007-01-01

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was ...

A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

DEFF Research Database (Denmark)

Boldyreff, B; Issinger, O G

1997-01-01

In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

DEFF Research Database (Denmark)

Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

2007-01-01

-linked lymphocytic choriomeningitis virus (LCMV)-derived epitopes was long-lived and protective. Notably, in contrast to full-length protein, the response elicited with the beta(2)-microglobulin-linked LCMV-derived epitope was CD4(+) T-cell independent. Furthermore, virus-specific CD8(+) T cells primed...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T-cell...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...

Clinical significance of serum beta2-microglobulin and carcinoembryonic antigen in patients with cervical cancer treated with irradiation

International Nuclear Information System (INIS)

Sato, Yoshiaki; Tottori, Kosei; Higuchi, Akira; Takeuchi, Shoshichi

1979-01-01

β 2 -microglobulin (β 2 -m) is a small polypeptide present in all body fluids. In recent years much attention has been paid to the high prevalence of elevated serum β 2 -m levels in malignancy. The purpose of this study was to investigate the clinical significance of β 2 -m in patients with cervical cancer treated with irradiation therapy. For comparison, as an established tumor marker, CEA was assayed in the same samples. The results were as follows: 1. Both β 2 -m and CEA values seemed to relate to the clinical stage of tumor growth. 2. The patients who ended in a poor prognosis retrospectively had the significantly elevated CEA and β 2 -m values compared to those of the patients with good prognosis. 3. CEA or β 2 -m assay by themselves had not so large value in the diagnosis of cervical cancer, but an increase in number of positive cases was obtained when the two markers were jointly considered. Because when the CEA was negative, the β 2 -m assay was capable of compensating for this deficiency. (author)

Radioimmunoassay of serum β2-microglobulin in donor's blood

International Nuclear Information System (INIS)

Yin Shihua; Song Shiyun; Li Kelin; Chen Guanglian; Liu Fengmin

1993-01-01

Serum β 2 -microglobulin (β 2 -MG) was tested by radioimmunoassay in 149 donors' and 54 healthy volunteers' blood. The results were 203 +- 33.0 nmol/l and 176 +- 26.2 nmol/l, respectively. There was significant difference statistically between them (P 2 -MG content. In order to increase the quality of donated blood and to keep the health of blood donor, it is suggested that the high content of serum β 2 -MG is the indicator of too frequent blood donating. The results also showed that the content of β 2 -MG in donor's blood is not a normal reference value

Serum beta2-microglobulin values among healthy Brazilians using a DPC IMMULITE® assay Valores de beta2-microglobulina em brasileiros saudáveis usando o sistema automatizado DPC IMMULITE®

Directory of Open Access Journals (Sweden)

Fabíola Branco Filippin

2005-02-01

Full Text Available PURPOSE: The present study was designed to determine the normal range of serum beta 2-microglobulin (Sb2M levels among healthy volunteers in Brazil. Levels of Sb2M are elevated in human immunodeficiency virus (HIV-infected patients and have been shown to be the best predictor of HIV infection status and of some malignant disorders, especially multiple myeloma. In order to achieve its optimal use in Brazilian clinical diagnosis, an adequate reference interval study was performed for Sb2M IMMULITE® assay, based on the fact that its reference range limits were evaluated among European populations. METHODS: Ninety-six healthy blood donors were evaluated, and Sb2M levels were measured by chemiluminescent enzyme immunoassay using the IMMULITE® automated analyzer. RESULTS: A normal range of Sb2M values, established by a nonparametric statistical method, was 1.05 to 3.9 mg/mL, with the upper limit being higher than that reported elsewhere. CONCLUSIONS: This study presented new data indicating that there is a significant difference between the current reference limits for Sb2M IMMULITE® assay and those found in Brazil, providing evidence that significant differences in range of normal values may occur among different populations, and that these new values should be considered for Brazilian individuals.OBJETIVO: O objetivo do presente estudo foi determinar valores de referência de beta2-Microglobulina sérica (Sb2M em voluntários saudáveis. Sabe-se que tal parâmetro apresenta-se elevado em pacientes infectados com o vírus da imunodeficiência humana (HIV e tem se mostrado melhor marcador da infecção por HIV e de desordens malignas, especialmente mieloma múltiplo. De forma a se obter o melhor diagnóstico clínico, um intervalo de referência adequado de Sb2M para a população brasileira foi determinado empregando-se o ensaio IMMULITE®; já que este tem como parâmetro uma faixa de referência determinada a partir de populações europ

Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

Science.gov (United States)

Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

2000-09-15

The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

Dimers of beta 2-glycoprotein I mimic the in vitro effects of beta 2-glycoprotein I-anti-beta 2-glycoprotein I antibody complexes

NARCIS (Netherlands)

Lutters, B. C.; Meijers, J. C.; Derksen, R. H.; Arnout, J.; de Groot, P. G.

2001-01-01

Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a

Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1.

Science.gov (United States)

Chang, Yang; Tesco, Giuseppina; Jeong, William J; Lindsley, Loren; Eckman, Elizabeth A; Eckman, Christopher B; Tanzi, Rudolph E; Guénette, Suzanne Y

2003-12-19

Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of alpha-secretase-derived APP (APPs alpha). Increased beta-amyloid (A beta) generation was also observed in cells showing the FE65-dependent increase in APPs alpha. To understand the mechanism for the observed increase in both A beta and APPs alpha given that alpha-secretase cleavage of a single APP molecule precludes A beta generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates gamma-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A beta 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on gamma-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

Increasing protein stability by improving beta-turns.

Science.gov (United States)

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

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Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Bani-Yaghoub, Mahmud [Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Taylor, Rod [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Johnston, Linda J., E-mail: Linda.Johnston@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada); Pezacki, John Paul, E-mail: John.Pezacki@nrc-cnrc.gc.ca [Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 (Canada)

2009-04-24

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

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Emily B Martin

Full Text Available Monoclonal free light chain (LC proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL amyloidosis and multiple myeloma (MM. Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM.We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05 than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains.The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

A novel system for continuous protein refolding and on-line capture by expanded bed adsorption

DEFF Research Database (Denmark)

Ferré, Henrik; Ruffet, E; Nielsen, L.L.B

2005-01-01

A novel two-step protein refolding strategy has been developed, where continuous renaturation-by-dilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta(2)-microglobulin (HAT......-h beta(2)m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time...... of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-h beta(2)m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions...

Determination of Clara cell protein urinary elimination as a marker of tubular dysfunction.

Science.gov (United States)

Martín-Granado, Ascensión; Vázquez-Moncholí, Carmen; Luis-Yanes, María Isabel; López-Méndez, Marisela; García-Nieto, Víctor

2009-04-01

Clara cell 16-kDa protein (CC16) is a protein expressed primarily by the bronchial cells. It is rapidly eliminated by glomerular filtration, reabsorbed almost entirely, and catabolized in proximal tubule cells. To date, normal values for urinary CC16 in healthy children have not been determined. We have studied 63 pediatric patients (mean age 8.17 +/- 3.91 years) and 31 healthy children (control group; mean age 8.83 +/- 3.65 years). In the control group, the CC16/creatinine ratio was 1.22 +/- 1.52 microg/g. In 16 out of 31 control children, the value of the ratio was zero. Fourteen patients (22.2%) showed a high CC16/creatinine ratio; in contrast, among these same patients, the ratio N-acetyl-beta-D: -glucosaminidase (NAG)/creatinine was elevated in seven cases (11.1%) and the ratio beta2-microglobulin/creatinine was elevated in seven cases (11.1%). The three parameters were in agreement in 51 patients (80.9%). Among the patients, the CC16/creatinine ratio was correlated with both the beta2-microglobulin/creatinina ratio (r = 0.76, P < 0.001) and the NAG/creatinine ratio (r = 0.6, P < 0.001). Our findings indicate that CC16 is a good marker of proximal tubular function in childhood. The highest observed values were in children with proximal tubulopathies, in children with chronic renal failure, and in those treated with cyclosporine.

Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

DEFF Research Database (Denmark)

Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

2009-01-01

Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin....... The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas...

In silico and in vitro characterization of anti-amyloidogenic activity of vitamin K3 analogues for Alzheimer's disease.

Science.gov (United States)

Huy, Pham Dinh Quoc; Yu, Yao-Chung; Ngo, Son Tung; Thao, Tran Van; Chen, Chin-Piao; Li, Mai Suan; Chen, Yi-Cheng

2013-04-01

Aggregation of amyloid-beta (Aβ) has been proposed as the main cause of Alzheimer's disease (AD). Vitamin K deficiency has been linked to the pathogenesis of AD. Therefore, 15 synthesized vitamin K3 (VK3) analogues were studied for their anti-amyloidogenic activity. Biological and spectroscopic assays were used to characterize the effect of VK3 analogues on amyloidogenic properties of Aβ, such as aggregation, free radical formation, and cell viability. Molecular dynamics simulation was used to calculate the binding affinity and mode of VK3 analogue binding to Aβ. Both numerical and experimental results showed that several VK3 analogues, including VK3-6, VK3-8, VK3-9, VK3-10, and VK3-224 could effectively inhibit Aβ aggregation and conformational conversion. The calculated inhibition constants were in the μM range for VK3-10, VK3-6, and VK3-9 which was similar to the IC50 of curcumin. Cell viability assays indicated that VK3-9 could effectively reduce free radicals and had a protective effect on cytotoxicity induced by Aβ. The results clearly demonstrated that VK3 analogues could effectively inhibit Aβ aggregation and protect cells against Aβ induced toxicity. Modified VK3 analogues can possibly be developed as effective anti-amyloidogenic drugs for the treatment of AD. VK3 analogues effectively inhibit Aβ aggregation and are highly potent as anti-amyloidogenic drugs for therapeutic treatment of AD. Copyright © 2012 Elsevier B.V. All rights reserved.

BetaTPred: prediction of beta-TURNS in a protein using statistical algorithms.

Science.gov (United States)

Kaur, Harpreet; Raghava, G P S

2002-03-01

beta-turns play an important role from a structural and functional point of view. beta-turns are the most common type of non-repetitive structures in proteins and comprise on average, 25% of the residues. In the past numerous methods have been developed to predict beta-turns in a protein. Most of these prediction methods are based on statistical approaches. In order to utilize the full potential of these methods, there is a need to develop a web server. This paper describes a web server called BetaTPred, developed for predicting beta-TURNS in a protein from its amino acid sequence. BetaTPred allows the user to predict turns in a protein using existing statistical algorithms. It also allows to predict different types of beta-TURNS e.g. type I, I', II, II', VI, VIII and non-specific. This server assists the users in predicting the consensus beta-TURNS in a protein. The server is accessible from http://imtech.res.in/raghava/betatpred/

Towards Alzheimer's beta-amyloid vaccination.

Science.gov (United States)

Frenkel, D; Solomon, B

2001-01-01

Beta-amyloid pathology, the main hallmark of Alzheimer's disease (AD), has been linked to its conformational status and aggregation. We recently showed that site-directed monoclonal antibodies (mAbs) towards the N-terminal region of the human beta-amyloid peptide bind to preformed beta-amyloid fibrils (Abeta), leading to disaggregation and inhibition of their neurotoxic effect. Here we report the development of a novel immunization procedure to raise effective anti-aggregating amyloid beta-protein (AbetaP) antibodies, using as antigen filamentous phages displaying the only EFRH peptide found to be the epitope of these antibodies. Due to the high antigenicity of the phage no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies in animals model, that exhibit identity to human AbetaP. Such antibodies are able to sequester peripheral AbetaP, thus avoiding passage through the blood brain barrier (BBB) and, as recently shown in a transgenic mouse model, to cross the BBB and dissolve already formed beta-amyloid plaques. To our knowledge, this is the first attempt to use as a vaccine a self-anti-aggregating epitope displayed on a phage, and this may pave the way to treat abnormal accumulation-peptide diseases, such as Alzheimer's disease or other amyloidogenic diseases. Copyright 2001 The International Association for Biologicals.

Anti-inflammatory and anti-amyloidogenic effects of a small molecule, 2,4-bis(p-hydroxyphenyl-2-butenal in Tg2576 Alzheimer’s disease mice model

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Jin Peng

2013-01-01

Full Text Available Abstract Background Alzheimer’s disease (AD is pathologically characterized by excessive accumulation of amyloid-beta (Aβ fibrils within the brain and activation of astrocytes and microglial cells. In this study, we examined anti-inflammatory and anti-amyloidogenic effects of 2,4-bis(p-hydroxyphenyl-2-butenal (HPB242, an anti-inflammatory compound produced by the tyrosine-fructose Maillard reaction. Methods 12-month-old Tg2576 mice were treated with HPB242 (5 mg/kg for 1 month and then cognitive function was assessed by the Morris water maze test and passive avoidance test. In addition, western blot analysis, Gel electromobility shift assay, immunostaining, immunofluorescence staining, ELISA and enzyme activity assays were used to examine the degree of Aβ deposition in the brains of Tg2576 mice. The Morris water maze task was analyzed using two-way ANOVA with repeated measures. Otherwise were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. Results Treatment of HPB242 (5 mg/kg for 1 month significantly attenuated cognitive impairments in Tg2576 transgenic mice. HPB242 also prevented amyloidogenesis in Tg2576 transgenic mice brains. This can be evidenced by Aβ accumulation, BACE1, APP and C99 expression and β-secretase activity. In addition, HPB242 suppresses the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 as well as activation of astrocytes and microglial cells. Furthermore, activation of nuclear factor-kappaB (NF-κB and signal transducer and activator of transcription 1/3 (STAT1/3 in the brain was potently inhibited by HPB242. Conclusions Thus, these results suggest that HPB242 might be useful to intervene in development or progression of neurodegeneration in AD through its anti-inflammatory and anti-amyloidogenic effects.

Interactions between two beta-sheets. Energetics of beta/beta packing in proteins.

Science.gov (United States)

Chou, K C; Némethy, G; Rumsey, S; Tuttle, R W; Scheraga, H A

1986-04-20

The analysis of the interactions between regularly folded segments of the polypeptide chain contributes to an understanding of the energetics of protein folding. Conformational energy-minimization calculations have been carried out to determine the favorable ways of packing two right-twisted beta-sheets. The packing of two five-stranded beta-sheets was investigated, with the strands having the composition CH3CO-(L-Ile)6-NHCH3 in one beta-sheet and CH3CO-(L-Val)6-NHCH3 in the other. Two distinct classes of low-energy packing arrangements were found. In the class with lowest energies, the strands of the two beta-sheets are aligned nearly parallel (or antiparallel) with each other, with a preference for a negative orientation angle, because this arrangement corresponds to the best complementary packing of the two twisted saddle-shaped beta-sheets. In the second class, with higher interaction energies, the strands of the two beta-sheets are oriented nearly perpendicular to each other. While the surfaces of the two beta-sheets are not complementary in this arrangement, there is good packing between the corner of one beta-sheet and the interior part of the surface of the other, resulting in a favorable energy of packing. Both classes correspond to frequently observed orientations of beta-sheets in proteins. In proteins, the second class of packing is usually observed when the two beta-sheets are covalently linked, i.e. when a polypeptide strand passes from one beta-sheet to the other, but we have shown here that a large contribution to the stabilization of this packing arrangement arises from noncovalent interactions.

Resveratrol and Amyloid-Beta: Mechanistic Insights

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Yongming Jia

2017-10-01

Full Text Available The amyloid-beta (Aβ hypothesis that dyshomeostasis between Aβ production and clearance is a very early, key molecular factor in the etiology of Alzheimer’s disease (AD has been proposed and examined in the AD research field. Scientists have focused on seeking natural products or drugs to influence the dynamic equilibrium of Aβ, targeting production and clearance of Aβ. There is emerging evidence that resveratrol (Res, a naturally occurring polyphenol mainly found in grapes and red wine, acts on AD in numerous in vivo and in vitro models. Res decreases the amyloidogenic cleavage of the amyloid precursor protein (APP, enhances clearance of amyloid beta-peptides, and reduces Aβ aggregation. Moreover, Res also protects neuronal functions through its antioxidant properties. This review discusses the action of Res on Aβ production, clearance and aggregation and multiple potential mechanisms, providing evidence of the useful of Res for AD treatment.

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

DEFF Research Database (Denmark)

Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

2007-01-01

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

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Spens, Erika; Häggström, Lena

2009-05-20

NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

Science.gov (United States)

Cheng, Sara Y; Chou, George; Buie, Creighton; Vaughn, Mark W; Compton, Campbell; Cheng, Kwan H

2016-03-01

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on

Beta-structures in fibrous proteins.

Science.gov (United States)

Kajava, Andrey V; Squire, John M; Parry, David A D

2006-01-01

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

OpenAIRE

Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

1995-01-01

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

OpenAIRE

Heyduk, T; Heyduk, E; Severinov, K; Tang, H; Ebright, R H

1996-01-01

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta ...

EGCG Inhibited Lipofuscin Formation Based on Intercepting Amyloidogenic β-Sheet-Rich Structure Conversion.

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Shuxian Cai

Full Text Available Lipofuscin (LF is formed during lipid peroxidation and sugar glycosylation by carbonyl-amino crosslinks with biomacrolecules, and accumulates slowly within postmitotic cells. The environmental pollution, modern dietary culture and lifestyle changes have been found to be the major sources of reactive carbonyl compounds in vivo. Irreversible carbonyl-amino crosslinks induced by carbonyl stress are essentially toxiferous for aging-related functional losses in modern society. Results show that (--epigallocatechin gallate (EGCG, the main polyphenol in green tea, can neutralize the carbonyl-amino cross-linking reaction and inhibit LF formation, but the underlying mechanism is unknown.We explored the mechanism of the neutralization process from protein, cell, and animal levels using spectrofluorometry, infrared spectroscopy, conformation antibodies, and electron microscopy. LF demonstrated an amyloidogenic β-sheet-rich with antiparallel structure, which accelerated the carbonyl-amino crosslinks formation and disrupted proteolysis in both PC12 cells and D-galactose (D-gal-induced brain aging mice models. Additionally, EGCG effectively inhibited the formation of the amyloidogenic β-sheet-rich structure of LF, and prevented its conversion into toxic and on-pathway aggregation intermediates, thereby cutting off the carbonyl-amino crosslinks.Our study indicated that the amyloidogenic β-sheet structure of LF may be the core driving force for carbonyl-amino crosslinks further formation, which mediates the formation of amyloid fibrils from native state of biomacrolecules. That EGCG exhibits anti-amyloidogenic β-sheet-rich structure properties to prevent the LF formation represents a novel strategy to impede the development of degenerative processes caused by ageing or stress-induced premature senescence in modern environments.

Interrelationship of βeta-2 microglobulin, blood urea nitrogen and creatinine in streptozotocin -induced diabetes mellitus in rabbits

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Shahram Javadi

2013-12-01

Full Text Available Measurement of serum creatinine (Cr and blood urea nitrogen (BUN are used as indicators of glomerular filtration rate. The increased levels of these biomarkers are usually detectable at advanced stages of kidney complications. The aim of this study was to find the interrelationship of beta-2 microglobulin (β2M, BUN and Cr in streptozotocin (STZ-induced diabetes mellitus in rabbits. Diabetes was induced by a single intraperitoneal (IP injection of 65 mg kg-1 of STZ in rabbits. The levels of serum insulin, glucose and three above mentioned biomarkers were measured one day before (day -1 and on days 1-3 after injection of STZ and continued weekly to the end of the experiment (12 weeks. A statistically significant increase of serum β2M, BUN, Cr and glucose levels, and a significant decrease of insulin levels were observed in diabetic animals. However, β2M levels increased as early as one day after STZ injection compared to Cr and BUN that elevated at day two, suggesting a probable diagnostic advantage of β2M over currently used biomarkers in diabetic related kidney complications.

Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis

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Bastus Neus

2008-01-01

Full Text Available Abstract Peptide-Gold nanoparticles selectively attached to β-amyloid protein (Aβ amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the Aβ aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer’s disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer’s disease.

Ranking beta sheet topologies of proteins

DEFF Research Database (Denmark)

Fonseca, Rasmus; Helles, Glennie; Winter, Pawel

2010-01-01

One of the challenges of protein structure prediction is to identify long-range interactions between amino acids. To reliably predict such interactions, we enumerate, score and rank all beta-topologies (partitions of beta-strands into sheets, orderings of strands within sheets and orientations...... of paired strands) of a given protein. We show that the beta-topology corresponding to the native structure is, with high probability, among the top-ranked. Since full enumeration is very time-consuming, we also suggest a method to deal with proteins with many beta-strands. The results reported...... in this paper are highly relevant for ab initio protein structure prediction methods based on decoy generation. The top-ranked beta-topologies can be used to find initial conformations from which conformational searches can be started. They can also be used to filter decoys by removing those with poorly...

The signaling cascades of Ganoderma lucidum extracts in stimulating non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y cell lines.

Science.gov (United States)

Pinweha, Sirinthorn; Wanikiat, Payong; Sanvarinda, Yupin; Supavilai, Porntip

2008-12-19

Ganoderma lucidum (GL) is a medicinal mushroom that possesses various pharmacological properties which are also documented in the ancient reports where GL is praised for its effects on the promotion of health and longevity. In this study, we have investigated the effect of GL mycelia extracts on the non-amyloidogenic protein secretion (sAPPalpha) and the amyloid precursor protein (APP) expression in SH-SY5Y neuroblastoma cells. In order to characterize the signaling pathway which mediates GL-enhanced sAPPalpha secretion, we used inhibitors of nerve growth factor (NGF) signaling pathways, phosphatidylinositol 3 kinase (PI3K), phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK1/2), to block GL-mediated sAPPalpha secretion as well as ERK1/2 and PKC activation by using Western blot analysis. Our results provided for the first time evidence that GL mycelia extracts increased APP expression and promoted sAPPalpha secretion. In addition, GL extracts activated ERK1/2 and PKC phosphorylation. The complex signaling cascades of PI3K and ERK may be responsible for GL-mediated sAPPalpha secretion.

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

DEFF Research Database (Denmark)

Raaf, Jennifer; Brunstein, Elena; Issinger, Olaf-Georg

2008-01-01

. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability......, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed...

Molecular simulations of beta-amyloid protein near hydrated lipids (PECASE).

Energy Technology Data Exchange (ETDEWEB)

Thompson, Aidan Patrick; Han, Kunwoo (Texas A& M University, College Station, TX); Ford, David M. (Texas A& M University, College Station, TX)

2005-12-01

We performed molecular dynamics simulations of beta-amyloid (A{beta}) protein and A{beta} fragment(31-42) in bulk water and near hydrated lipids to study the mechanism of neurotoxicity associated with the aggregation of the protein. We constructed full atomistic models using Cerius2 and ran simulations using LAMMPS. MD simulations with different conformations and positions of the protein fragment were performed. Thermodynamic properties were compared with previous literature and the results were analyzed. Longer simulations and data analyses based on the free energy profiles along the distance between the protein and the interface are ongoing.

The proteome response to amyloid protein expression in vivo.

Directory of Open Access Journals (Sweden)

Ricardo A Gomes

Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin

OpenAIRE

Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

2012-01-01

A crystal of nurse shark β2-microglobulin diffracted to 2.3 Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å3 Da−1, corresponding to 62.5% solvent content.

Exploring the structural diversity in inhibitors of α-synuclein amyloidogenic folding, aggregation and neurotoxicity

Science.gov (United States)

Das, Sukanya; Pukala, Tara L.; Smid, Scott D.

2018-05-01

Aggregation of α-Synuclein (αS) protein to amyloid fibrils is a neuropathological hallmark of Parkinson’s disease (PD). Growing evidence suggests that extracellular αS aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular αS aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of αSA53T, a more aggregation-prone αS mutant found in familial PD. For the first time, we investigated the αS anti-amyloid activity of semi-synthetic flavonoid 2', 3', 4' trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favourable binding with αSA53T. All seven compounds inhibited αSA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native αSA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of αSA53T following 48 hrs incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated αSA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated αSA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of αSA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that i

Expression, purification, crystallization and preliminary X-ray diffraction analysis of grass carp β2-microglobulin

International Nuclear Information System (INIS)

Chen, Weihong; Chu, Fuliang; Peng, Hao; Zhang, Jianhua; Qi, Jianxun; Jiang, Fan; Xia, Chun; Gao, Feng

2008-01-01

Grass carp β 2 -microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. β 2 -Microglobulin (β 2 m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β 2 m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β 2 m (Ctid-β 2 m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da −1 and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β 2 m as a model

Ellagic acid promotes A{beta}42 fibrillization and inhibits A{beta}42-induced neurotoxicity

Energy Technology Data Exchange (ETDEWEB)

Feng, Ying [Department of Histology and Embryology, College of Basic Medical Science, China Medical University, Shenyang 110001 (China); Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China); Yang, Shi-gao; Du, Xue-ting; Zhang, Xi; Sun, Xiao-xia; Zhao, Min [Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China); Sun, Gui-yuan, E-mail: sungy2004@sohu.com [Department of Histology and Embryology, College of Basic Medical Science, China Medical University, Shenyang 110001 (China); Liu, Rui-tian, E-mail: rtliu@tsinghua.edu.cn [Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China)

2009-12-25

Smaller, soluble oligomers of {beta}-amyloid (A{beta}) play a critical role in the pathogenesis of Alzheimer's disease (AD). Selective inhibition of A{beta} oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against A{beta} neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on A{beta}42 aggregation and neurotoxicity in vitro. EA promoted A{beta} fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited A{beta} aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in A{beta}42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic A{beta} aggregates to render them harmless, our MTT results showed that EA could significantly reduce A{beta}42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.

The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

NARCIS (Netherlands)

Nilsson, Maria; Wasylik, Sylwia; Mörgelin, Matthias; Olin, Anders I.; Meijers, Joost C. M.; Derksen, Ronald H. W. M.; de Groot, Philip G.; Herwald, Heiko

2008-01-01

During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices.

Science.gov (United States)

Zanuy, David; Gunasekaran, Kannan; Lesk, Arthur M; Nussinov, Ruth

2006-04-21

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

Science.gov (United States)

Albers, Shawn; Inthathirath, Fatima; Gill, Sandeep K; Winick-Ng, Warren; Jaworski, Ewa; Wong, Daisy Y L; Gros, Robert; Rylett, R Jane

2014-09-01

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to

Role of residual kidney function and convective volume on change in β2-microglobulin levels in hemodiafiltration patients

NARCIS (Netherlands)

E.L. Penne (Lars); N.C. van der Weerd (Neelke); P.J. Blankestijn (Peter); M.A. van den Dorpel (Marinus); M.P.C. Grooteman (Muriel); M.J. Nubé (Menso); P.M. ter Wee (Piet); R. Lévesque (Renée); M.L. Bots (Michiel)

2010-01-01

textabstractBackground and objectives: Removal of β2-microglobulin (β2M) can be increased by adding convective transport to hemodialysis (HD). The aim of this study was to investigate the change in β2M levels after 6-mo treatment with hemodiafiltration (HDF) and to evaluate the role of residual

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

Energy Technology Data Exchange (ETDEWEB)

Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

2011-11-15

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

Directory of Open Access Journals (Sweden)

Mohammed Akli eAyoub

2013-12-01

Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

Predictive value of plasma β2-microglobulin on human body function and senescence.

Science.gov (United States)

Dong, X-M; Cai, R; Yang, F; Zhang, Y-Y; Wang, X-G; Fu, S-L; Zhang, J-R

2016-06-01

To explore the correlation between plasma β2-microglobulin (β2-MG) as senescence factor with age, heart, liver and kidney function as well as the predictive value of β2-MG in human metabolism function and senescence. 387 cases of healthy people of different ages were selected and the automatic biochemical analyzer was used to test β2-MG in plasma based on immunoturbidimetry and also all biochemical indexes. The correlation between β2-MG and age, gender and all biochemical indexes was analyzed. β2-MG was positively correlated to age, r = 0.373; and the difference was of statistical significance (p human body function and anti-senescence and have significant basic research and clinical guidance values.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin

International Nuclear Information System (INIS)

Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

2012-01-01

A crystal of nurse shark β 2 -microglobulin diffracted to 2.3 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å 3 Da −1 , corresponding to 62.5% solvent content. β 2 -Microglobulin (β 2 m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide–MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β 2 m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β 2 m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3 2 21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β 2 m and IgSF in the AIS

Plasma pre beta-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus : Role of phospholipid transfer protein

NARCIS (Netherlands)

Dallinga-Thie, G. M.; van Tol, A.; Dullaart, R. P. F.

Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-beta-HDL, generation in vitro. We determined the effect of atorvastatin on pre-beta-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-beta-HDL formation as well as

Beta-2 Microglobulin Tumor Marker

Science.gov (United States)

... T. J. (© 2011). Mosby's Diagnostic and Laboratory Test Reference 10th Edition: Mosby, Inc., Saint Louis, MO. Pp 680-682. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics . Burtis CA, Ashwood ER, Bruns ...

A novel venom protein of the Asian bee (Apis cerana indica with an affinity to human α1-microglobulin

Directory of Open Access Journals (Sweden)

Rosdiana Natzir

1999-01-01

Full Text Available Bee stings are a common health problem throughout the world and can sometimes result in fatal anaphylactic reactions. We have studied Asian bee (Apis cerana indica, Apis cerana nigrocincta and Apis dorsata venoms and have discovered a novel protein with a molecular size of 50 kDa (p50, as shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which has not been reported in the venom of the Western honey-bee, Apis mellifera (AM. The p50 protein showed a unique affinity to human α1-microglobulin (α1-m. As a result, p50 was purified using an affinity column with α1-m. The p50 protein was further purified by an affinity column with a monoclonal antibody raised against p50 in mice. The p50 protein induced an inflammatory reaction following injection into mouse ear; that is, degranulation of mast cells, edema, hyperemia and hyperpermeation of the local capillaries were observed. The reaction was very similar to that seen when phospholipase A2 of AM, a representative bee venom, was administered by injection. The inflammatory reaction induced by p50 was completely inhibited by mixing p50 with α1-m prior to injection. These results indicate that p50 is a unique venom component of the Asian bee that induces the inflammatory reaction and that human α1-m may be involved as a protective mechanism against bee stings of at least some Asian bee species.

beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

DEFF Research Database (Denmark)

Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

2000-01-01

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta).

NARCIS (Netherlands)

Drake, P.G.; Peters, G.H.; Andersen, H.S.; Hendriks, W.J.A.J.; Moller, N.P.

2003-01-01

Islet-cell antigen 512 (IA-2) and phogrin (IA-2beta) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent

Immunolocalization of keratin-associated beta-proteins (beta-keratins) in pad lamellae of geckos suggest that glycine-cysteine-rich proteins contribute to their flexibility and adhesiveness.

Science.gov (United States)

Alibardi, Lorenzo

2013-03-01

The epidermis of digital pads in geckos comprises superficial microornamentation from the oberhautchen layer that form long setae allowing these lizards to climb vertical surfaces. The beta-layer is reduced in pad lamellae but persists up to the apical free margin. Setae are made of different proteins including keratin-associated beta-proteins, formerly indicated as beta-keratins. In order to identify specific setal proteins the present ultrastructural study on geckos pad lamellae analyzes the immunolocalization of three beta-proteins previously found in the epidermis and adhesive setae of the green anolis. A protein rich in glycine but poor in cysteine (HgG5-like) is absent or masked in gecko pad lamellae. Another protein rich in glycine and cysteine (HgGC3-like) is weakly present in setae, oberhautchen and beta-layer. A glycine and cysteine medium rich beta-protein (HgGC10-like) is present in the lower part of the beta-layer but is absent in the oberhautchen, setae, and mesos layer. The latter two proteins may form intermolecular bonds that contribute to the flexibility of the corneous material sustaining the setae. The pliable alpha-layer present beneath the thin beta-layer and in the hinge region of the pad lamellae also contains HgGC10-like proteins. Based on the possibility that some HgGC3-like or other cys-rich beta-proteins are charged in the setae it is suggested that their charges influence the mechanism of adhesion increasing the induction of dipoles on the substrate and enhancing attractive van der Waals forces. Copyright © 2013 Wiley Periodicals, Inc.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of grass carp β{sub 2}-microglobulin

Energy Technology Data Exchange (ETDEWEB)

Chen, Weihong [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Chu, Fuliang [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Peng, Hao [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Zhang, Jianhua [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Qi, Jianxun; Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Xia, Chun, E-mail: xiachun@cau.edu.cn [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Gao, Feng, E-mail: xiachun@cau.edu.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China)

2008-03-01

Grass carp β{sub 2}-microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. β{sub 2}-Microglobulin (β{sub 2}m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β{sub 2}m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β{sub 2}m (Ctid-β{sub 2}m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da{sup −1} and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β{sub 2}m as a model.

Substitution of proline32 by α-methylproline preorganizes β2-microglobulin for oligomerization but not for aggregation into amyloids.

Science.gov (United States)

Torbeev, Vladimir; Ebert, Marc-Olivier; Dolenc, Jozica; Hilvert, Donald

2015-02-25

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human β2-microglobulin (β2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a β2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]β2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type β2m, [MePro32]β2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type β2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]β2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial β2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of β2m amyloid structures.

Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions.

Science.gov (United States)

Nyström, Sofie; Hammarström, Per

2015-05-11

Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

Kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin as prognostic markers in idiopathic membranous nephropathy.

Science.gov (United States)

Maas, Rutger Jh; van den Brand, Jan Ajg; Waanders, Femke; Meijer, Esther; Goor van, Harry; Peters, Hilde P; Hofstra, Julia M; Wetzels, Jack Fm

2016-01-01

Urinary excretion of alpha-1-microglobulin and beta-2-microglobulin reflects tubular damage and predicts outcome in patients with idiopathic membranous nephropathy with reasonable accuracy. Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin are novel biomarkers of tubular damage. We investigated if these markers could improve prediction of outcome in idiopathic membranous nephropathy. We measured kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in urine samples from patients with idiopathic membranous nephropathy, who had nephrotic proteinuria and normal renal function. Excretion of alpha-1-microglobulin and beta-2-microglobulin had been measured previously. Progression was defined as a serum creatinine rise >30%, a rise in serum creatinine to an absolute value of ≥135 µmol/L, or a clinical decision to start immunosuppressive therapy. Remission was defined as proteinuria 50% reduction from baseline. Sixty-nine patients were included. Median follow-up was 35 months (interquartile range 18-63 months). Progression occurred in 30 patients (44%), and spontaneous remission in 36 (52%). Kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin excretion rates were significantly correlated with each other, and with alpha-1-microglobulin and beta-2-microglobulin. The areas under the receiver operating characteristic curves for progression were 0.75 (0.62-0.87) for kidney injury molecule-1 and 0.74 (0.62-0.87) for neutrophil gelatinase-associated lipocalin. In multivariate analysis with either alpha-1-microglobulin and beta-2-microglobulin, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin did not independently predict outcome. Kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin excretion rates correlated with excretion rates of other tubular damage markers and predicted outcome in patients with idiopathic membranous nephropathy. They did not add prognostic value

Increased serum ß2-microglobulin is associated with clinical and immunological markers of disease activity in systemic lupus erythematosus patients

DEFF Research Database (Denmark)

Hermansen, M-L F; Hummelshøj, L; Lundsgaard, Dorte

2012-01-01

The objective of this study was to explore the relationship between serum levels of ß2-microglobulin (ß2MG), which some studies suggest reflect disease activity in systemic lupus erythematosus (SLE), and various clinical and immunological markers of disease activity in SLE. Twenty-six SLE patients...

Coagulation Factors Test

Science.gov (United States)

... Blood Testing Alpha-1 Antitrypsin Alpha-fetoprotein (AFP) Tumor Marker AMAS Aminoglycoside Antibiotics Ammonia Amniocentesis Amylase ANCA/MPO/ ... Beta-2 Microglobulin Kidney Disease Beta-2 Microglobulin Tumor Marker Bicarbonate (Total CO2) Bilirubin Blood Culture Blood Gases ...

Blood and urine β2-microglobulin in pyelonephritic patients with diabetes mellituS

International Nuclear Information System (INIS)

Vasyukova, E.A.; Turiev, G.S.; Gerasimova, N.P.; Ametov, A.S.

1983-01-01

Blood and wine β 2 -microglobulin (B 2 MG) was determined in pyelonephritic patients with diabetes mellitus using the radioimmunoassay method. Different lesions of urogenital system such as pyelonephrit in 27 cases, isolated nephro microangiopathy in 25 cases, Urinary tracks infection in 13 cases Were revealed on the base of a complex examination (radioisotopic renography, dynamic nephroscintigraphy, extretory urography etc). No pathologic changes were discovered in 12 patients. It has been shown that simultaneous determination of blood and wine β 2 -MG allows one to specify a place of primary nephron damage. An increase of β 2 -MG concentration in blood serum is characteristic for violation of glomenular function of kidneys but an increase of its excretion with urine indicates a damage of proximal small channels

Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

DEFF Research Database (Denmark)

Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B

1995-01-01

A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta......-subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

DEFF Research Database (Denmark)

Benedikz, Eirikur; Merz, G S; Schwenk, V

1999-01-01

of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C...... (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders...

Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

Directory of Open Access Journals (Sweden)

Olgica Trenchevska

Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

DEFF Research Database (Denmark)

Sasaki, T; Brakebusch, C; Engel, J

1998-01-01

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

Cellular expression or binding of desLys58-beta2 microglobulin is not dependent on the presence of the tri-molecular MHC class I complex

DEFF Research Database (Denmark)

Wang, M; Corlin, D B; Heegaard, N H H

2008-01-01

-exposure to dbeta2m. Binding of 332-01 antibody could not be displaced by addition of high concentrations of native beta2m. In conclusion, our data indicate that dbeta2m - in contrast to native beta2m - binds to a hitherto unknown cell surface receptor independent of classical MHC class I molecules. As beta2m has...... previously been shown to display biological activities such as the induction of both growth promotion and apoptosis, C1 complement activity, shown to mediate cleavage of beta2m, could be involved in these processes....

Primordial linkage of β2-microglobulin to the MHC.

Science.gov (United States)

Ohta, Yuko; Shiina, Takashi; Lohr, Rebecca L; Hosomichi, Kazuyoshi; Pollin, Toni I; Heist, Edward J; Suzuki, Shingo; Inoko, Hidetoshi; Flajnik, Martin F

2011-03-15

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.

Human amyloid beta protein gene locus: HaeIII RFLP

Energy Technology Data Exchange (ETDEWEB)

Taylor, J E; Gonzalez-DeWhitt, P A; Fuller, F; Cordell, B; Frossard, P M [California Biotechnology Inc., Mountain View (USA); Tinklenberg, J R; Davies, H D; Eng, L F; Yesavage, J A [Stanford Univ. School of Medicine, Palo Alto, CA (USA)

1988-07-25

A 2.2 kb EcoRI-EcoRI fragment from the 5{prime} end of the human amyloid beta protein cDNA was isolated from a human fibroblast cDNA library and subcloned into pGEM3. HaeIII (GGCC) detects 6 invariant bands at 0.5 kb, 1.0 kb, 1.1 kb, 1.3 kb, 1.4 kb and 1.6 kb and a two-allele polymorphism with bands at either 1.9 kb or 2.1 kb. Its frequency was studied in 50 North Americans. Human amyloid beta protein gene mapped to the long arm of chromosome 21 (21q11.2-21q21) by Southern blot analysis of human-rodent somatic cell hybrids. Co-dominant segregation was observed in two families (15 individuals).

S100A7, a novel Alzheimer's disease biomarker with non-amyloidogenic alpha-secretase activity acts via selective promotion of ADAM-10.

Directory of Open Access Journals (Sweden)

Weiping Qin

Full Text Available Alzheimer's disease (AD is the most common cause of dementia among older people. At present, there is no cure for the disease and as of now there are no early diagnostic tests for AD. There is an urgency to develop a novel promising biomarker for early diagnosis of AD. Using surface-enhanced laser desorption ionization-mass spectrometry SELDI-(MS proteomic technology, we identified and purified a novel 11.7-kDa metal- binding protein biomarker whose content is increased in the cerebrospinal fluid (CSF and in the brain of AD dementia subjects as a function of clinical dementia. Following purification and protein-sequence analysis, we identified and classified this biomarker as S100A7, a protein known to be involved in immune responses. Using an adenoviral-S100A7 expression system, we continued to examine the potential role of S100A7 in AD amyloid neuropathology in in vitro model of AD. We found that the expression of exogenous S100A7 in primary cortico-hippocampal neuron cultures derived from Tg2576 transgenic embryos inhibits the generation of beta-amyloid (Abeta(1-42 and Abeta(1-40 peptides, coincidental with a selective promotion of "non- amyloidogenic" alpha-secretase activity via promotion of ADAM (a disintegrin and metalloproteinase-10. Finally, a selective expression of human S100A7 in the brain of transgenic mice results in significant promotion of alpha-secretase activity. Our study for the first time suggests that S100A7 may be a novel biomarker of AD dementia and supports the hypothesis that promotion of S100A7 expression in the brain may selectively promote alpha-secretase activity in the brain of AD precluding the generation of amyloidogenic peptides. If in the future we find that S1000A7 protein content in CSF is sensitive to drug intervention experimentally and eventually in the clinical setting, S100A7 might be developed as novel surrogate index (biomarker of therapeutic efficacy in the characterization of novel drug agents for

Beta2- and beta3-adrenoceptors activate glucose uptake in chick astrocytes by distinct mechanisms: a mechanism for memory enhancement?

Science.gov (United States)

Hutchinson, Dana S; Summers, Roger J; Gibbs, Marie E

2007-11-01

Isoprenaline, acting at beta-adrenoceptors (ARs), enhances memory formation in single trial discriminated avoidance learning in day-old chicks by mechanisms involving alterations in glucose and glycogen metabolism. Earlier studies of memory consolidation in chicks indicated that beta3-ARs enhanced memory by increasing glucose uptake, whereas beta2-ARs enhance memory by increasing glycogenolysis. This study examines the ability of beta-ARs to increase glucose uptake in chick forebrain astrocytes. The beta-AR agonist isoprenaline increased glucose uptake in a concentration-dependent manner, as did insulin. Glucose uptake was increased by the beta2-AR agonist zinterol and the beta3-AR agonist CL316243, but not by the beta1-AR agonist RO363. In chick astrocytes, reverse transcription-polymerase chain reaction studies showed that beta1-, beta2-, and beta3-AR mRNA were present, whereas radioligand-binding studies showed the presence of only beta2- and beta3-ARs. beta-AR or insulin-mediated glucose uptake was inhibited by phosphatidylinositol-3 kinase and protein kinase C inhibitors, suggesting a possible interaction between the beta-AR and insulin pathways. However beta2- and beta3-ARs increase glucose uptake by two different mechanisms: beta2-ARs via a Gs-cAMP-protein kinase A-dependent pathway, while beta3-ARs via interactions with Gi. These results indicate that activation of beta2- and beta3-ARs causes glucose uptake in chick astrocytes by distinct mechanisms, which may be relevant for memory enhancement.

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

DEFF Research Database (Denmark)

Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

2003-01-01

quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...... synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins...

Quantitative modelling of amyloidogenic processing and its influence by SORLA in Alzheimer's disease.

Science.gov (United States)

Schmidt, Vanessa; Baum, Katharina; Lao, Angelyn; Rateitschak, Katja; Schmitz, Yvonne; Teichmann, Anke; Wiesner, Burkhard; Petersen, Claus Munck; Nykjaer, Anders; Wolf, Jana; Wolkenhauer, Olaf; Willnow, Thomas E

2012-01-04

The extent of proteolytic processing of the amyloid precursor protein (APP) into neurotoxic amyloid-β (Aβ) peptides is central to the pathology of Alzheimer's disease (AD). Accordingly, modifiers that increase Aβ production rates are risk factors in the sporadic form of AD. In a novel systems biology approach, we combined quantitative biochemical studies with mathematical modelling to establish a kinetic model of amyloidogenic processing, and to evaluate the influence by SORLA/SORL1, an inhibitor of APP processing and important genetic risk factor. Contrary to previous hypotheses, our studies demonstrate that secretases represent allosteric enzymes that require cooperativity by APP oligomerization for efficient processing. Cooperativity enables swift adaptive changes in secretase activity with even small alterations in APP concentration. We also show that SORLA prevents APP oligomerization both in cultured cells and in the brain in vivo, eliminating the preferred form of the substrate and causing secretases to switch to a less efficient non-allosteric mode of action. These data represent the first mathematical description of the contribution of genetic risk factors to AD substantiating the relevance of subtle changes in SORLA levels for amyloidogenic processing as proposed for patients carrying SORL1 risk alleles.

GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

DEFF Research Database (Denmark)

Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A

2007-01-01

The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its cr...

Molecular Origin of Gerstmann-Str ussler-Scheinker Syndrome: Insight from Computer Simulation of an Amyloidogenic Prion Peptide

Energy Technology Data Exchange (ETDEWEB)

Diadone, Isabella [University of L' Aquila, L' Aquila, Italy; DiNola, Alfredo [University of Rome; Smith, Jeremy C [ORNL

2011-01-01

Prion proteins become pathogenic through misfolding. Here, we characterize the folding of a peptide consisting of residues 109 122 of the Syrian hamster prion protein (the H1 peptide) and of a more amyloidogenic A117V point mutant that leads in humans to an inheritable form of the Gerstmann-Straeussler-Scheinker syndrome. Atomistic molecular dynamics simulations are performed for 2.5 s. Both peptides lose their -helical starting conformations and assume a -hairpin that is structurally similar in both systems. In each simulation several unfolding/refolding events occur, leading to convergence of the thermodynamics of the conformational states to within 1 kJ/mol. The similar stability of the -hairpin relative to the unfolded state is observed in the two peptides. However, substantial differences are found between the two unfolded states. A local minimum is found within the free energy unfolded basin of the A117V mutant populated by misfolded collapsed conformations of comparable stability to the -hairpin state, consistent with increased amyloidogenicity. This population, in which V117 stabilizes a hydrophobic core, is absent in the wild-type peptide. These results are supported by simulations of oligomers showing a slightly higher stability of the associated structures and a lower barrier to association for the mutated peptide. Hence, a single point mutation carrying only two additional methyl groups is here shown to be responsible for rather dramatic differences of structuring within the unfolded (misfolded) state.

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

Directory of Open Access Journals (Sweden)

Rita Costa

Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta in the brain. Transthyretin (TTR is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14 and (15-42 showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

[Structure analysis of disease-related proteins using vibrational spectroscopy].

Science.gov (United States)

Hiramatsu, Hirotsugu

2014-01-01

Analyses of the structure and properties of identified pathogenic proteins are important for elucidating the molecular basis of diseases and in drug discovery research. Vibrational spectroscopy has advantages over other techniques in terms of sensitivity of detection of structural changes. Spectral analysis, however, is complicated because the spectrum involves a substantial amount of information. This article includes examples of structural analysis of disease-related proteins using vibrational spectroscopy in combination with additional techniques that facilitate data acquisition and analysis. Residue-specific conformation analysis of an amyloid fibril was conducted using IR absorption spectroscopy in combination with (13)C-isotope labeling, linear dichroism measurement, and analysis of amide I band features. We reveal a pH-dependent property of the interacting segment of an amyloidogenic protein, β2-microglobulin, which causes dialysis-related amyloidosis. We also reveal the molecular mechanisms underlying pH-dependent sugar-binding activity of human galectin-1, which is involved in cell adhesion, using spectroscopic techniques including UV resonance Raman spectroscopy. The decreased activity at acidic pH was attributed to a conformational change in the sugar-binding pocket caused by protonation of His52 (pKa 6.3) and the cation-π interaction between Trp68 and the protonated His44 (pKa 5.7). In addition, we show that the peak positions of the Raman bands of the C4=C5 stretching mode at approximately 1600 cm(-1) and the Nπ-C2-Nτ bending mode at approximately 1405 cm(-1) serve as markers of the His side-chain structure. The Raman signal was enhanced 12 fold using a vertical flow apparatus.

Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues

DEFF Research Database (Denmark)

Kaufman, J; Andersen, R; Avila, D

1992-01-01

molecules and the MHC-encoded nonclassical molecules more than CD1 or the class I-like FcR. In contrast, the chicken alpha 3 domain is equally homologous to all alpha 3 domains, to beta 2m and to class II beta 2 domains. For each pair of extracellular domains (alpha 1 vs alpha 2, alpha 3 vs beta 2m...... of small exons in the cytoplasmic region. The cDNA sequences were compared to turkey beta 2m, the apparent allele B-F12 alpha and other vertebrate homologs, using the 2.6 A structure of the human HLA-A2 molecule as a model. Both chicken alpha 1 and alpha 2 domains resemble mammalian classical class I...... the ends of the peptide, two residues that bind CD8, and three residues that are phosphorylated. The positions of the allelic residues are conserved. There are other patches of invariant residues on alpha 1, alpha 2, and beta 2m; these might bind TCR or other molecules involved in class I function...

A novel proteomic biomarker panel as a diagnostic tool for patients with ovarian cancer

DEFF Research Database (Denmark)

Høgdall, Claus; Fung, Eric T; Christensen, Ib J

2011-01-01

Previous reports have shown that the proteomic markers apolipoprotein A1, hepcidin, transferrin, inter-alpha trypsin IV internal fragment, transthyretin, connective-tissue activating protein 3 and beta-2 microglobulin may discriminate between a benign pelvic mass and ovarian cancer (OC). The aim...

Deletion of beta-2-microglobulin ameliorates spinal cord lesion load and promotes recovery of brainstem NAA levels in a murine model of multiple sclerosis.

Science.gov (United States)

Denic, Aleksandar; Pirko, Istvan; Wootla, Bharath; Bieber, Allan; Macura, Slobodan; Rodriguez, Moses

2012-09-01

We used genetic deletion of β2-microglobulin to study the influence of CD8(+) T cells on spinal cord demyelination, remyelination, axonal loss and brainstem N-acetyl aspartate levels during the acute and chronic phases of Theiler's murine encephalomyelitis virus (TMEV) infection. We used β2m(-/-) and β2m(+/+) B10.Q mice (of H-2(q) background) normally susceptible to TMEV-induced demyelination. Over the disease course, β2m(+/+) mice had increasing levels of demyelination and minimal late-onset remyelination. In contrast, β2m(-/-) mice had steady levels of demyelination from 45-390 dpi and remyelination was extensive and more complete. Early in the disease, brainstem NAA levels drop in both strains, but accordingly with remyelination and axonal preservation, NAA recover in β2m(-/-) mice despite equivalent brainstem pathology. At 270 dpi, β2m(+/+) mice had significantly fewer spinal cord axons than β2m(-/-) mice (up to 28% less). In addition, β2m(+/+) mice lost axons of all calibers, whereas β2m(-/-) mice had a modest loss of only medium- and large-caliber axons. This study further supports the hypothesis that CD8(+) T cells are involved in demyelination, and axonal loss following Theiler's virus-induced demyelination. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

Science.gov (United States)

Myre, Michael A.; Washicosky, Kevin; Moir, Robert D.; Tesco, Giuseppina; Tanzi, Rudolph E.; Wasco, Wilma

2013-01-01

The detection of cell cycle proteins in Alzheimer’s disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells

Czech Academy of Sciences Publication Activity Database

Ježek, Petr; Olejár, Tomáš; Smolková, Katarína; Ježek, Jan; Dlasková, Andrea; Plecitá-Hlavatá, Lydie; Zelenka, Jaroslav; Špaček, Tomáš; Engstová, Hana; Reguera Pajuelo, David; Jabůrek, Martin

2014-01-01

Roč. 63, Suppl.1 (2014), S73-S91 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GAP305/12/1247; GA ČR(CZ) GPP304/10/P204; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondria * uncoupling protein UCP2 * pancreatic beta-cells * reactive oxygen species * glucose-stimulated insulin secretion Subject RIV: EA - Cell Biology Impact factor: 1.293, year: 2014

Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database

DEFF Research Database (Denmark)

Andersen, H U; Fey, S J; Larsen, Peter Mose

1997-01-01

as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins...... may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii......) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15...

Structure modeling and mutational analysis of gap junction beta 2 ...

African Journals Online (AJOL)

Yomi

2012-04-03

Apr 3, 2012 ... Three dimensional (3 D) structure is very useful for understanding biological functions. Gap junction beta 2 (GJB2), human gene encoding for gap junction beta 2 protein is involved in ... Research in deafness became real.

[Study of beta-turns in globular proteins].

Science.gov (United States)

Amirova, S R; Milchevskiĭ, Iu V; Filatov, I V; Esipova, N G; Tumanian, V G

2005-01-01

The formation of beta-turns in globular proteins has been studied by the method of molecular mechanics. Statistical method of discriminant analysis was applied to calculate energy components and sequences of oligopeptide segments, and after this prediction of I type beta-turns has been drawn. The accuracy of true positive prediction is 65%. Components of conformational energy considerably affecting beta-turn formation were delineated. There are torsional energy, energy of hydrogen bonds, and van der Waals energy.

Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

OpenAIRE

Horvath, Istvan; Wittung-Stafshede, Pernilla

2016-01-01

Protein assembly into ordered so-called amyloid fibers is a process that promotes several neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (PD). Also type-2 diabetes (T2D) is a disease involving amyloid formation, although it occurs in the pancreas. Since the protein that forms amyloids in PD, α-synuclein (aS), is also expressed in the pancreas, we investigated whether it could affect aggregation of the peptide involved in T2D, and vice versa. Using in vitro methods an...

LHCb: $2\\beta_s$ measurement at LHCb

CERN Multimedia

Conti, G

2009-01-01

A measurement of $2\\beta_s$, the phase of the $B_s-\\bar{B_s}$ oscillation amplitude with respect to that of the ${\\rm b} \\rightarrow {\\rm c^{+}}{\\rm W^{-}}$ tree decay amplitude, is one of the key goals of the LHCb experiment with first data. In the Standard Model (SM), $2\\beta_s$ is predicted to be $0.0360^{+0.0020}_{-0.0016} \\rm rad$. The current constraints from the Tevatron are: $2\\beta_{s}\\in[0.32 ; 2.82]$ at 68$\\%$CL from the CDF experiment and $2\\beta_{s}=0.57^{+0.24}_{-0.30}$ from the D$\\oslash$ experiment. Although the statistical uncertainties are large, these results hint at the possible contribution of New Physics in the $B_s-\\bar{B_s}$ box diagram. After one year of data taking at LHCb at an average luminosity of $\\mathcal{L}\\sim2\\cdot10^{32}\\rm cm^{-2} \\rm s^{-1}$ (integrated luminosity $\\mathcal{L}_{\\rm int}\\sim 2 \\rm fb^{-1}$), the expected statistical uncertainty on the measurement is $\\sigma(2\\beta_s)\\simeq 0.03$. This uncertainty is similar to the $2\\beta_s$ value predicted by the SM.

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

DEFF Research Database (Denmark)

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

2002-01-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

Changes in glomerular filtration rate, lithium clearance and plasma protein clearances in the early phase after unilateral nephrectomy in living healthy renal transplant donors

DEFF Research Database (Denmark)

Strandgaard, S; Kamper, A; Skaarup, P

1988-01-01

1. Glomerular and tubular function was studied before and 2 months after unilateral nephrectomy in 14 healthy kidney donors by measurement of the clearances of 51Cr-labelled ethylenediaminetetra-acetate, lithium, beta 2-microglobulin, albumin and immunoglobulin G. 2. The glomerular filtration rat...

Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of Beta-hydroxy-Beta-methylbutyrate

Science.gov (United States)

Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite Beta-hydroxy-Beta-methylbutyrate (HMB). To determine the effects of HMB on protein synthesi...

Do post-translational beta cell protein modifications trigger type 1 diabetes?

DEFF Research Database (Denmark)

Størling, Joachim; Overgaard, Anne Julie; Brorsson, Caroline Anna

2013-01-01

beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading...... diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from...... the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1...

Beta-carotene encapsulated in food protein nanoparticles reduces peroxyl radical oxidation in Caco-2 cells

Science.gov (United States)

Beta-carotene (BC) was encapsulated by sodium caseinate (SC), whey protein isolate (WPI), and soybean protein isolate (SPI) by the homogenization-evaporation method forming nanoparticles of 78, 90 and 370 nm diameter. Indices of the chemical antioxidant assays, the reducing power, DPPH radical scave...

β2-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence.

Science.gov (United States)

Gao, Na; Wang, Ying; Zheng, Chun-Ming; Gao, Yan-Li; Li, Hui; Li, Yan; Fu, Ting-Ting; Xu, Li-Li; Wang, Wei; Ying, Sun; Huang, Kewu

2017-05-01

β 2 -Microglobulin (β 2 M), the light chain of the major histocompatibility complex class I (MHC I), has been identified as a proaging factor and is involved in the pathogenesis of neurodegenerative disorders by driving cognitive and regenerative impairments. However, little attention has focused on the effect of β 2 M in the development of lung emphysema. Here, we found that concentrations of β 2 M in plasma were significantly elevated in patients with lung emphysema than those in normal control subjects (1.89 ± 0.12 vs. 1.42 ± 0.06 mg/l, P lung tissue of emphysema (39.90 ± 1.97 vs. 23.94 ± 2.11%, P lung emphysema through induction of lung epithelial cell senescence and inhibition. Copyright © 2017 the American Physiological Society.

Dataset of the molecular dynamics simulations of bilayers consisting of short amyloidogenic peptide VDSWNVLVAG from Bgl2p–glucantransferase of S. cerevisiae cell wall

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Anna V. Glyakina

2016-12-01

Full Text Available The amyloidogenic peptide VDSWNVLVAG from Bgl2p–glucantransferase of Saccharomyces cerevisiae cell wall and its modifying analog VESWNVLVAG were taken for the construction of four types of bilayers which differ by orientation of the peptides in the layers and of the layers relative to each other. These bilayers were used as starting models for the molecular dynamics (MD at three charge states (neutral, pH3, and pH5. The changes of the fraction of secondary structure during 1 ns simulations were received for 96 MD trajectories. The data article contains the necessary information for the construction of models of β-strands organization in the oligomer structure. These results were used in the associated research article “Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p–glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils” (Selivanova et al., 2016 [1].

Human interleukin 1. beta. stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca sup 2+ handling

Energy Technology Data Exchange (ETDEWEB)

Welsh, N.; Nilsson, T.; Hallberg, A.; Arkhammar, P.; Berggren, P.-O.; Sandler, S.

1989-01-01

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1{beta} in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca{sup 2+} handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In shortterm incubations (<1 min) interleukin 1{beta} did not affect the production of inositoltrisphosphate. Addition of interleukin 1{beta} affected neither the cytoplasmic free Ca{sup 2+} concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a {sup 32}P-labelled substrate for this enzyme, was not altered by interleukin 1{beta}. Separation of {sup 32}P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1{beta} are not caused by acute activation of phospholipase C and protein kinase C or by an alternation of islet Ca{sup 2+} handling of the B-cells. (author).

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

DEFF Research Database (Denmark)

Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

2005-01-01

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

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Jiang-Li Tan

Full Text Available BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP, which represents the first step in the production of amyloid β (Aβ peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer's disease (AD. The association between oxidative stress (OS and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10-40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS, as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

DEFF Research Database (Denmark)

Yde, C W; Olsen, B B; Meek, D

2008-01-01

25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

2012-08-10

Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

Identification of group specific motifs in Beta-lactamase family of proteins

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Saxena Akansha

2009-12-01

Full Text Available Abstract Background Beta-lactamases are one of the most serious threats to public health. In order to combat this threat we need to study the molecular and functional diversity of these enzymes and identify signatures specific to these enzymes. These signatures will enable us to develop inhibitors and diagnostic probes specific to lactamases. The existing classification of beta-lactamases was developed nearly 30 years ago when few lactamases were available. DLact database contain more than 2000 beta-lactamase, which can be used to study the molecular diversity and to identify signatures specific to this family. Methods A set of 2020 beta-lactamase proteins available in the DLact database http://59.160.102.202/DLact were classified using graph-based clustering of Best Bi-Directional Hits. Non-redundant (> 90 percent identical protein sequences from each group were aligned using T-Coffee and annotated using information available in literature. Motifs specific to each group were predicted using PRATT program. Results The graph-based classification of beta-lactamase proteins resulted in the formation of six groups (Four major groups containing 191, 726, 774 and 73 proteins while two minor groups containing 50 and 8 proteins. Based on the information available in literature, we found that each of the four major groups correspond to the four classes proposed by Ambler. The two minor groups were novel and do not contain molecular signatures of beta-lactamase proteins reported in literature. The group-specific motifs showed high sensitivity (> 70% and very high specificity (> 90%. The motifs from three groups (corresponding to class A, C and D had a high level of conservation at DNA as well as protein level whereas the motifs from the fourth group (corresponding to class B showed conservation at only protein level. Conclusion The graph-based classification of beta-lactamase proteins corresponds with the classification proposed by Ambler, thus there is

Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

International Nuclear Information System (INIS)

Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

2010-01-01

Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

Binding of human beta 2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells

DEFF Research Database (Denmark)

Claësson, M H; Nissen, Mogens Holst

1994-01-01

line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h beta 2m. In two of the T lymphomas, EL4 and BW5147, binding of h beta 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis....... EL4 cells which had bound h beta 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest the binding of h beta 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both...

The prion protein as a receptor for amyloid-beta

NARCIS (Netherlands)

Kessels, Helmut W.; Nguyen, Louis N.; Nabavi, Sadegh; Malinow, Roberto

2010-01-01

Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic

Prediction and analysis of beta-turns in proteins by support vector machine.

Science.gov (United States)

Pham, Tho Hoan; Satou, Kenji; Ho, Tu Bao

2003-01-01

Tight turn has long been recognized as one of the three important features of proteins after the alpha-helix and beta-sheet. Tight turns play an important role in globular proteins from both the structural and functional points of view. More than 90% tight turns are beta-turns. Analysis and prediction of beta-turns in particular and tight turns in general are very useful for the design of new molecules such as drugs, pesticides, and antigens. In this paper, we introduce a support vector machine (SVM) approach to prediction and analysis of beta-turns. We have investigated two aspects of applying SVM to the prediction and analysis of beta-turns. First, we developed a new SVM method, called BTSVM, which predicts beta-turns of a protein from its sequence. The prediction results on the dataset of 426 non-homologous protein chains by sevenfold cross-validation technique showed that our method is superior to the other previous methods. Second, we analyzed how amino acid positions support (or prevent) the formation of beta-turns based on the "multivariable" classification model of a linear SVM. This model is more general than the other ones of previous statistical methods. Our analysis results are more comprehensive and easier to use than previously published analysis results.

Immunological predictors of survival in HIV type 2-infected rural villagers in Guinea-Bissau

DEFF Research Database (Denmark)

Jaffar, Shabbar; Van der Loeff, Maarten Schim; Eugen-Olsen, Jesper

2005-01-01

We investigated the association between beta2-microglobulin, neopterin, serum levels of soluble urokinase-type plasminogen activator receptor (suPAR), CD4 count, and plasma viremia with survival in 133 HIV-2-infected villagers and 160 controls living in rural Guinea-Bissau. Subjects were recruite...

Neuromodulatory Effect of Thymoquinone in Attenuating Glutamate-Mediated Neurotoxicity Targeting the Amyloidogenic and Apoptotic Pathways

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Ibram Amin Fouad

2018-04-01

Full Text Available Overexposure of the glutamatergic N-methyl-d-aspartate (NMDA receptor to the excitatory neurotransmitter l-glutamic acid leads to neuronal cell death by excitotoxicity as a result of increased intracellular Ca2+, mitochondrial dysfunction, and apoptosis. Moreover, it was previously reported that prolonged activation of the NMDA receptor increased beta-amyloid (Aβ levels in the brain. Thymoquinone (TQ, the active constituent of Nigella sativa seeds, has been shown to have potent antioxidant and antiapoptotic effects. The aim of the present study was to explore the neuromodulatory effects of different doses of TQ (2.5 and 10 mg/kg against apoptotic cell death and Aβ formation resulting from glutamate administration in rats using vitamin E as a positive control. Behavioral changes were assessed using Y-maze and Morris water maze tests for evaluating spatial memory and cognitive functions. Caspase-3, Lactate dehydrogenase, Aβ-42, and cytochrome c gene expression were determined. TQ-treated groups showed significant decreases in the levels of all tested biochemical and behavioral parameters compared with the glutamate-treated group. These findings demonstrated that TQ has a promising neuroprotective activity against glutamate-induced neurotoxicity and this effect is mediated through its anti-amyloidogenic, antioxidant, and antiapoptotic activities.

Using support vector machine to predict beta- and gamma-turns in proteins.

Science.gov (United States)

Hu, Xiuzhen; Li, Qianzhong

2008-09-01

By using the composite vector with increment of diversity, position conservation scoring function, and predictive secondary structures to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta- and gamma-turns in the proteins is proposed. The 426 and 320 nonhomologous protein chains described by Guruprasad and Rajkumar (Guruprasad and Rajkumar J. Biosci 2000, 25,143) are used for training and testing the predictive model of the beta- and gamma-turns, respectively. The overall prediction accuracy and the Matthews correlation coefficient in 7-fold cross-validation are 79.8% and 0.47, respectively, for the beta-turns. The overall prediction accuracy in 5-fold cross-validation is 61.0% for the gamma-turns. These results are significantly higher than the other algorithms in the prediction of beta- and gamma-turns using the same datasets. In addition, the 547 and 823 nonhomologous protein chains described by Fuchs and Alix (Fuchs and Alix Proteins: Struct Funct Bioinform 2005, 59, 828) are used for training and testing the predictive model of the beta- and gamma-turns, and better results are obtained. This algorithm may be helpful to improve the performance of protein turns' prediction. To ensure the ability of the SVM method to correctly classify beta-turn and non-beta-turn (gamma-turn and non-gamma-turn), the receiver operating characteristic threshold independent measure curves are provided. (c) 2008 Wiley Periodicals, Inc.

Nonlinear analysis of sequence symmetry of beta-trefoil family proteins

Energy Technology Data Exchange (ETDEWEB)

Li Mingfeng [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Huang Yanzhao [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Xu Ruizhen [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China); Xiao Yi [Biomolecular Physics and Modeling Group, Department of Physics, Huazhong University of Science and Technology, Wuhan 430074, Hubei (China)]. E-mail: yxiao@mail.hust.edu.cn

2005-07-01

The tertiary structures of proteins of beta-trefoil family have three-fold quasi-symmetry while their amino acid sequences appear almost at random. In the present paper we show that these amino acid sequences have hidden symmetries in fact and furthermore the degrees of these hidden symmetries are the same as those of their tertiary structures. We shall present a modified recurrence plot to reveal hidden symmetries in protein sequences. Our results can explain the contradiction in sequence-structure relations of proteins of beta-trefoil family.

Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

Energy Technology Data Exchange (ETDEWEB)

Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

1985-10-07

Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

In silico sequence analysis and homology modeling of predicted beta-amylase 7-like protein in Brachypodium distachyon L.

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ERTUĞRUL FILIZ

2014-04-01

Full Text Available Beta-amylase (β-amylase, EC 3.2.1.2 is an enzyme that catalyses hydrolysis of glucosidic bonds in polysaccharides. In this study, we analyzed protein sequence of predicted beta-amylase 7-like protein in Brachypodium distachyon. pI (isoelectric point value was found as 5.23 in acidic character, while the instability index (II was found as 50.28 with accepted unstable protein. The prediction of subcellular localization was revealed that the protein may reside in chloroplast by using CELLO v.2.5. The 3D structure of protein was performed using comparative homology modeling with SWISS-MODEL. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 95.4% in favored region. The results of our study contribute to understanding of β-amylase protein structure in grass species and will be scientific base for 3D modeling of beta-amylase proteins in further studies.

Relation between the 2{nu}{beta}{beta} and 0{nu}{beta}{beta} nuclear matrix elements

Energy Technology Data Exchange (ETDEWEB)

Vogel, Petr [Kellogg Radiation Laboratory, Caltech, Pasadena, CA 91125 (United States); Simkovic, Fedor [Department of Nuclear Physics and Biophysics, Comenius University, Mlynska dolina F1, SK-84248 Bratislava (Slovakia)

2011-12-16

A formal relation between the GT part of the nuclear matrix elements M{sub GT}{sup 0{nu}} of 0{nu}{beta}{beta} decay and the closure matrix elements M{sub cl}{sup 2{nu}} of 2{nu}{beta}{beta} decay is established. This relation is based on the integral representation of these quantities in terms of their dependence on the distance r between the two nucleons undergoing transformation. We also discuss the difficulties in determining the correct values of the closure 2{nu}{beta}{beta} decay matrix elements.

Urinary Markers of Tubular Injury in Early Diabetic Nephropathy

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Temesgen Fiseha

2016-01-01

Full Text Available Diabetic nephropathy (DN is a common and serious complication of diabetes associated with adverse outcomes of renal failure, cardiovascular disease, and premature mortality. Early and accurate identification of DN is therefore of critical importance to improve patient outcomes. Albuminuria, a marker of glomerular involvement in early renal damage, cannot always detect early DN. Thus, more sensitive and specific markers in addition to albuminuria are needed to predict the early onset and progression of DN. Tubular injury, as shown by the detection of tubular injury markers in the urine, is a critical component of the early course of DN. These urinary tubular markers may increase in diabetic patients, even before diagnosis of microalbuminuria representing early markers of normoalbuminuric DN. In this review we summarized some new and important urinary markers of tubular injury, such as neutrophil gelatinase associated lipocalin (NGAL, kidney injury molecule-1 (KIM-1, liver-type fatty acid binding protein (L-FABP, N-acetyl-beta-glucosaminidase (NAG, alpha-1 microglobulin (A1M, beta 2-microglobulin (B2-M, and retinol binding protein (RBP associated with early DN.

Combining Cystatin C and Creatinine Yields a Reliable Glomerular Filtration Rate Estimation in Older Adults in Contrast to β-Trace Protein and β2-Microglobulin.

Science.gov (United States)

Werner, Karin; Pihlsgård, Mats; Elmståhl, Sölve; Legrand, Helen; Nyman, Ulf; Christensson, Anders

2017-01-01

The glomerular filtration rate (GFR) is the most important measure of kidney function and chronic kidney disease (CKD). This study aims to validate commonly used equations for estimated GFR (eGFR) based on creatinine (cr), cystatin C (cys), β-trace protein (BTP), and β2-microglobulin (B2M) in older adults. We conducted a validation study with 126 participants aged between 72 and 98 with a mean measured GFR (mGFR) by iohexol clearance of 54 mL/min/1.73 m2. The eGFR equations (CKD-Epidemiology collaboration [CKD-EPI], Berlin Initiative Study [BIS], Full Age Spectrum [FAS], Modification of Diet in Renal Disease [MDRD]cr, Caucasian-Asian-Pediatric-Adult [CAPA]cys, Lund-Malmö Revised [LM-REV]cr, and MEAN-LM-CAPAcr-cys), were assessed in terms of bias (median difference: eGFR-mGFR), precision (interquartile range of the differences), and accuracy (P30: percentage of estimates ±30% of mGFR). The equations were compared to a benchmark equation: CKD-EPIcr-cys. All cystatin C-based equations underestimated the GFR compared to mGFR, whereas bias was mixed for the equations based only on creatinine. Accuracy was the highest for CKD-EPIcr-cys (98%) and lowest for MDRD (82%). Below mGFR 45 mL/min/1.73 m2 only equations incorporating cystatin C reached P30 accuracy >90%. CKD-EPIcr-cys was not significantly more accurate than the other cystatin C-based equations. In contrast, CKD-EPIcr-cys was significantly more accurate than all creatinine-based equations except LM-REVcr. This study confirms that it is reasonable to use equations incorporating cystatin C and creatinine in older patients across a wide spectrum of GFR. However, the results call into question the use of creatinine alone below mGFR 45 mL/min/1.73 m2. B2M and BTP do not demonstrate additional value in eGFR determination in older adults. © 2017 S. Karger AG, Basel.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

Energy Technology Data Exchange (ETDEWEB)

Pavlikova, Nela, E-mail: nela.pavlikova@lf3.cuni.cz [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic); Smetana, Pavel [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic); Halada, Petr [Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague (Czech Republic); Kovar, Jan [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic)

2015-10-15

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

International Nuclear Information System (INIS)

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-01-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin

Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

Science.gov (United States)

Siese, A; Jaros, P P; Willig, A

1999-02-01

In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

Cartilage acidic protein 1, a new member of the beta-propeller protein family with amyloid propensity.

Science.gov (United States)

Anjos, Liliana; Morgado, Isabel; Guerreiro, Marta; Cardoso, João C R; Melo, Eduardo P; Power, Deborah M

2017-02-01

Cartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Serum TGF-beta2 and TGF-beta3 are increased and positively correlated to pain, functionality, and radiographic staging in osteoarthritis.

Science.gov (United States)

Kapetanakis, Stilianos; Drygiannakis, Ioannis; Kazakos, Kostantinos; Papanas, Nikolaos; Kolios, George; Kouroumalis, Elias; Verettas, Dionysios-Alexandros

2010-08-11

The goal of this study was to verify or reject the hypothesis that systematic differences exist in various profibrotic or antifibrotic factors between osteoarthritic patients and controls, as well as between different stages of osteoarthritis. The study group comprised 63 patients with knee osteoarthritis and 18 controls. Transforming growth factor-beta (TGF-beta)1, -2, -3; tissue inhibitor of metalloproteinase (TIMP)-1 protein levels; and gelatinolytic activity of matrix metalloproteinase (MMP)-1, -2, -3, -9 activities were measured by enzyme-linked immunosorbent assay and gelatin zymography, respectively. Visual analog scale scores, Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores, Lequesne clinical osteoarthritis scales, and Kellgren-Lawrence radiographic grading were recorded for each patient.Transforming growth factor-beta2 and -3 (in contrast to TGF-beta1 and TIMP-1) serum protein levels were significantly higher in osteoarthritic patients compared to controls (210%+/-14% [P<.001] and 232%+/-7% [P<10(-7)], respectively). Additionally, TGF-beta2 and -3 were strongly positively correlated to Kellgren-Lawrence radiographic grading of the disease (P<10(-5) and P<10(-7), respectively). Moreover, TGF-beta2 correlated positively with the WOMAC scale (P=.007). However, TIMP-1 decreased as osteoarthritis progressed clinically, but remained irrelevant to radiographic staging. Furthermore, activities of MMP-2 and -9, but not MMP-1+/-3, were lower in patients with osteoarthritis. Copyright 2010, SLACK Incorporated.

Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

Energy Technology Data Exchange (ETDEWEB)

Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

2011-09-09

Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

Science.gov (United States)

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Loss of function of ATXN1 increases amyloid beta-protein levels by potentiating beta-secretase processing of beta-amyloid precursor protein.

Science.gov (United States)

Zhang, Can; Browne, Andrew; Child, Daniel; Divito, Jason R; Stevenson, Jesse A; Tanzi, Rudolph E

2010-03-19

Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date approximately 80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Abeta, the proteolytic product of beta-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Abeta and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Abeta40 and Abeta42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Abeta levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Abeta levels is modulated via beta-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating beta-secretase cleavage of APP and Abeta levels.

Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein.

Science.gov (United States)

Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E

2010-09-10

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

DEFF Research Database (Denmark)

Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

2004-01-01

elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene...... of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8...

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

DEFF Research Database (Denmark)

Bless, N M; Huber-Lang, M; Guo, R F

2000-01-01

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...... were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response....... Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti...

Interactions between an alpha-helix and a beta-sheet. Energetics of alpha/beta packing in proteins.

Science.gov (United States)

Chou, K C; Némethy, G; Rumsey, S; Tuttle, R W; Scheraga, H A

1985-12-05

Conformational energy computations have been carried out to determine the favorable ways of packing a right-handed alpha-helix on a right-twisted antiparallel or parallel beta-sheet. Co-ordinate transformations have been developed to relate the position and orientation of the alpha-helix to the beta-sheet. The packing was investigated for a CH3CO-(L-Ala)16-NHCH3 alpha-helix interacting with five-stranded beta-sheets composed of CH3CO-(L-Val)6-NHCH3 chains. All internal and external variables for both the alpha-helix and the beta-sheet were allowed to change during energy minimization. Four distinct classes of low-energy packing arrangements were found for the alpha-helix interacting with both the parallel and the anti-parallel beta-sheet. The classes differ in the orientation of the axis of the alpha-helix relative to the direction of the strands of the right-twisted beta-sheet. In the class with the most favorable arrangement, the alpha-helix is oriented along the strands of the beta-sheet, as a result of attractive non-bonded side-chain-side-chain interactions along the entire length of the alpha-helix. A class with nearly perpendicular orientation of the helix axis to the strands is also of low energy, because it allows similarly extensive attractive interactions. In the other two classes, the helix is oriented diagonally relative to the strands of the beta-sheet. In one of them, it interacts with the convex surface near the middle of the saddle-shaped twisted beta-sheet. In the other, it is oriented along the concave diagonal of the beta-sheet and, therefore, it interacts only with the corner regions of the sheet, so that this packing is energetically less favorable. The packing arrangements involving an antiparallel and a parallel beta-sheet are generally similar, although the antiparallel beta-sheet has been found to be more flexible. The major features of 163 observed alpha/beta packing arrangements in 37 proteins are accounted for in terms of the computed

Support vector machines for prediction and analysis of beta and gamma-turns in proteins.

Science.gov (United States)

Pham, Tho Hoan; Satou, Kenji; Ho, Tu Bao

2005-04-01

Tight turns have long been recognized as one of the three important features of proteins, together with alpha-helix and beta-sheet. Tight turns play an important role in globular proteins from both the structural and functional points of view. More than 90% tight turns are beta-turns and most of the rest are gamma-turns. Analysis and prediction of beta-turns and gamma-turns is very useful for design of new molecules such as drugs, pesticides, and antigens. In this paper we investigated two aspects of applying support vector machine (SVM), a promising machine learning method for bioinformatics, to prediction and analysis of beta-turns and gamma-turns. First, we developed two SVM-based methods, called BTSVM and GTSVM, which predict beta-turns and gamma-turns in a protein from its sequence. When compared with other methods, BTSVM has a superior performance and GTSVM is competitive. Second, we used SVMs with a linear kernel to estimate the support of amino acids for the formation of beta-turns and gamma-turns depending on their position in a protein. Our analysis results are more comprehensive and easier to use than the previous results in designing turns in proteins.

Machine learning methods can replace 3D profile method in classification of amyloidogenic hexapeptides

Directory of Open Access Journals (Sweden)

Stanislawski Jerzy

2013-01-01

Full Text Available Abstract Background Amyloids are proteins capable of forming fibrils. Many of them underlie serious diseases, like Alzheimer disease. The number of amyloid-associated diseases is constantly increasing. Recent studies indicate that amyloidogenic properties can be associated with short segments of aminoacids, which transform the structure when exposed. A few hundreds of such peptides have been experimentally found. Experimental testing of all possible aminoacid combinations is currently not feasible. Instead, they can be predicted by computational methods. 3D profile is a physicochemical-based method that has generated the most numerous dataset - ZipperDB. However, it is computationally very demanding. Here, we show that dataset generation can be accelerated. Two methods to increase the classification efficiency of amyloidogenic candidates are presented and tested: simplified 3D profile generation and machine learning methods. Results We generated a new dataset of hexapeptides, using more economical 3D profile algorithm, which showed very good classification overlap with ZipperDB (93.5%. The new part of our dataset contains 1779 segments, with 204 classified as amyloidogenic. The dataset of 6-residue sequences with their binary classification, based on the energy of the segment, was applied for training machine learning methods. A separate set of sequences from ZipperDB was used as a test set. The most effective methods were Alternating Decision Tree and Multilayer Perceptron. Both methods obtained area under ROC curve of 0.96, accuracy 91%, true positive rate ca. 78%, and true negative rate 95%. A few other machine learning methods also achieved a good performance. The computational time was reduced from 18-20 CPU-hours (full 3D profile to 0.5 CPU-hours (simplified 3D profile to seconds (machine learning. Conclusions We showed that the simplified profile generation method does not introduce an error with regard to the original method, while

Machine learning methods can replace 3D profile method in classification of amyloidogenic hexapeptides.

Science.gov (United States)

Stanislawski, Jerzy; Kotulska, Malgorzata; Unold, Olgierd

2013-01-17

Amyloids are proteins capable of forming fibrils. Many of them underlie serious diseases, like Alzheimer disease. The number of amyloid-associated diseases is constantly increasing. Recent studies indicate that amyloidogenic properties can be associated with short segments of aminoacids, which transform the structure when exposed. A few hundreds of such peptides have been experimentally found. Experimental testing of all possible aminoacid combinations is currently not feasible. Instead, they can be predicted by computational methods. 3D profile is a physicochemical-based method that has generated the most numerous dataset - ZipperDB. However, it is computationally very demanding. Here, we show that dataset generation can be accelerated. Two methods to increase the classification efficiency of amyloidogenic candidates are presented and tested: simplified 3D profile generation and machine learning methods. We generated a new dataset of hexapeptides, using more economical 3D profile algorithm, which showed very good classification overlap with ZipperDB (93.5%). The new part of our dataset contains 1779 segments, with 204 classified as amyloidogenic. The dataset of 6-residue sequences with their binary classification, based on the energy of the segment, was applied for training machine learning methods. A separate set of sequences from ZipperDB was used as a test set. The most effective methods were Alternating Decision Tree and Multilayer Perceptron. Both methods obtained area under ROC curve of 0.96, accuracy 91%, true positive rate ca. 78%, and true negative rate 95%. A few other machine learning methods also achieved a good performance. The computational time was reduced from 18-20 CPU-hours (full 3D profile) to 0.5 CPU-hours (simplified 3D profile) to seconds (machine learning). We showed that the simplified profile generation method does not introduce an error with regard to the original method, while increasing the computational efficiency. Our new dataset

A Redox-Active, Compact Molecule for Cross-Linking Amyloidogenic Peptides into Nontoxic, Off-Pathway Aggregates: In Vitro and In Vivo Efficacy and Molecular Mechanisms

Energy Technology Data Exchange (ETDEWEB)

Derrick, Jeffrey S.; Kerr, Richard A.; Nam, Younwoo; Oh, Shin Bi; Lee, Hyuck Jin; Earnest, Kaylin G.; Suh, Nayoung; Peck, Kristy L.; Ozbil, Mehmet; Korshavn, Kyle J.; Ramamoorthy, Ayyalusamy; Prabhakar, Rajeev; Merino, Edward J.; Shearer, Jason; Lee, Joo-Yong; Ruotolo, Brandon T.; Lim, Mi Hee

2015-11-25

Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand–peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer’s disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

Kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin as prognostic markers in idiopathic membranous nephropathy

NARCIS (Netherlands)

Maas, Rutger J. H.; van den Brand, Jan A. J. G.; Waanders, Femke; Meijer, Esther; van Goor, Harry; Peters, Hilde P.; Hofstra, Julia M.; Wetzels, Jack F. M.

Background: Urinary excretion of alpha-1-microglobulin and beta-2-microglobulin reflects tubular damage and predicts outcome in patients with idiopathic membranous nephropathy with reasonable accuracy. Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin are novel

Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

NARCIS (Netherlands)

Wilhelmus, M.M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Kusters, B.; Maat-Schieman, M.L.; Waal, R.M.W. de; Verbeek, M.M.

2006-01-01

Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major

Beta-defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin.

Directory of Open Access Journals (Sweden)

Patrick A M Jansen

Full Text Available BACKGROUND: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2 is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. METHODOLOGY/PRINCIPAL FINDINGS: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. CONCLUSIONS/SIGNIFICANCE: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

Science.gov (United States)

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-10-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. Copyright © 2015 Elsevier Inc. All rights reserved.

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

DEFF Research Database (Denmark)

Sennvik, Kristina; Bogdanovic, N; Volkmann, Inga

2004-01-01

beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP...... the beta-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal beta-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. beta-sAPP was also found surrounding senile plaques...

Quantitative protein and fat metabolism in bull calves treated with beta-adrenergic agonist

DEFF Research Database (Denmark)

Chwalibog, André; Jensen, K; Thorbek, G

1996-01-01

Protein and energy utilization and quantitative retention of protein, fat and energy was investigated with 12 Red Danish bulls during two subsequent 6 weeks trials (Sections A and B) at a mean live weight of 195 and 335 kg respectively. Treatments were control (Group 1) and beta-agonist (L-644...... matter, metabolizable energy and digestible protein was of the same magnitude for all groups. The beta-agonist had no significant effect on protein digestibility and metabolizability of energy, but daily live weight gain was significantly higher in the treated bulls. The utilization of digested protein...

Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

OpenAIRE

Wong, S K; Martin, B R; Tolkovsky, A M

1985-01-01

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guano...

Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

Directory of Open Access Journals (Sweden)

Christine Pampeno

Full Text Available The laminin receptor (LamR is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C and PrP(Sc. Indeed, LamR is a receptor for PrP(C. Whether LamR interacts with PrP(Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin.

Science.gov (United States)

Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

2012-04-01

β(2)-Microglobulin (β(2)m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide-MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β(2)m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β(2)m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3(2)21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β(2)m and IgSF in the AIS. © 2012 International Union of Crystallography. All rights reserved.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

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Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

Science.gov (United States)

White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

2014-08-01

The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

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Gajewska, Małgorzata; Motyl, Tomasz

2004-10-01

TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway.

Application of path analysis to urinary findings of cadmium-induced renal dysfunction.

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Abe, T; Kobayashi, E; Okubo, Y; Suwazono, Y; Kido, T; Shaikh, Z A; Nogawa, K

2001-01-01

In order to identify some causal relations among various urinary indices of cadmium-induced renal dysfunction, such as glucose, total protein, amino nitrogen, beta 2-microglobulin (beta 2-m), metallothionein (MT), and cadmium (Cd), we applied path analysis method to previous epidemiological studies targeting the residents of the Cd-polluted Kakehashi River basin of Ishikawa Prefecture, Japan. We obtained a diagram-termed path model, representing some causal relations among the above urinary indices. It shows that urinary Cd is located at the beginning point in the diagram, and Cd-induced renal dysfunction develops in the following order: Cd exposure-->increase of beta 2-m and/or MT excretion-->increase of amino-N and/or total protein excretion-->increase of glucose excretion. It was proved mathematically, that in the case of both males and females, increased excretions of beta 2-m and/or MT were the most sensitive urinary indices of the early stage of chronic Cd-induced renal dysfunction.

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

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Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

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Guruprasad, K; Rajkumar, S

2000-06-01

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

Effect of beta-hydroxy-beta-methylbutyrate (HMB) on protein metabolism in whole body and in selected tissues.

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Holecek, M; Muthny, T; Kovarik, M; Sispera, L

2009-01-01

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effect. The aim of the study was to examine the role of exogenous HMB on leucine and protein metabolism in whole body and selected tissues. Rats were administered by HMB (0.1 g/kg b.w.) or by saline. The parameters of whole-body protein metabolism were evaluated 24 h later using L-[1-14C]leucine and L-[3,4,5-3H]phenylalanine. Changes in proteasome dependent proteolysis and protein synthesis were determined according the "chymotrypsin-like" enzyme activity and labeled leucine and phenylalanine incorporation into the protein. A decrease in leucine clearance and whole-body protein turnover (i.e., a decrease in whole-body proteolysis and protein synthesis) was observed in HMB treated rats. Proteasome-dependent proteolysis decreased significantly in skeletal muscle, changes in heart, liver, jejunum, colon, kidney, and spleen were insignificant. Decrease in protein synthesis was observed in the heart, colon, kidney, and spleen, while an increase was observed in the liver. There were no significant changes in leucine oxidation. We conclude that protein anabolic effect of HMB in skeletal muscle is related to inhibition of proteolysis in proteasome. Alterations in protein synthesis in visceral tissues may affect several important functions and the metabolic status of the whole body.

Estimation of benchmark dose as the threshold levels of urinary cadmium, based on excretion of total protein, β 2-microglobulin, and N-acetyl-β-D-glucosaminidase in cadmium nonpolluted regions in Japan

International Nuclear Information System (INIS)

Kobayashi, Etsuko; Suwazono, Yasushi; Uetani, Mirei; Inaba, Takeya; Oishi, Mitsuhiro; Kido, Teruhiko; Nishijo, Muneko; Nakagawa, Hideaki; Nogawa, Koji

2006-01-01

Previously, we investigated the association between urinary cadmium (Cd) concentration and indicators of renal dysfunction, including total protein, β 2 -microglobulin (β 2 -MG), and N-acetyl-β-D-glucosaminidase (NAG). In 2778 inhabitants ≥50 years of age (1114 men, 1664 women) in three different Cd nonpolluted areas in Japan, we showed that a dose-response relationship existed between renal effects and Cd exposure in the general environment without any known Cd pollution. However, we could not estimate the threshold levels of urinary Cd at that time. In the present study, we estimated the threshold levels of urinary Cd as the benchmark dose low (BMDL) using the benchmark dose (BMD) approach. Urinary Cd excretion was divided into 10 categories, and an abnormality rate was calculated for each. Cut-off values for urinary substances were defined as corresponding to the 84% and 95% upper limit values of the target population who have not smoked. Then we calculated the BMD and BMDL using a log-logistic model. The values of BMD and BMDL for all urinary substances could be calculated. The BMDL for the 84% cut-off value of β 2 -MG, setting an abnormal value at 5%, was 2.4 μg/g creatinine (cr) in men and 3.3 μg/g cr in women. In conclusion, the present study demonstrated that the threshold level of urinary Cd could be estimated in people living in the general environment without any known Cd-pollution in Japan, and the value was inferred to be almost the same as that in Belgium, Sweden, and China

A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome

Energy Technology Data Exchange (ETDEWEB)

A Kolyada; C Lee; A De Biasio; N Beglova

2011-12-31

{beta}2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of {beta}2GPI generated by anti-{beta}2GPI antibodies is pathologically important, in contrast to monomeric {beta}2GPI which is abundant in plasma. We created a dimeric inhibitor, A1-A1, to selectively target {beta}2GPI in {beta}2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of {beta}2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of {beta}2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of {beta}2GPI present in human serum, {beta}2GPI purified from human plasma and the individual domain V of {beta}2GPI. We demonstrated that when {beta}2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of {beta}2GPI to cardiolipin, regardless of the source of {beta}2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of {beta}2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-{beta}2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric {beta}2GPI to cardiolipin. Our results suggest that the approach of using a dimeric inhibitor to block {beta}2GPI in the pathological multivalent {beta}2GPI/antibody complexes holds significant promise. The novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.

The effect of short-term glucagon infusion on kidney function in normal man

DEFF Research Database (Denmark)

Parving, H H; Noer, J; Kehlet, H

1977-01-01

Kidney function was studied in six normal males before and during a 2 h glucagon (10 ng/kg/min) infusion. The following variables were determined during each 20 min clearance period; glomerular filtration rate (GFR), renal plasma-flow (RPF) , filtration fraction (FF), urinary albumin and beta2......-microglobulin-excretion rates. Glucagon infusion resulted in a fourfold increase in plasma glucagon concentration. The infusion induced a significant increase in GFR (+9%), FF (+9%) and urinary beta2-microglobulin excretion rate (+32%), (p less than 0.01). RPF and urinary albumin excretion rates were...

Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

DEFF Research Database (Denmark)

Brockdorff, J; Kanner, S B; Nielsen, M

1998-01-01

beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...

Amyloidosis associated with dialysis. Dialyseassoziierte Amyloidosteopathie - radiologische Aspekte

Energy Technology Data Exchange (ETDEWEB)

Schadmand, S.; Klose, K.J. (Mainz Univ. (Germany, F.R.). Inst. fuer Klinische Strahlenkunde); Wandel, E. (Mainz Univ. (Germany, F.R.). 1. Medizinische Klinik und Poliklinik)

1991-06-01

Amongst the complications of dialysis, amyloid osteopathy is getting increasingly significant. It is due to deposition of {beta}2-microglobulin. To determine the incidence and time of development of this complication, the skeletal radiographs of 185 patients undergoing dialysis, some for up to ten years, were analysed retrospectively. In about 10% of patients, the presence of {beta}2-microglobulin osteopathy may be expected. The radiological features, sites of predilection and differential diagnosis of amyloid osteopathy and of other skeletal changes due to dialysis are discussed. (orig.).

Protein kinase CK2 structure-function relationship

DEFF Research Database (Denmark)

Boldyreff, B; Meggio, F; Pinna, L A

1994-01-01

Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... subunit have been prepared and assayed for their ability to assemble with the catalytic alpha subunit to give a fully competent CK2 holoenzyme. The beta subunit contains an acidic stretch (amino acid 55-64), which is obviously responsible for a negative control of enzyme activity since mutations...

Local sequence information in cellular retinoic acid-binding protein I: specific residue roles in beta-turns.

Science.gov (United States)

Rotondi, Kenneth S; Gierasch, Lila M

2003-01-01

We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein. Copyright 2004 Wiley Periodicals, Inc.

Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

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Olsson, J E; Sandberg, M [Department of Neurology, University Hospital, S-221 85 Lund, Sweden

1975-01-01

The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of /sup 14/C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed.

Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey

International Nuclear Information System (INIS)

Olsson, J.-E.; Sandberg, M.

1975-01-01

The sites of synthesis of the low molwculat weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissue were cultured in Eagle's minium essential medium in the presence of 14 C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed. (author)

Common ADRB2 haplotypes derived from 26 polymorphic sites direct beta2-adrenergic receptor expression and regulation phenotypes.

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Alfredo Panebra

2010-07-01

Full Text Available The beta2-adrenergic receptor (beta2AR is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes, while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes. An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

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Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

2007-02-01

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

Comparison between Amnisure Placental Alpha Microglobulin-1 Rapid Immunoassay and Standard Diagnostic Methods for Detection of Rupture of Membranes

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Beng Kwang Ng

2013-01-01

Full Text Available Objective. To determine the diagnostic accuracy of placental alpha microglobulin-1 assay and standard diagnostic methods for detecting rupture of membrane. Study Design. Prospective diagnostic study, between June 2011 to November 2011 at a tertiary centre. Initial evaluation included both the standard diagnostic methods for rupture of membranes and placental alpha microglobulin-1 immunoassay. The actual rupture of membranes was diagnosed on review of the medical records after delivery (absence of membrane or a positive pad chart. Main Outcome Measures. Placental alpha microglobulin-1 immunoassay and standard diagnostic methods for diagnosis of rupture of membrane. Results. A total of 211 patients were recruited. At initial presentation, 187 patients (88.6% had ruptured membranes, while 24 patients (11.4% had intact membranes. Placental alpha microglobulin-1 immunoassay confirmed rupture of membranes at initial presentation with a sensitivity of 95.7% (179 of 187, specificity of 100% (24 of 24, positive predictive value of 100% (179 of 179, and negative predictive value of 75.0% (24 of 32. By comparison, the conventional standard diagnostic methods had a sensitivity of 78.1% (146 of 187, specificity of 100% (24 of 24, positive predictive value of 100% (146 of 146, and negative predictive value of 36.9% (24 of 65 in diagnosing rupture of membrane. Conclusion. Placental alpha-microglobulin-1 immunoassay is a rapid and accurate method for confirming the diagnosis of rupture of membrane. It was superior to conventional standard diagnostic methods (pooling, nitrazine, and ferning, the nitrazine test alone or fern test alone.

Fetal hemoglobin, α1-microglobulin and hemopexin are potential predictive first trimester biomarkers for preeclampsia.

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Anderson, Ulrik Dolberg; Gram, Magnus; Ranstam, Jonas; Thilaganathan, Basky; Kerström, Bo; Hansson, Stefan R

2016-04-01

Overproduction of cell-free fetal hemoglobin (HbF) in the preeclamptic placenta has been recently implicated as a new etiological factor of preeclampsia. In this study, maternal serum levels of HbF and the endogenous hemoglobin/heme scavenging systems were evaluated as predictive biomarkers for preeclampsia in combination with uterine artery Doppler ultrasound. Case-control study including 433 women in early pregnancy (mean 13.7weeks of gestation) of which 86 subsequently developed preeclampsia. The serum concentrations of HbF, total cell-free hemoglobin, hemopexin, haptoglobin and α1-microglobulin were measured in maternal serum. All patients were examined with uterine artery Doppler ultrasound. Logistic regression models were developed, which included the biomarkers, ultrasound indices, and maternal risk factors. There were significantly higher serum concentrations of HbF and α1-microglobulin and significantly lower serum concentrations of hemopexin in patients who later developed preeclampsia. The uterine artery Doppler ultrasound results showed significantly higher pulsatility index values in the preeclampsia group. The optimal prediction model was obtained by combining HbF, α1-microglobulin and hemopexin in combination with the maternal characteristics parity, diabetes and pre-pregnancy hypertension. The optimal sensitivity for all preeclampsia was 60% at 95% specificity. Overproduction of placentally derived HbF and depletion of hemoglobin/heme scavenging mechanisms are involved in the pathogenesis of preeclampsia. The combination of HbF and α1-microglobulin and/or hemopexin may serve as a prediction model for preeclampsia in combination with maternal risk factors and/or uterine artery Doppler ultrasound. Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin.

Science.gov (United States)

Yasuda, Satoshi; Kai, Masahiro; Imai, Shin-ichi; Kanoh, Hideo; Sakane, Fumio

2008-01-01

DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.

Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome

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Kondo, Akira; Miyamoto, Toshiaki; Yonekawa, Osamu

2009-01-01

beta2-glycoprotein I (beta2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. beta2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed t...

The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.

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Kanamori, Mutsumi; Sandy, Peter; Marzinotto, Stefania; Benetti, Roberta; Kai, Chikatoshi; Hayashizaki, Yoshihide; Schneider, Claudio; Suzuki, Harukazu

2003-10-03

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

Energy Technology Data Exchange (ETDEWEB)

Yu,P.

2007-01-01

-FTIR absorption intensity), increased the percentage of {beta}-sheets (from 37.2% to 49.8%: S-FTIR absorption intensity) and reduced the {alpha}-helix to {beta}-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.

IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

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Sparre, T; Bjerre-Christensen, Ulla; Mose Larsen, P

2002-01-01

of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen......Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression...

Oxidative Modification of Blood Serum Proteins in Multiple Sclerosis after Interferon Beta and Melatonin Treatment

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Monika Adamczyk-Sowa

2017-01-01

Full Text Available Multiple sclerosis (MS is a disease involving oxidative stress (OS. This study was aimed at examination of the effect of melatonin supplementation on OS parameters, especially oxidative protein modifications of blood serum proteins, in MS patients. The study included 11 control subjects, 14 de novo diagnosed MS patients with the relapsing-remitting form of MS (RRMS, 36 patients with RRMS receiving interferon beta-1b (250 μg every other day, and 25 RRMS patients receiving interferon beta-1b plus melatonin (5 mg daily. The levels of N′-formylkynurenine, kynurenine, dityrosine, carbonyl groups, advanced glycation products (AGEs, advanced oxidation protein products (AOPP, and malondialdehyde were elevated in nontreated RRSM patients. N′-Formylkynurenine, kynurenine, AGEs, and carbonyl contents were decreased only in the group treated with interferon beta plus melatonin, while dityrosine and AOPP contents were decreased both in the group of patients treated with interferon beta and in the group treated with interferon beta-1b plus melatonin. These results demonstrate that melatonin ameliorates OS in MS patients supporting the view that combined administration of interferon beta-1b and melatonin can be more effective in reducing OS in MS patients than interferon beta-1b alone.

Protein kinase CK2 in human diseases

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Guerra, Barbara; Issinger, Olaf-Georg

2008-01-01

Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

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Rian de Laat

2015-03-01

Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

International Nuclear Information System (INIS)

Pi Jingbo; Zhang Qiang; Fu Jingqi; Woods, Courtney G.; Hou Yongyong; Corkey, Barbara E.; Collins, Sheila; Andersen, Melvin E.

2010-01-01

This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H 2 O 2 , act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

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Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Urinary β-2 Microglobulin Levels Sensitively Altered in an Osteomalacia Patient Receiving Add-on Adefovir Dipivoxil Therapy for Hepatitis B Virus Infection.

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Takagi, Junko; Morita, Hiroyuki; Ito, Kiyoaki; Ohashi, Tomohiko; Hirase, Sho; Ito, Tatsuo; Morishima, Takkan; Otake, Kazuo; Yoneda, Masashi

2016-01-01

Adefovir dipivoxil (ADV) is effective for hepatitis B virus (HBV) infection; however, ADV may provoke renal injury resulting in osteomalacia, and this side effect is seldom recognized until bone fractures emerge. We herein present a 66-year-old woman with HBV infection who received ADV for 6 years. Although she exhibited no sign of bone fractures, her urinary β-2 microglobulin (β2MG) level increased to 83,837 μg/L and scintigraphy revealed minimal fractures of the third rib. ADV was subsequently reduced and her urinary β2MG rapidly fell to 3,637 μg/L. Conversely, her urinary N-acetyl-β-D-glucosaminidase, and serum phosphate, alkaline phosphatase levels did not respond.

A novel proteomic biomarker panel as a diagnostic tool for patients with ovarian cancer

DEFF Research Database (Denmark)

Høgdall, Claus; Fung, Eric T; Christensen, Ib J

2011-01-01

Previous reports have shown that the proteomic markers apolipoprotein A1, hepcidin, transferrin, inter-alpha trypsin IV internal fragment, transthyretin, connective-tissue activating protein 3 and beta-2 microglobulin may discriminate between a benign pelvic mass and ovarian cancer (OC). The aim...... was to determine if these serum proteomic biomarkers alone as well as in combination with age and serum CA125, could be helpful in triage of women with a pelvic mass....

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic beta cell.

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Kowluru, Anjaneyulu

2008-01-15

Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the beta cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The "newly formed" phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the beta cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet beta cell; (iii) propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet beta cell function.

Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2 Agonists

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Fabio Cattaneo

2013-04-01

Full Text Available The formyl peptide receptor 2 (FPR2 is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ-42 and prion protein (Prp106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP and pituitary adenylate cyclase activating polypeptide (PACAP-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC, protein kinase C (PKC isoforms, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt pathway, the mitogen-activated protein kinase (MAPK pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2

Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

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Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

2008-10-23

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

Characterization of binding of human alpha 2-macroglobulin to group G streptococci

International Nuclear Information System (INIS)

Chhatwal, G.S.; Mueller, H.P.; Blobel, H.

1983-01-01

An interaction was observed between human alpha 2-macroglobulin (alpha 2M) and streptococci belonging to group A, C, and G. Of 27 group C and 19 group G streptococcal cultures, 13 and 14, respectively, bound 125 I-labeled alpha 2M. Some group A streptococci also interacted with alpha 2M. A number of other bacterial species tested did not react with alpha 2M. The binding of 125 I-labeled alpha 2M to group G streptococci was time dependent, saturable, and could be inhibited by unlabeled alpha 2M. Inhibition experiments indicated that the streptococcal binding site for alpha 2M differed from the receptors for immunoglobulin G, fibrinogen, aggregated beta 2-microglobulin, albumin, and fibronectin. The alpha 2M binding activity was remarkably sensitive to trypsin and heat treatment indicating its protein nature. Kinetic analysis indicated a homogenous population of binding sites. The number of binding sites per bacterial cell was estimated to be approximately 20,000

H2O2-Activated Mitochondrial Phospholipase iPLA2 gamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells

Czech Academy of Sciences Publication Activity Database

Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin; Ježek, Petr

2015-01-01

Roč. 23, č. 12 (2015), s. 958-972 ISSN 1523-0864 R&D Projects: GA ČR(CZ) GPP303/11/P320; GA ČR(CZ) GA13-02033S; GA ČR(CZ) GA13-06666S; GA ČR GA15-02051S Institutional support: RVO:67985823 Keywords : mitochondrial phospholipase iPLA2 gamma * uncoupling protein UCP2 * G-protein coupled receptor - 40 * glucose-stimulated insulin secretion * pancreatic beta cells Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 7.093, year: 2015

Joint detection and clinical analyse of urine β2-microglobulin, albumin and immunoglobulin G in patients with hyperthyroidism

International Nuclear Information System (INIS)

Cui Liqun; Yang Baojun

2009-01-01

Objective: To evaluate the clinical significance of joint detection of urine β 2 -microglobulin (β 2 MG), albumin (Alb) and immunoglobulin G (IgG) in patients with hyperthyroidism Methods: Urine β 2 -MG, Alb, IgG and serum thyroid hormone free trilute (FT 3 ), free thyroxin (FT 4 ) of 45 healthy volunteers (as control group) and 120 patients with hyperthyroidism were measured by radioimmunoassay (RIA). Results: The urine β 2 -MG, Alb and IgG in the hyperthyroidism group (including the primary group (group A) and uncured group (group B)) were distinctly higher than those in control group and hyperthyroidism cured group (group C) (t=6.682, P 2 -MG, Alb and IgG with serum FT 3 and FT 4 (the related coefficients were 0.98, 0.88, 0.93, 0.87, 0.94, and 0.85 respectively). Conclusion: It is important to measure urine β 2 -MG together with Alb and IgG in early judgment of the location and degree of kidney injury, as well as the severity of disease in patients with hyperthyroidism. (authors)

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber.

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Papanikolopoulou, Katerina; Schoehn, Guy; Forge, Vincent; Forsyth, V Trevor; Riekel, Christian; Hernandez, Jean-François; Ruigrok, Rob W H; Mitraki, Anna

2005-01-28

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

Science.gov (United States)

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

LRP-mediated clearance of Abeta is inhibited by KPI-containing isoforms of APP.

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Moir, Robert D; Tanzi, Rudolph E

2005-04-01

The pathogenesis of Alzheimer's disease (AD) involves the abnormal accumulation and deposition of beta-amyloid in cerebral blood vessels and in the brain parenchyma. Critical in modulating beta-amyloid deposition in brain is the flux of Abeta across the blood brain barrier. The low-density lipoprotein receptor-related protein (LRP), is a large endocytic receptor that mediates the efflux of Abeta out of brain and into the periphery. The first step in the LRP-mediated clearance of Abeta involves the formation of a complex between Abeta and the LRP ligands apolipoprotein E (apoE) or alpha(2)-macroglobulin (alpha(2)M). The Abeta/chaperone complexes then bind to LRP via binding sites on apoE or alpha(2)M. The efflux of Abeta/chaperone complexes out of the neuropil and into the periphery may be attenuated by LRP-ligands that compete with apoE or alpha(2)M for LRP binding. LRP is also the cell surface receptor for Kunitz Protease Inhibitor (KPI) containing isoforms of Abeta's parent protein, the amyloid protein precursor (APP). Protein and mRNA levels of KPI-containing APP isoforms (APP-KPI) are elevated in AD brain and are associated with increased Abeta production. In this study we show that soluble non-amyloidogenic APP-KPI can also inhibit the uptake of Abeta/alpha(2)M in a cell culture model of LRP mediated Abeta clearance. Clearance of Abeta/apoE complexes was not inhibited by APP-KPI. Our findings are consistent with studies showing that apoE and alpha(2)M have discrete binding sites on LRP. Most significantly, our data suggests that the elevated levels of APP-KPI in AD brain may attenuate the clearance of Abeta, the proteins own amyloidogenic catabolic product.

AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

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Maayan Shaked

Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

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Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC

In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

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Incilay Sinici

Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750. Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1 and a new more extensive hybrid (H2, with our documented in cellulo (live cell- based assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila.

Science.gov (United States)

Frenkel-Pinter, Moran; Stempler, Shiri; Tal-Mazaki, Sharon; Losev, Yelena; Singh-Anand, Avnika; Escobar-Álvarez, Daniela; Lezmy, Jonathan; Gazit, Ehud; Ruppin, Eytan; Segal, Daniel

2017-08-01

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., β-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein degradation in skeletal muscle during experimental hyperthyroidism in rats and the effect of beta-blocking agents.

Science.gov (United States)

Angerås, U; Hasselgren, P O

1987-04-01

beta-Blocking agents are increasingly used in the management of hyperthyroid patients. The effect of this treatment on increased muscle protein breakdown in the hyperthyroid state is not known. In the present study, experimental hyperthyroidism was induced in rats by daily ip injections of T3 (100 micrograms/100 g BW) during a 10-day period. Control animals received corresponding volumes of solvent. In groups of rats the selective beta-1-blocking agent metoprolol or the nonselective beta-blocker propranolol was infused by miniosmotic pumps implanted sc on the backs of the animals. Protein degradation was measured in incubated intact soleus and extensor digitorum longus muscles by determining tyrosine release into the incubation medium. The protein degradation rate in incubated extensor digitorum longus and soleus muscles was increased by 50-60% during T3 treatment. Metoprolol or propranolol did not influence muscle protein breakdown in either T3-treated or control animals. The results suggest that T3-induced increased muscle proteolysis is not mediated by beta-receptors, and muscle weakness and wasting in hyperthyroidism might not be affected by beta-blockers.

Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

Energy Technology Data Exchange (ETDEWEB)

Boehme, K.A.

2006-12-15

Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

Science.gov (United States)

Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

2016-02-01

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies

C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Bregenholt, S; Nording, J A

1998-01-01

We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

Science.gov (United States)

Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

Science.gov (United States)

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

A role for 12/15 lipoxygenase in the amyloid beta precursor protein metabolism.

Science.gov (United States)

Succol, Francesca; Praticò, Domenico

2007-10-01

12/15 Lipoxygenase (12/15LO) protein levels and activity are increased in pathologically affected regions of Alzheimer's disease (AD) brains, compared with controls. Its metabolic products are elevated in cerebrospinal fluid of patients with AD and individuals with mild cognitive impairment, suggesting that this enzyme may be involved early in AD pathogenesis. Herein, we investigate the effect of pharmacologic inhibition of 12/15LO on the amyloid beta precursor protein (APP) metabolism. To this end, we used CHO and N2A cells stably expressing human APP with the Swedish mutant, and two structurally distinct and selective 12/15LO inhibitors, PD146176 and CDC. Our results demonstrated that both drugs dose-dependently reduced Abeta formation without affecting total APP levels. Interestingly, in the same cells we observed a significant reduction in secreted (s)APPbeta and beta-secretase (BACE), but not sAPPalpha and ADAM10 protein levels. Together, these data show for the first time that this enzymatic pathway influences Abeta formation whereby modulating the BACE proteolytic cascade. We conclude that specific pharmacologic inhibition of 12/15LO could represent a novel therapeutic target for treating or preventing AD pathology in humans.

PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.

Science.gov (United States)

Chevalier, Christophe; Al Bazzal, Ali; Vidic, Jasmina; Février, Vincent; Bourdieu, Christiane; Bouguyon, Edwige; Le Goffic, Ronan; Vautherot, Jean-François; Bernard, Julie; Moudjou, Mohammed; Noinville, Sylvie; Chich, Jean-François; Da Costa, Bruno; Rezaei, Human; Delmas, Bernard

2010-04-23

The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.

Assessment of response to beta-blockers by expression of βArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients

DEFF Research Database (Denmark)

Trebicka, Jonel; von Heydebrand, Matthias; Lehmann, Jennifer

2016-01-01

BACKGROUND & AIMS: Non-selective beta-blockers (NSBB) are first choice for prevention of variceal bleeding. But possible deleterious effects in refractory ascites and frequent non-response are clinical drawbacks. Since levels of vasoactive proteins in antrum mucosa reflect vascular dysfunction...... and protein expression of Ras homolog family member A (RhoA), Rho-kinase (ROCK)2, beta-arrestin2 (βArr2), endothelial nitric oxide synthase (eNOS) and the phosphorylation of downstream effectors VASP and moesin were analyzed using PCR and Western blot. Further 21 patients on NSBB were evaluated...

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

Science.gov (United States)

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Expression, refolding and crystallization of murine MHC class I H-2Db in complex with human β2-microglobulin

International Nuclear Information System (INIS)

Sandalova, Tatyana; Michaëlsson, Jakob; Harris, Robert A.; Ljunggren, Hans-Gustaf; Kärre, Klas; Schneider, Gunter; Achour, Adnane

2005-01-01

Mouse MHC class I H-2Db in complex with human β2m and the LCMV-derived peptide gp33 has been produced and crystallized. Resolution of the structure of this complex combined with the structural comparison with the previously solved crystal structure of H-2Db/mβ2m/gp33 should lead to a better understanding of how the β2m subunit affects the overall conformation of MHC complexes as well as the stability of the presented peptides. β 2 -Microglobulin (β 2 m) is non-covalently linked to the major histocompatibility (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I can bind human β 2 m (hβ 2 m) and such hybrid molecules are often used in structural and functional studies. The replacement of mouse β 2 m (mβ 2 m) by hβ 2 m has important functional consequences for MHC class I complex stability and specificity, but the structural basis for this is unknown. To investigate the impact of species-specific β 2 m subunits on MHC class I conformation, murine MHC class I H-2D b in complex with hβ 2 m and the peptide gp33 derived from lymphocytic choriomeningitis virus (LCMV) has been expressed, refolded in vitro and crystallized. Crystals containing two complexes per asymmetric unit and belonging to the space group P2 1 , with unit-cell parameters a = 68.1, b = 65.2, c = 101.9 Å, β = 102.4°, were obtained

Secondary Structure Preferences of Mn2+ Binding Sites in Bacterial Proteins

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Tatyana Aleksandrovna Khrustaleva

2014-01-01

Full Text Available 3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used. Major Mn2+ binders are aspartic acid (6.82% of Asp residues, histidine (14.76% of His residues, and glutamic acid (3.51% of Glu residues. We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88% does not depend on genomic GC-content.

Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched alpha-lactalbumin and beta-lactoglobulin food ingredients

Science.gov (United States)

A potentially economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (SCO2) as an acid to produce enriched fractions of alpha-lactalbumin (a-LA) and beta-lactoglobulin (b-LG) from whey protein isolate. To prepare the fractions, so...

Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

DEFF Research Database (Denmark)

Niefind, K; Raaf, J; Issinger, Olaf-Georg

2009-01-01

the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP......Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

Conformational interconversions in peptide beta-turns: analysis of turns in proteins and computational estimates of barriers.

Science.gov (United States)

Gunasekaran, K; Gomathi, L; Ramakrishnan, C; Chandrasekhar, J; Balaram, P

1998-12-18

The two most important beta-turn features in peptides and proteins are the type I and type II turns, which differ mainly in the orientation of the central peptide unit. Facile conformational interconversion is possible, in principle, by a flip of the central peptide unit. Homologous crystal structures afford an opportunity to structurally characterize both possible conformational states, thus allowing identification of sites that are potentially stereochemically mobile. A representative data set of 250 high-resolution (turns that are assigned different conformational types (type I/type II) in related structures. A total of 55 examples of beta-turns were identified as possible candidates for a stereochemically mobile site. Of the 55 examples, 45 could be classified as a potential site for interconversion between type I and type II beta-turns, while ten correspond to flips from type I' to type II' structures. As a further check, the temperature factors of the central peptide unit carbonyl oxygen atom of the 55 examples were examined. The analysis reveals that the turn assignments are indeed reliable. Examination of the secondary structures at the flanking positions of the flippable beta-turns reveals that seven examples occur in the loop region of beta-hairpins, indicating that the formation of ordered secondary structures on either side of the beta-turn does not preclude local conformational variations. In these beta-turns, Pro (11 examples), Lys (nine examples) and Ser (seven examples) were most often found at the i+1 position. Glycine was found to occur overwhelmingly at position i+2 (28 examples), while Ser (seven examples) and Asn (six examples) were amongst the most frequent residues. Activation energy barriers for the interconversion between type I and type II beta-turns were computed using the peptide models Ac-Pro-Aib-NHMe and Ac-Pro-Gly-NHMe within the framework of the AM1 semi-empirical molecular orbital procedure. In order to have a uniform basis for

DMPD: Immunoreceptor-like signaling by beta 2 and beta 3 integrins. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17913496 Immunoreceptor-like signaling by beta 2 and beta 3 integrins. Jakus Z, Fod...) Show Immunoreceptor-like signaling by beta 2 and beta 3 integrins. PubmedID 17913496 Title Immunoreceptor-...like signaling by beta 2 and beta 3 integrins. Authors Jakus Z, Fodor S, Abram CL

Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

Energy Technology Data Exchange (ETDEWEB)

Selmi, Anna [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Malinowski, Mariusz [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Brutkowski, Wojciech [Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw (Poland); Bednarek, Radoslaw [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland)

2012-08-15

Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Complete deficiency of mitochondrial trifunctional protein due to a novel mutation within the beta-subunit of the mitochondrial trifunctional protein gene leads to failure of long-chain fatty acid beta-oxidation with fatal outcome

NARCIS (Netherlands)

Schwab, Karl Otfried; Ensenauer, Regina; Matern, Dietrich; Uyanik, Gökhan; Schnieders, Birgit; Wanders, Ronald A.; Lehnert, Willy

2003-01-01

The mitochondrial trifunctional protein (MTP) is a multienzyme complex which catalyses three of the four chain-shortening reactions in the beta-oxidation of long-chain fatty acids. Clinically, failure of long-chain fatty acid beta-oxidation leads to hypoketotic hypoglycaemia associated with coma,

Reduced glomerular size- and charge-selectivity in clinically healthy individuals with microalbuminuria

DEFF Research Database (Denmark)

Jensen, J S; Borch-Johnsen, K; Deckert, T

1995-01-01

The pathophysiologic mechanism behind microalbuminuria, a potential atherosclerotic risk factor, was explored by measuring fractional clearances of four endogenous plasma proteins of different size and electric charge (albumin, beta 2-microglobulin, immunoglobulin G, and immunoglobulin G4). Twenty......-eight clinically healthy individuals with microalbuminuria, defined as a urinary albumin excretion of 6.6-150 micrograms min-1, and 60 matched control subjects were studied. Fractional immunoglobulin G clearance was higher (geometric means (95% confidence intervals)) 3.0 (2.3-3.9) x 10(-6), n = 28, vs. 2.1 (1...

Chicken type II collagen induced immune tolerance of mesenteric lymph node lymphocytes by enhancing beta2-adrenergic receptor desensitization in rats with collagen-induced arthritis.

Science.gov (United States)

Zhao, Wei; Tong, Tong; Wang, Ling; Li, Pei-Pei; Chang, Yan; Zhang, Ling-Ling; Wei, Wei

2011-01-01

Chicken type II collagen (CCII) is a protein extracted from the cartilage of chicken breast and exhibits intriguing possibilities for the treatment of autoimmune diseases by inducing oral tolerance. In this study, we investigated the effects of CCII on inflammatory and immune responses to the mesenteric lymph node lymphocytes (MLNLs) and the mechanisms by which CCII regulates beta2-adrenergic receptor (beta2-AR) signal transduction in collagen-induced arthritis (CIA) rats. The onset of secondary arthritis in rats appeared around day 14 after injection of CCII emulsion. Remarkable secondary inflammatory response and lymphocytes proliferation were observed in CIA rats. The administration of CCII (10, 20, 40μgkg(-1)day(-1), days 15-22) could significantly reduce synovial hyperplasia, lymphatic follicle hyperplasia, inflammatory cells infiltration of MLNLs in CIA rats. CCII (10, 20, 40μgkg(-1)day(-1), days 15-22) restored the previously decreased level of cAMP of MLNLs of CIA rats. Meanwhile, CCII increased total protein expressions of beta2-AR, GRK2 and decreased that of beta-arrestin1, 2 of MLNLs in CIA rats but had an slight effect on GRK3. CCII further increased plasmatic protein expressions of GRK2, G(α)s and decreased that of beta-arrestin1, 2, beta2-AR, and increased membrane protein expressions of beta2-AR, GRK2, G(α)s and decreased that of beta-arrestin1, 2 of MLNLs in CIA rats. These results demonstrate that the mechanisms of CCII on beta2-AR desensitization and beta2-AR-AC-cAMP transmembrane signal transduction of MLNLs play crucial roles in pathogenesis of this disease. Copyright © 2010 Elsevier B.V. All rights reserved.

Role of TGF-beta1-independent changes in protein neosynthesis, p38alphaMAPK, and cdc42 in hydrogen peroxide-induced senescence-like morphogenesis

DEFF Research Database (Denmark)

Chrétien, Aline; Dierick, Jean-François; Delaive, Edouard

2008-01-01

for p38(MAPK) activation, in turn triggering phosphorylation of L-caldesmon and HSP27. Cdc42 was also shown to be mainly responsible for the increase in TGF-beta1 mRNA level observed at 24 h after treatment with H(2)O(2) and onward. This study further clarified the mechanisms of senescence......The role of TGF-beta1 in hydrogen peroxide-induced senescence-like morphogenesis has been described. The aim of this work was to investigate whether TGF-beta1-independent changes in protein synthesis are involved in this morphogenesis and to study possible mechanisms occurring earlier than TGF-beta......1 overexpression. Among the multiple TGF-beta1-independent changes in protein neosynthesis, followed or not by posttranslational modifications, identified by proteomic analysis herein, those of ezrin, L-caldesmon, and HSP27 were particularly studied. Rho-GTPase cdc42 was shown to be responsible...

Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

Directory of Open Access Journals (Sweden)

Serena Battaglia

Full Text Available BACKGROUND: Chronic hepatitis C virus (HCV infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT, a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.

Progesterone-associated proteins PP12 and PP14 in the human endometrium.

Science.gov (United States)

Rutanen, E M; Koistinen, R; Seppälä, M; Julkunen, M; Suikkari, A M; Huhtala, M L

1987-01-01

Two proteins, designated as PP12 and PP14 were originally isolated from soluble extracts of the human placenta and its adjacent membranes. We have shown that they are synthesized by decidualized/secretory endometrium and not by placenta. Both proteins occur at high concentrations in human amniotic fluid, which is therefore an excellent source for purification. PP12 is a 34-kDa glycoprotein, which has an N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala. This is identical with that of somatomedin-binding protein purified from the amniotic fluid. PP12 too binds somatomedin-C, or IGF-I (insulin-like growth factor-I). Human secretory endometrium synthesizes and secretes PP12, and progesterone stimulates its secretion. PP14 is a 28-kDa glycoprotein. Its N-terminal sequence shows homology to that of beta-lactoglobulins from various species. We have found PP14 in the human endometrium, serum and milk. Immunologically, PP14 is related to progestagen-associated endometrial protein (PEP), alpha-2 pregnancy-associated endometrial protein (alpha-2, PEG), endometrial protein 15 (EP15), alpha-uterine protein (AUP) and chorionic alpha-2 microglobulin (CAG-2). In ovulatory menstrual cycles, the concentration of PP14 increases in endometrial tissue as the secretory changes advance. In serum, the PP14 concentration begins to rise later than the progesterone levels, and high serum PP14 levels are maintained for the first days of the next cycle. By contrast, no elevation of serum PP14 level is seen in anovulatory cycles. Our results show that progesterone-associated proteins are synthesized by the human endometrium and appear in the peripheral circulation, where they can be quantitatively measured using immunochemical techniques.

HLA-B27 and human β2-microglobulin affect the gut microbiota of transgenic rats.

Directory of Open Access Journals (Sweden)

Phoebe Lin

Full Text Available The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m, compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

Prediction of the location and type of beta-turns in proteins using neural networks.

OpenAIRE

Shepherd, A. J.; Gorse, D.; Thornton, J. M.

1999-01-01

A neural network has been used to predict both the location and the type of beta-turns in a set of 300 nonhomologous protein domains. A substantial improvement in prediction accuracy compared with previous methods has been achieved by incorporating secondary structure information in the input data. The total percentage of residues correctly classified as beta-turn or not-beta-turn is around 75% with predicted secondary structure information. More significantly, the method gives a Matthews cor...

Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

DEFF Research Database (Denmark)

Claesson, M H; Endel, B; Ulrik, J

1994-01-01

Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b...

Distance-Based Configurational Entropy of Proteins from Molecular Dynamics Simulations.

Science.gov (United States)

Fogolari, Federico; Corazza, Alessandra; Fortuna, Sara; Soler, Miguel Angel; VanSchouwen, Bryan; Brancolini, Giorgia; Corni, Stefano; Melacini, Giuseppe; Esposito, Gennaro

2015-01-01

Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements;the conformational entropy of folding of β2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state;backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy);the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of β2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic.

Urinary trehalase activity as an indicator of kidney injury due to environmental cadmium exposure

Energy Technology Data Exchange (ETDEWEB)

Iwata, Kohkichi; Katoh, Terutaka; Morikawa, Yuuko; Aoshima, Keiko; Nishijo, Muneko; Teranishi, Hidetoyo; Kasuya, Minoru

1988-12-01

One hundred and seventy-eight subjects, patients with Itai-itai disease and their family members, aged 12-87 years living in a cadmium (Cd)-polluted area in the Jinzu River basin (Cd-exposed group) and 176 controls (control group) were examined. In the Cd-exposed group urinary trehalase increased with increasing age, urinary /beta/2-microglobulin (/beta/2-m) and retinol-binding protein. Although urinary cadmium was higher in the Cd-exposed group, no particular correlation was found between urinary trehalase and urinary cadmium. Seventeen men and 11 women showed raised urinary trehalase activities despite normal values of urinary /beta/2-m (<300 /mu/g/g.creatinine), suggesting that urinary trehalase increases earlier than urinary /beta/2-m. In 19 patients with Itai-itai disease included in the Cd-exposed group, urinary trehalase decreased with decreasing reciprocal of serum creatinine, suggesting that urinary trehalase decreases in the most advanced cases of chronic cadmium nephropathy due to reduced tubular cell mass. (orig.).

Lead induces chondrogenesis and alters transforming growth factor-beta and bone morphogenetic protein signaling in mesenchymal cell populations.

Science.gov (United States)

Zuscik, Michael J; Ma, Lin; Buckley, Taylor; Puzas, J Edward; Drissi, Hicham; Schwarz, Edward M; O'Keefe, Regis J

2007-09-01

It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton. The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways. We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), beta-catenin, AP-1, and nuclear factor-kappa B (NF-kappaB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis in vivo. Pb-exposed MSCs showed enhanced basal and TGF-beta/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-beta but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/beta-catenin pathway activity, it induced NFkappaB signaling and inhibited AP-1 signaling. The in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-beta, BMP, AP-1, and NFkappaB.

Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

Science.gov (United States)

Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

1999-01-01

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

Global regulator SATB1 recruits beta-catenin and regulates T(H2 differentiation in Wnt-dependent manner.

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Dimple Notani

2010-01-01

Full Text Available In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1, the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H2 specific transcription factor that regulates production of T(H2 cytokines and functions as T(H2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+ T cells, suggesting that SATB1 influences T(H2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1, an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H2 cells in a Wnt-dependent manner. SATB1 coordinates T(H2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1

Identification of beta 2-adrenoceptors on guinea pig alveolar macrophages using (-)-3-[125I]iodocyanopindolol

International Nuclear Information System (INIS)

Leurs, R.; Beusenberg, F.D.; Bast, A.; Van Amsterdam, J.G.; Timmerman, H.

1990-01-01

The beta-adrenoceptor antagonist (-)-3-[ 125 I]iodocyanopindolol ([ 125 I]ICYP) binds with high affinity and in saturable way to membranes of guinea pig alveolar macrophages. The equilibrium dissociation constant for [ 125 I]ICYP is 24.3 +/- 1.2 pM, and the number of binding sites is 166.3 +/- 13.7 fmol/mg protein (N = 4, +/- SEM). Displacement studies with selective antagonists showed that [ 125 I]ICYP labels beta 2-adrenoceptors on guinea pig alveolar macrophages

The change of transforming growth factor {beta} 1 (TGF- {beta} 1) expression by melatonin in irradiated lung

Energy Technology Data Exchange (ETDEWEB)

Jang, Seong Soon; Choi, Ihl Bohng [College of Medicine, The Catholic University of Korea, Seoul (Korea, Republic of)

2005-09-15

The changed expressions of TGF- {beta} 1, as a key cytokine in the fibrotic process, due to melatonin with potent antioxidative effects, were investigated in the irradiated lung using fibrosis-sensitive C57BL/6 mice. Female C57BL/6 mice were divided into control irradiation-only, and melatonin (300 mg/kg i.p. 1 hr before irradiation) pretreatment groups. The thoraces of the mice were irradiated with a single dose of 12 Gy. The mRNA expressions of TGF-{beta} 1 in the lung tissue 2 and 4 weeks after irradiation were quantified using semiquantitive RT-PCR, and the cellular origin and expression levels of TGF- {beta} 1 protein were identified using immunohistochemical staining. The relative mRNA expression levels in the irradiation-only and melatonin pretreatment group 2 and 4 weeks after irradiation were 1.92- and 1.80-fold ({rho} = 0.064) and 2.38- and 1.94-fold ({rho} = 0.004) increased, respectively compared to those in the control group. Increased expressions of TGF- {beta} 1 protein were prominently detected in regions of histopathological radiation injury, with alveolar macrophages and septal epithelial cells serving as important sources of TGF- {beta} 1 expression. At 2 and 4 weeks after irradiation, the expression levels of protein were 15.8% vs. 16.9% ({rho} = 0.565) and 36.1% vs. 25.7% ({rho} = 0.009), respectively. The mRNA and protein expressions of TGF- {beta} 1 in the lung tissue following thoracic irradiation with 12 Gy were significantly decreased by melatonin pretreatment at 4 weeks. These results indicate that melatonin may have a possible application as an antifibrotic agent in radiation-induced lung injury.

Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents

DEFF Research Database (Denmark)

Christensen, Gitte L.; Jacobsen, Maria L. B.; Wendt, Anna

2015-01-01

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression...

Isolation and characterization of ScGluD2, a new sugarcane beta-1,3-glucanase D family gene induced by Sporisorium scitamineum, ABA, H2O2, NaCl, and CdCl2 stresses

Directory of Open Access Journals (Sweden)

Yachun Su

2016-09-01

Full Text Available Beta-1,3-glucanases (EC 3.2.1.39, commonly known as pathogenesis-related (PR proteins, play an important role not only in plant defense against fungal pathogens but also in plant physiological and developmental processes. However, only a limited number of sugarcane beta-1,3-glucanase genes have been isolated. In the present study, we identified and characterized a new beta-1,3-glucanase gene ScGluD2 (GenBank Acc No. KF664181 from sugarcane. An X8 domain was present at the C terminal region of ScGluD2, suggesting beta-1,3-glucan-binding function. Phylogenetic analysis showed that the predicted ScGluD2 protein was classified into subfamily D beta-1,3-glucanase. Localization of the ScGluD2 protein in the plasma membrane was determined by tagging it with green fluorescent protein. The expression of ScGluD2 was more up-regulated in sugarcane smut-resistant cultivars in the early stage (1 d or 3 d than in the susceptible ones after being challenged by the smut pathogen, revealing that ScGluD2 may be involved in defense against the invasion of Sporisorium scitamineum. Transient overexpression of ScGluD2 in Nicotiana benthamiana leaves induced a defense response and exhibited antimicrobial action on the tobacco pathogens Pseudomonas solanacearum and Botrytis cinerea, further demonstrating that ScGluD2 was related to the resistance to plant pathogens. However, the transcripts of ScGluD2 partially increased (12 h under NaCl stress, and were steadily up-regulated from 6 h to 24 h upon ABA, H2O2, and CdCl2 treatments, suggesting that ABA may be a signal molecule regulating oxidative stress and play a role in the salt and heavy metal stress-induced stimulation of ScGluD2 transcripts. Taken together, ScGluD2, a novel member of subfamily D beta-1,3-glucanase, was a stress-related gene of sugarcane involved in plant defense against smut pathogen attack and salt and heavy metal stresses.

Phosphatidic acid regulates signal output by G protein coupled receptors through direct interaction with phospholipase C-beta(1).

Science.gov (United States)

Litosch, Irene; Pujari, Rajeshree; Lee, Shawn J

2009-09-01

Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-beta(1) (PLC-beta(1)) and potentiates stimulation of PLC-beta(1) activity by Galpha(q) in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944-957, lying within the PLC-beta(1) C-terminus alphaA helix and flexible loop of the Galpha(q) binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Galpha(q) or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr(952) and Ile(955) as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr(952) and Ile(955), PLC-beta(1) (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-beta(1) in vitro. COS 7 cells co-transfected with PLC-beta(1) (Y952G/I955G) demonstrate a 10-fold increase in the EC(50) for stimulation and a 60% decrease in maximum stimulation by carbachol via Galpha(q) linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-beta(1) but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-beta(1) and PLC-beta(1) (Y952G/I955G) activity is increased comparably by co-transfection with Galpha(q) and neither is markedly affected by co-transfection with Gbeta(1)gamma(2). Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC(50) for agonist stimulation of WT-PLC-beta(1) by 10-fold

Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics.

Science.gov (United States)

Shen, Hongbo; Xu, Feng; Hu, Hairong; Wang, Feifei; Wu, Qi; Huang, Qiang; Wang, Honghai

2008-12-01

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (beta/alpha)(8)-barrel proteins-the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (betaalpha)(8)-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (beta/alpha)(8)-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (beta/alpha)(8)-barrel scaffold.

The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

Energy Technology Data Exchange (ETDEWEB)

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

2012-06-01

Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V{alpha}/V{beta

[Effect of ginseng polysaccharide-induced wnt/beta-catenin signal transduction pathway on apoptosis of human nasopharyngeal cancer cells CNE-2].

Science.gov (United States)

Fan, Jia-Ming; Liu, Ze-Hong; Li, Jing; Wang, Ya-Ping; Yang, Lv-Yuan; Huang, Jiang-Ju

2013-10-01

To observe the effect of ginseng polysaccharide (GPS) on the proliferation and apoptosis of human nasopharyngeal cancer cells CNE-2, and discuss the possible mechanism. The effect of GPS on the growth of CNE-2 cells was observed by CCK8 assay. CNE-2 cells in the logarithmic phase were collected and processed respectively with different concentrations (0, 0. 1, 0. 2, 0. 3. 0. 4 g L-1) of GPS for 48 h. The flow cytometry was used to detect its induction effect on CNE-2 cell apoptosis. Hoechst-33258 cell staining and electron microscope were used to observe the morphological changes of cells. The beta-catenin mRNA expression was detected by Real-time PCR. The protein localizations and expressions of beta-catenin and TCF4 were tested by the immunofluorescence staining. The expressions of beta-catenin, Bcl-2 and Bax proteins were detected by Western blot. CCK8 assay results showed that GPS could remarkably inhibit the proliferation of CNE-2 cells, with dose-time dependence. IC50 of cells induced with GPS for 48 h was 0. 39 g L-1. After being processed with GPS with concentrations of 0.1, 0. 2, 0. 3, 0. 4 g L-1 for 48 h, the cell apoptosis rates of human nasopharyngeal cancer cells CNE-2 were (5. 69 +/- 0. 29)% , (10. 3 +/- 0. 63)% , (15. 4 +/- 0. 74 ) % and (35. 7 +/- 1. 86)% , respectively. Significant difference was observed compared with the control group (2. 08 +/- 0. 11) % (P GPS with the concentration of 0. 4 g L -1 for 48 h. The results of Real-time PCR showed a significant reduction in beta-catenin mRNA expression. The results of laser confocal microscopy revealed notable decrease of beta-catenin and TCF4 expression in nucleus and transfer from nucleus to cell membranes in beta-catenin expression areas after being processed with GPS for 48 h. Western blot showed significant decrease in the expressions of beta-catenin and anti-apoptosis protein Bcl-2, with an increasing expression in apoptosis-promoting protein Bax (P GPS could significantly inhibit the

Renal effects of chronic exposure to organic solvents. A clinical controlled trial

Energy Technology Data Exchange (ETDEWEB)

Krusell, L.; Nielsen, H.K.; Baelum, J.; Lundqvist, G.; Omland, O.; Vaeth, M.; Husted, S.E.; Mogensen, C.E.; Geday, E.

1985-01-01

Chronic effects of organic solvents on renal function were measured by creatinine clearances and urinary excretion rates of beta 2-microglobulin and albumin. Forty-three male printing trade workers occupationally exposed to different organic solvents for 9-25 years were compared with 43 age-matched male controls. No differences were found either in creatinine clearances or average basal levels of beta 2-microglobulin and albumin excretion rates, whereas a positive relation could be demonstrated between alcohol consumption on the day before the trial and urinary excretion rate of albumin. This investigation did not reveal any adverse renal effects of moderate chronic exposure to organic solvents in a group of active trade workers.

Renal effects of chronic exposure to organic solvents. A clinical controlled trial

DEFF Research Database (Denmark)

Krusell, Lars Romer; Nielsen, H K; Bælum, Jesper

1985-01-01

Chronic effects of organic solvents on renal function were measured by creatinine clearances and urinary excretion rates of beta 2-microglobulin and albumin. Forty-three male printing trade workers occupationally exposed to different organic solvents for 9-25 years were compared with 43 age......-matched male controls. No differences were found either in creatinine clearances or average basal levels of beta 2-microglobulin and albumin excretion rates, whereas a positive relation could be demonstrated between alcohol consumption on the day before the trial and urinary excretion rate of albumin....... This investigation did not reveal any adverse renal effects of moderate chronic exposure to organic solvents in a group of active trade workers....

Role of beta-adrenoceptors in memory consolidation: beta3-adrenoceptors act on glucose uptake and beta2-adrenoceptors on glycogenolysis.

Science.gov (United States)

Gibbs, Marie E; Hutchinson, Dana S; Summers, Roger J

2008-09-01

Noradrenaline, acting via beta(2)- and beta(3)-adrenoceptors (AR), enhances memory formation in single trial-discriminated avoidance learning in day-old chicks by mechanisms involving changes in metabolism of glucose and/or glycogen. Earlier studies of memory consolidation in chicks implicated beta(3)- rather than beta(2)-ARs in enhancement of memory consolidation by glucose, but did not elucidate whether stimulation of glucose uptake or of glycolysis was responsible. This study examines the role of glucose transport in memory formation using central injection of the nonselective facilitative glucose transporter (GLUT) inhibitor cytochalasin B, the endothelial/astrocytic GLUT-1 inhibitor phloretin and the Na(+)/energy-dependent endothelial glucose transporter (SGLT) inhibitor phlorizin. Cytochalasin B inhibited memory when injected into the mesopallium (avian cortex) either close to or between 25 and 45 min after training, whereas phloretin and phlorizin only inhibited memory at 30 min. This suggested that astrocytic/endothelial (GLUT-1) transport is critical at the time of consolidation, whereas a different transporter, probably the neuronal glucose transporter (GLUT-3), is important at the time of training. Inhibition of glucose transport by cytochalasin B, phloretin, or phlorizin also interfered with beta(3)-AR-mediated memory enhancement 20 min posttraining, whereas inhibition of glycogenolysis interfered with beta(2)-AR agonist enhancement of memory. We conclude that in astrocytes (1) activities of both GLUT-1 and SGLT are essential for memory consolidation 30 min posttraining; (2) neuronal GLUT-3 is essential at the time of training; and (3) beta(2)- and beta(3)-ARs consolidate memory by different mechanisms; beta(3)-ARs stimulate central glucose transport, whereas beta(2)-ARs stimulate central glycogenolysis.

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2alpha/CK2beta interaction

DEFF Research Database (Denmark)

Hochscherf, Jennifer; Lindenblatt, Dirk; Steinkrueger, Michaela

2015-01-01

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2alpha and CK2beta in addition to established a...

Apolipoprotein M predicts pre-beta-HDL formation: studies in type 2 diabetic and nondiabetic subjects

DEFF Research Database (Denmark)

Plomgaard, P; Dullaart, R P F; de Vries, R

2009-01-01

protein (PLTP) activity and the ability of plasma to promote cholesterol efflux from cultured fibroblasts. RESULTS: ApoM was approximately 9% lower in patients with type 2 diabetes compared to controls (0.025 +/- 0.006 vs. 0.027 +/- 0.007 g L(-1), P = 0.01). The difference in apoM was largely attributable...... diabetes. Pre-beta-HDL and pre-beta-HDL formation are positively associated with apoM, supporting the hypothesis that apoM plays a role in HDL remodelling in humans. Lower apoM may provide a mechanism to explain why pre-beta-HDL formation is not increased in type 2 diabetes despite elevated PLTP activity.......OBJECTIVE: Studies in mice suggest that plasma apoM is lowered in hyperinsulinaemic diabetes and that apoM stimulates formation of pre-beta-HDL. Pre-beta-HDL is an acceptor of cellular cholesterol and may be critical for reverse cholesterol transport. Herein, we examined whether patients with type...

β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+ pathway in xenopus gastrulation.

Directory of Open Access Journals (Sweden)

Katharina Seitz

Full Text Available β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+ cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+ signaling cascade upstream of Protein Kinase C (PKC and Ca(2+/Calmodulin-dependent Protein Kinase II (CamKII. We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

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Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 is not responsible for sodium retention in nephrotic rats

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Bistrup, C; Thiesson, H C; Jensen, B L

2005-01-01

AIM: In mineralocorticoid target cells 11-beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) converts glucocorticoids into non-active metabolites thereby protecting the mineralocorticoid receptor (MR) from stimulation by glucocorticoids. In nephrotic syndrome, a decreased activity of 11betaHSD2...... has been suggested to allow glucocorticoids to stimulate MR, thereby contributing to sodium retention. We tested this hypothesis in the puromycin aminonucleoside model of nephrotic syndrome in rats. METHODS: Complete sodium and potassium intakes and excretions (faeces and urine) were measured in rats......)] to suppress endogenous glucocorticoids in the proteinuric stage during active sodium retention. RESULTS: Nephrotic rats developed proteinuria, positive sodium balance, decreased plasma aldosterone concentration, and decreased urinary Na(+)/K(+) ratio. 11betaHSD2 mRNA expression was down-regulated but protein...

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

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van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

2002-06-21

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

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Stalter, G; Siemer, S; Becht, E

1994-01-01

of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

Polymorphisms within novel risk loci for type 2 diabetes determine beta-cell function.

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Harald Staiger

Full Text Available BACKGROUND: Type 2 diabetes arises when insulin resistance-induced compensatory insulin secretion exhausts. Insulin resistance and/or beta-cell dysfunction result from the interaction of environmental factors (high-caloric diet and reduced physical activity with a predisposing polygenic background. Very recently, genetic variations within four novel genetic loci (SLC30A8, HHEX, EXT2, and LOC387761 were reported to be more frequent in subjects with type 2 diabetes than in healthy controls. However, associations of these variations with insulin resistance and/or beta-cell dysfunction were not assessed. METHODOLOGY/PRINCIPAL FINDINGS: By genotyping of 921 metabolically characterized German subjects for the reported candidate single nucleotide polymorphisms (SNPs, we show that the major alleles of the SLC30A8 SNP rs13266634 and the HHEX SNP rs7923837 associate with reduced insulin secretion stimulated by orally or intravenously administered glucose, but not with insulin resistance. In contrast, the other reported type 2 diabetes candidate SNPs within the EXT2 and LOC387761 loci did not associate with insulin resistance or beta-cell dysfunction, respectively. CONCLUSIONS/SIGNIFICANCE: The HHEX and SLC30A8 genes encode for proteins that were shown to be required for organogenesis of the ventral pancreas and for insulin maturation/storage, respectively. Therefore, the major alleles of type 2 diabetes candidate SNPs within these genetic loci represent crucial alleles for beta-cell dysfunction and, thus, might confer increased susceptibility of beta-cells towards adverse environmental factors.

Insights into alternative prion protein topologies induced under high hydrostatic pressure

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Torrent, Joan [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Alvarez-Martinez, Maria Teresa [INSERM U431, IFR 122, Place Eugene Bataillon, F-34095 Montpellier cedex 5 (France); Heitz, Frederic [CRBM, CNRS-UPR 1086, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Liautard, Jean-Pierre [INSERM U431, IFR 122, Place Eugene Bataillon, F-34095 Montpellier cedex 5 (France); Balny, Claude [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France); Lange, Reinhard [INSERM U128, IFR 122, 1919 Route de Mende, F-34293 Montpellier cedex 5 (France)

2004-04-14

The critical step in the pathogenesis of transmissible spongiform encephalopathies (TSEs) appears to be a conformational transition of a normal prion protein (PrP{sup C}) into a misfolded isoform (PrP{sup Sc}). To gain insight into the structural conversion of the prion protein we have exploited the use of high hydrostatic pressure combined with various spectroscopic techniques. In vitro transitions of the recombinant PrP to a scrapie-like form have never resulted in an infectious structure. It is our hypothesis that the acquisition of the disease-causing conformation depends on folding pathways which are difficult to attain. We attempt to favour, via specific reaction conditions at high pressure, alternative routes of misfolding leading to a stable infectious amyloidogenic conformer. Our results have demonstrated the potential of high pressure to reveal various prion structural changes, which are inaccessible by conventional methods. Especially, we have characterized a pressure-induced conformer in which the normal {alpha}-helical structure is changed into a highly aggregated {beta}-sheet conformation showing markedly increased resistance to proteolysis (key markers of potential infectious agents). Our work may have important implications, not only for ultimately proving the protein-only hypothesis and for understanding the basic mechanism of the disease, but also for developing preventative and therapeutic measures.

Increased expression of the auxiliary beta(2-subunit of ventricular L-type Ca(2+ channels leads to single-channel activity characteristic of heart failure.

Directory of Open Access Journals (Sweden)

Roger Hullin

2007-03-01

Full Text Available Increased activity of single ventricular L-type Ca(2+-channels (L-VDCC is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation.By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1 or beta(3 isoforms, beta(2a and beta(2b induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V1.2 also reveal increased single-channel activity and sarcolemmal beta(2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase", reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2 expression. Additional evidence for the cause-effect relationship between beta(2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V1.2 and inducible beta(2 cardiac overexpression. Here in non-failing hearts induction of beta(2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure.Our study presents evidence of the pathobiochemical relevance of beta(2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Molecular cloning and characterization of a human beta-Gal-3'-sulfotransferase that acts on both type 1 and type 2 (Gal beta 1-3/1-4GlcNAc-R) oligosaccharides.

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Honke, K; Tsuda, M; Koyota, S; Wada, Y; Iida-Tanaka, N; Ishizuka, I; Nakayama, J; Taniguchi, N

2001-01-05

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing beta-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal beta 1-3GalNAc alpha-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO(3)-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1- 4Glc-PA by two-dimensional (1)H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal beta 1-3GlcNAc-R) and type 2 (Gal beta 1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel beta-Gal-3'-sulfotransferase gene family.

CPAD, Curated Protein Aggregation Database: A Repository of Manually Curated Experimental Data on Protein and Peptide Aggregation.

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Thangakani, A Mary; Nagarajan, R; Kumar, Sandeep; Sakthivel, R; Velmurugan, D; Gromiha, M Michael

2016-01-01

Accurate distinction between peptide sequences that can form amyloid-fibrils or amorphous β-aggregates, identification of potential aggregation prone regions in proteins, and prediction of change in aggregation rate of a protein upon mutation(s) are critical to research on protein misfolding diseases, such as Alzheimer's and Parkinson's, as well as biotechnological production of protein based therapeutics. We have developed a Curated Protein Aggregation Database (CPAD), which has collected results from experimental studies performed by scientific community aimed at understanding protein/peptide aggregation. CPAD contains more than 2300 experimentally observed aggregation rates upon mutations in known amyloidogenic proteins. Each entry includes numerical values for the following parameters: change in rate of aggregation as measured by fluorescence intensity or turbidity, name and source of the protein, Uniprot and Protein Data Bank codes, single point as well as multiple mutations, and literature citation. The data in CPAD has been supplemented with five different types of additional information: (i) Amyloid fibril forming hexa-peptides, (ii) Amorphous β-aggregating hexa-peptides, (iii) Amyloid fibril forming peptides of different lengths, (iv) Amyloid fibril forming hexa-peptides whose crystal structures are available in the Protein Data Bank (PDB) and (v) Experimentally validated aggregation prone regions found in amyloidogenic proteins. Furthermore, CPAD is linked to other related databases and resources, such as Uniprot, Protein Data Bank, PUBMED, GAP, TANGO, WALTZ etc. We have set up a web interface with different search and display options so that users have the ability to get the data in multiple ways. CPAD is freely available at http://www.iitm.ac.in/bioinfo/CPAD/. The potential applications of CPAD have also been discussed.

17 beta-estradiol and tamoxifen upregulate estrogen receptor beta expression and control podocyte signaling pathways in a model of type 2 diabetes.

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Catanuto, Paola; Doublier, Sophie; Lupia, Enrico; Fornoni, Alessia; Berho, Mariana; Karl, Michael; Striker, Gary E; Xia, Xiaomei; Elliot, Sharon

2009-06-01

Diabetic nephropathy remains one of the most important causes of end-stage renal disease. This is particularly true for women from racial/ethnic minorities. Although administration of 17beta-estradiol to diabetic animals has been shown to reduce extracellular matrix deposition in glomeruli and mesangial cells, effects on podocytes are lacking. Given that podocyte injury has been implicated as a factor leading to the progression of proteinuria and diabetic nephropathy, we treated db/db mice, a model of type 2 diabetic glomerulosclerosis, with 17beta-estradiol or tamoxifen to determine whether these treatments reduce podocyte injury and decrease glomerulosclerosis. We found that albumin excretion, glomerular volume, and extracellular matrix accumulation were decreased in these mice compared to placebo treatment. Podocytes isolated from all treatment groups were immortalized and these cell lines were found to express the podocyte markers WT-1, nephrin, and the TRPC6 cation channel. Tamoxifen and 17beta-estradiol treatment decreased podocyte transforming growth factor-beta mRNA expression but increased that of the estrogen receptor subtype beta protein. 17beta-estradiol, but not tamoxifen, treatment decreased extracellular-regulated kinase phosphorylation. These data, combined with improved albumin excretion, reduced glomerular size, and decreased matrix accumulation, suggest that both 17beta-estradiol and tamoxifen may protect podocytes against injury and therefore ameliorate diabetic nephropathy.

The cleavage product of amyloid-β protein precursor sAβPPα modulates BAG3-dependent aggresome formation and enhances cellular proteasomal activity.

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Renziehausen, Jana; Hiebel, Christof; Nagel, Heike; Kundu, Arpita; Kins, Stefan; Kögel, Donat; Behl, Christian; Hajieva, Parvana

2015-01-01

Alzheimer's disease (AD) is the major age-associated form of dementia characterized by gradual cognitive decline. Aberrant cleavage of the amyloid-β protein precursor (AβPP) is thought to play an important role in the pathology of this disease. Two principal AβPP processing pathways exist: amyloidogenic cleavage of AβPP resulting in production of the soluble N-terminal fragment sAβPPβ, amyloid-β (Aβ), which accumulates in AD brain, and the AβPP intracellular domain (AICD) sAβPPα, p3 and AICD are generated in the non-amyloidogenic pathway. Prevalence of amyloidogenic versus non-amyloidogenic processing leads to depletion of sAβPPα and an increase in Aβ. Although sAβPPα is a well-accepted neurotrophic protein, molecular effects of this fragment remains unknown. Different studies reported impaired protein degradation pathways in AD brain, pointing to a role of disturbed proteasomal activity in the pathogenesis of this disease. Here we studied the possible role of sAβPPα in Bag3-mediated selective macroautophagy and proteasomal degradation. Employing human IMR90 cells, HEK 293 cells, and primary neurons, we demonstrate that sAβPPα prevents the proteotoxic stress-induced increase of Bag3 at the protein and at the mRNA level indicating a transcriptional regulation. Intriguingly, p62 and LC3, two other key players of autophagy, were not affected. Moreover, the formation and the accumulation of disease-related protein aggregates were significantly reduced by sAβPPα. Interestingly, there was a significant increase of proteasomal activity by sAβPPα as demonstrated by using various proteasome substrates. Our findings demonstrate that sAβPPα modulates Bag3 expression, aggresome formation, and proteasomal activity, thereby providing first evidence for a function of sAβPPα in the regulation of proteostasis.

Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

International Nuclear Information System (INIS)

Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

2014-01-01

Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro

Mapping of the gene encoding the. beta. -amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21

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Patterson, D.; Gardiner, K.; Kao, F.T.; Tanzi, R.; Watkins, P.; Gusella, J.F. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (USA))

1988-11-01

The gene encoding the {beta}-amyloid precursor protein has been assigned to human chromosome 21, as has a gene responsible for at least some cases of familial Alzheimer disease. Linkage studies strongly suggest that the {beta}-amyloid precursor protein and the product corresponding to familial Alzheimer disease are from two genes, or at least that several million base pairs of DNA separate the markers. The precise location of the {beta}-amyloid precursor protein gene on chromosome 21 has not yet been determined. Here the authors show, by using a somatic-cell/hybrid-cell mapping panel, in situ hybridization, and transverse-alternating-field electrophoresis, that the {beta}-amyloid precursor protein gene is located on chromosome 21 very near the 21q21/21q/22 border and probably within the region of chromosome 21 that, when trisomic, results in Down syndrome.

Detection of occupational lead nephropathy using early renal markers.

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Kumar, B D; Krishnaswamy, K

1995-01-01

Automotive use of leaded gasoline continues to be an important source of occupational exposure to lead in India and other countries. The present study assessed the renal function and markers of early renal damage of 22 mechanics at three automobile garages. Urinary N-acetyl-3-D-glucosaminidase activity and beta-2-microglobulin levels were significantly increased in auto garage mechanics with blood leads of 30-69 micrograms/dL. A significant correlation was observed between blood lead levels and urinary N-acetyl-3-D-glucosaminidase activity but not with urine beta-2-microglobulin levels. A marginal impairment in creatinine clearance was not statistically significant. Urinary N-acetyl-3-D-glucosaminidase activity offers a sensitive monitor of blood lead and renal tubular injury.

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

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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.

Transforming growth factor-beta messenger RNA and protein in murine colitis

DEFF Research Database (Denmark)

Whiting, C V; Williams, A M; Claesson, Mogens Helweg

2001-01-01

Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-beta transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-beta1 and TGF-beta3 mRNAs, but both were expressed significantly...... farther along the crypt axis in disease. Control lamina propria cells transcribed little TGF-beta1 or TGF-beta3 mRNA, but in inflamed tissues many cells expressed mRNA for both isoforms. No TGF-beta2 message was detected in either control or inflamed tissues. Immunohistochemistry for latent and active TGF...

Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

DEFF Research Database (Denmark)

Damstrup, L; Rygaard, K; Spang-Thomsen, M

1993-01-01

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....