Characterization and biodistribution of recombinant and recombinant/chimeric constructs of monoclonal antibody B72.3

International Nuclear Information System (INIS)

Colcher, D.; Milenic, D.; Roselli, M.

1989-01-01

Radiolabeled B72.3 has been administered both i.v. and i.p. in patients with colorectal and ovarian cancer as well as other carcinomas and has been shown to selectively bind to approximately 70-80% of metastatic lesions. Greater than 50% of the patients that have been treated with B72.3 have developed an immunological response to murine IgG after a single injection. In an attempt to minimize the immune response of these patients to the administered murine monoclonal antibody, we developed a recombinant form of the murine B72.3 as well as a recombinant/chimeric antibody, using the variable regions of the murine B72.3 and human heavy chain (gamma 4) and light chain (kappa) constant regions. We report here that both the recombinant B72.3 [rB72.3] and the recombinant/chimeric B72.3 [cB72.3(gamma 4)] IgGs maintain the tissue binding and idiotypic specificity of the native murine IgG. The native B72.3, rB72.3, and cB72.3(gamma 4) IgGs were radiolabeled and the biodistribution of these IgGs was studied in athymic mice bearing human colon carcinoma xenografts (LS-174T). Differences were observed between the cB72.3(gamma 4) and the native B72.3 in the percentage of injected dose/g that localized in the tumor. The somewhat lower absolute amounts of the cB72.3(gamma 4) in the tumor are mostly likely due to the observed more rapid clearance from the blood and body of the mouse as compared to the native B72.3 and rB72.3. All three forms [native B72.3, rB72.3, and cB72.3(gamma 4)] of the IgG, however, were able to localize the colon tumor with similar radiolocalization indices [percentage of injected dose/g in tumor divided by the percentage of injected dose/g in normal tissue

Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain.

Science.gov (United States)

Saxena, Ashima; Hur, Regina S; Luo, Chunyuan; Doctor, Bhupendra P

2003-12-30

Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.

12 CFR 723.2 - What are the prohibited activities?

Science.gov (United States)

2010-01-01

... MEMBER BUSINESS LOANS § 723.2 What are the prohibited activities? (a) Who is ineligible to receive a... senior management employees is tied to the profit or sale of the business or commercial endeavor for... 12 Banks and Banking 6 2010-01-01 2010-01-01 false What are the prohibited activities? 723.2...

Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

Science.gov (United States)

Maruthamuthu, Murali Kannan; Hong, Jiyeon; Arulsamy, Kulandaisamy; Somasundaram, Sivachandiran; Hong, SoonHo; Choe, Woo-Seok; Yoo, Ik-Keun

2018-04-01

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Characterization of monomeric intermediates during VSV glycoprotein structural transition.

Directory of Open Access Journals (Sweden)

Aurélie A Albertini

2012-02-01

Full Text Available Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV glycoprotein G ectodomain (G(th, aa residues 1-422, the fragment that was previously crystallized. While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

A technique to prepare boronated B72.3 monoclonal antibody for boron neutron capture therapy

International Nuclear Information System (INIS)

Ranadive, G.N.; Rosenzweig, H.S.; Epperly, M.W.

1993-01-01

B72.3 monoclonal antibody has been successfully boronated using mercaptoundecahydro-closo-dodecaborate (boron cage compound). The reagent was incorporated by first reacting the lysine residues of the antibody with m-maleimidobenzoyl succinimide ester (MBS), followed by Michael addition to the maleimido group by the mercapto boron cage compound to form a physiologically stable thioether linkage. Boron content of the antibody was determined by atomic absorption spectroscopy. For biodistribution studies, boronated antibody was radioiodinated with iodogen. 125 I-labeled and boronated B72.3 monoclonal antibody demonstrated clear tumor localization when administered via tail vein injections to athymic nude mice bearing LS174-T tumor xenografts. Boronated antibody was calculated to deliver 10 6 boron atoms per tumor cell. Although this falls short of the specific boron content originally proposed as necessary for boron neutron capture therapy (BNCT), recent calculations suggest that far fewer atoms of 10 B per tumor cell would be necessary to effect successful BNCT when the boron is targeted to the tumor cell membrane. (author)

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

Science.gov (United States)

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Hydrogen behaviour in the aged low activation martensitic steel F82H for fusion reactor applications

International Nuclear Information System (INIS)

Benamati, G.

1997-10-01

A time dependent permeation method is used to measure the permeability, diffusivity and solubility of hydrogen in the low activation martensitic steel F82H aged for 2000 h under vacuum at 773 K. The measurements cover the temperature range from 373 to 723 K which includes the onset of hydrogen trapping effects on diffusivity and solubility. The results are interpreted using a trapping model. The number of trap sites and their average energies for hydrogen in the aged F82H steel are determined. These data are compared with those obtained for deuterium in F82H steel

25 CFR 700.723 - Control of livestock disease and parasites.

Science.gov (United States)

2010-04-01

... 25 Indians 2 2010-04-01 2010-04-01 false Control of livestock disease and parasites. 700.723... RELOCATION PROCEDURES New Lands Grazing § 700.723 Control of livestock disease and parasites. Whenever livestock within the New Lands become infected with contagious or infectious disease or parasites or have...

Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Mieskes, G; Issinger, O G

1993-01-01

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylat......The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation...... and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All...... kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR...

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

Science.gov (United States)

Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs

International Nuclear Information System (INIS)

Takagaki, Yoshio; Manley, J.L.; MacDonald, C.C.; Shenk, T.

1992-01-01

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. The authors have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-cross linked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. They have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long α-helix stabilized by salt bridges is predicted. An ∼270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (∼20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs

BSN723T Prevents Atherosclerosis and Weight Gain in ApoE Knockout Mice Fed a Western Diet.

Science.gov (United States)

Williams, Jarrod; Ensor, Charles; Gardner, Scott; Smith, Rebecca; Lodder, Robert

This study tests the hypothesis that BSN723T can prevent the development of hyperlipidemia and atherosclerosis in ApoE -/- knockout mice fed a Western (high fat, high cholesterol, and high sucrose) diet. BSN723T is a combination drug therapy consisting of D-tagatose and dihydromyricetin (BSN723). D-tagatose has an antihyperglycemic effect in animal and human studies and shows promise as a treatment for type 2 diabetes and obesity. Many claims regarding BSN723's pharmacological activities have been made including anti-cancer, anti-diabetic, anti-hypertensive, anti-inflammatory, and anti-atherosclerotic effects. To our knowledge this is the first study that combines D-tagatose and BSN723 for the treatment of hyperlipidemia and the prevention of atherosclerosis. ApoE-deficient mice were randomized into five groups with equivalent mean body weights. The mice were given the following diets for 8 weeks: Group 1 - Standard diet; Group 2 - Western diet; Group 3 - Western diet formulated with D-tagatose; Group 4 - Western diet formulated with BSN723; Group 5 - Western diet formulated with BSN723T. Mice were measured for weight gain, tissue and organ weights, total serum cholesterol and triglycerides and formation of atherosclerosis. The addition of D-tagatose, either alone or in combination with BSN723, prevented the increase in adipose tissue and weight gain brought on by the Western diet. Both D-tagatose and BSN723 alone reduced total cholesterol and the formation of atherosclerosis in the aorta compared to mice on the Western diet. Addition of BSN723 to D-tagatose (BSN723T) did not increase efficacy in prevention of increases in cholesterol or atherosclerosis compared to D-tagatose alone. Addition of either D-tagatose or BSN723 alone to a Western diet prevented weight gain, increases in total serum cholesterol and triglycerides, and the formation of atherosclerosis. However, there was no additive or synergistic effect on the measured parameters with the combination BSN

Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

International Nuclear Information System (INIS)

Chu, J.J.H.; Ng, M.L.

2003-01-01

This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, α-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with β-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus

Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

Directory of Open Access Journals (Sweden)

Pernilla Lång

2008-03-01

Full Text Available Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer.Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity.Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

Use of monoclonal antibody B72.3 in the management of gynecologic malignancies

International Nuclear Information System (INIS)

Simpson, J.; Schlom, J.

1988-01-01

Monoclonal antibodies are currently used in the diagnosis of gynecologic malignancies by way of immunohistochemical assays, serum assays, and in situ radiolocalization of carcinoma lesions. Among them is MAb B72.3, generated against a human tumor-associated antigen (TAG-72). Using immunohistochemical techniques, MAb B72.3 has shown reactivity with 100 percent of common epithelial ovarian carcinomas and endometrial carcinomas and non-reactivity with normal adult tissues, with the exception of normal secretory endometrium. B72.3 appears to be a valuable immunocytologic adjunct, with greater than 90 percent of effusions and fine-needle aspiration biopsies from gynecologic carcinomas showing reactivity. Using a serum assay developed to detect the presence of the TAG-72 antigen, 48 percent of patients with ovarian carcinoma demonstrated TAG-72-positive sera versus 1 percent of control sera. 131 I-labeled MAb B72.3 IgG and gamma scanning have been used for the in situ detection of metastatic carcinoma. Twelve of 15 patients with ovarian carcinoma showed positive gamma scans, and approximately 80 percent of the lesions demonstrated specific localization of the antibody. These studies indicate the potential utility of MAb B72.3 in the diagnosis of gynecologic carcinoma. 57 references

SYNTHESIS AND STRUCTURE OF BIS(PHENYLTETRAMETHYLCYCLOPENTADIENYL)TITANIUM(III) HYDRIDE - THE FIRST MONOMERIC BIS(CYCLOPENTADIENYL)TITANIUM(III) HYDRIDE : The First Monomeric Bis(cyclopentadienyl)titanium(III) Hydride

NARCIS (Netherlands)

de Wolf, J.M.; Meetsma, A.; Teuben, J.H

1995-01-01

The first structurally characterized monomeric bis(cyclopentadienyl)titanium(III) hydride, (C(5)PhMe(4))(2)TiH (4), was synthesized by hydrogenolysis of (C(5)PhMe(4))(2)TiMe (5). Hydride 4 was found to be a monomeric bent sandwich by X-ray diffraction methods, and the pentamethylcyclopentadienyl

Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease Nsp15 Suggests a Role for Hexamerization As An Allosteric Switch

Energy Technology Data Exchange (ETDEWEB)

Joseph, J.S.; Saikatendu, K.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

2007-07-09

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn{sup 2+}-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Angstroms. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by {approx}120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

12 CFR 723.5 - How do you implement a member business loan program?

Science.gov (United States)

2010-01-01

... with the type of lending the credit union will be engaging in. The experience must provide the credit.... However, the actual decision to grant a loan must reside with the credit union. (b) Conflicts of Interest... program? 723.5 Section 723.5 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING...

BSN723T Prevents Atherosclerosis and Weight Gain in ApoE Knockout Mice Fed a Western Diet

OpenAIRE

Williams, Jarrod; Ensor, Charles; Gardner, Scott; Smith, Rebecca; Lodder, Robert

2015-01-01

Objective This study tests the hypothesis that BSN723T can prevent the development of hyperlipidemia and atherosclerosis in ApoE-/- knockout mice fed a Western (high fat, high cholesterol, and high sucrose) diet. BSN723T is a combination drug therapy consisting of D-tagatose and dihydromyricetin (BSN723). Background D-tagatose has an antihyperglycemic effect in animal and human studies and shows promise as a treatment for type 2 diabetes and obesity. Many claims regarding BSN723's pharmacolog...

200 kDa and 160 kDa neurofilament protein phosphatase resistance following in vivo aluminum chloride exposure.

Science.gov (United States)

Strong, M J; Jakowec, D M

1994-01-01

We have used time-course dephosphorylation experiments and two dimensional isoelectric focusing to assess the phosphorylation state of neurofilament (NF) proteins following the intracisternal inoculation of AlCl3. Littermates of New Zealand white rabbits, age 5-6 weeks, were inoculated with either 1000, 750, 500, 250 or 100 micrograms AlCl3 in 0.9% NaCl or 0.9% NaCl alone, killed 48 hours later and the NF-enriched cytoskeletal fraction isolated from the spinal cord. Neurofilamentous inclusions did not occur following inoculums of 100 or 250 micrograms AlCl3, but thereafter developed in increasing quantities in a dosage-dependent manner. Incubation of the NF-enriched fraction with E. Coli. alkaline phosphatase (enzyme: substrate 1:50) induced a replacement of the highly phosphorylated 200 kDa isoform of NFH with a more poorly phosphorylated 170 kDa isoform, confirmed by immunoblot analysis. This reaction was complete within 20 minutes with NF derived from NaCl, 100 or 250 micrograms AlCl3 inoculated rabbits and within 30 minutes for 500 micrograms AlCl3 inoculums. However, residual highly phosphorylated NFH isoforms persisted at 60 minutes for 750 micrograms inoculums and 90 minutes for that derived from 1000 micrograms AlCl3 inoculums. A similar inhibition of phosphatase activity was observed for NFM. Following two dimensional electrophoresis of the NF-enriched isolate, no alteration in the net phosphorylation state of individual NF subunit proteins was observed--regardless of the inoculum. These results demonstrate a dose-dependent induction of neurofilamentous inclusions in spinal motor neurons following intracisternal AlCl3 inoculation accompanied by increasing phosphatase resistance without a demonstrable alteration in NF net phosphorylation state.

Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35-kDa membrane protein

International Nuclear Information System (INIS)

Palamarczyk, G.; Drake, R.; Haley, B.; Lennarz, W.J.

1990-01-01

In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase, yeast microsomal membranes were allowed to react with 5-azido[β- 32 P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32 P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[β- 32 P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase

A monomeric variant of insulin degrading enzyme (IDE loses its regulatory properties.

Directory of Open Access Journals (Sweden)

Eun Suk Song

2010-03-01

Full Text Available Insulin degrading enzyme (IDE is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure.IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

Enhanced coagulation for improving coagulation performance and reducing residual aluminum combining polyaluminum chloride with diatomite.

Science.gov (United States)

Hu, Wenchao; Wu, Chunde

2016-01-01

The feasibility of using enhanced coagulation, which combined polyaluminum chloride (PAC) with diatomite for improving coagulation performance and reducing the residual aluminum (Al), was discussed. The effects of PAC and diatomite dosage on the coagulation performance and residual Al were mainly investigated. Results demonstrated that the removal efficiencies of turbidity, dissolved organic carbon (DOC), and UV254 were significantly improved by the enhanced coagulation, compared with PAC coagulation alone. Meaningfully, the five forms of residual Al (total Al (TAl), total dissolved Al (TDAl), dissolved organic Al (DOAl), dissolved monomeric Al (DMAl), and dissolved organic monomeric Al (DOMAl)) all had different degrees of reduction in the presence of diatomite and achieved the lowest concentrations (0.185, 0.06, 0.053, 0.014, and 0 mg L(-1), respectively) at a PAC dose of 15 mg L(-1) and diatomite dose of 40 mg L(-1). In addition, when PAC was used as coagulant, the majority of residual Al existed in dissolved form (about 31.14-70.16%), and the content of DOMAl was small in the DMAl.

Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

International Nuclear Information System (INIS)

Ditzel, H.; Erb, K.; Rasmussen, J.W.; Jensenius, J.C.

1992-01-01

Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-1 at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients. (orig.)

12 CFR 723.20 - How can a state supervisory authority develop and enforce a member business loan regulation?

Science.gov (United States)

2010-01-01

... state regulation minimizes the risk and accomplishes the overall objectives of NCUA's member business... and enforce a member business loan regulation? 723.20 Section 723.20 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS MEMBER BUSINESS LOANS § 723.20 How can a state...

A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA.

Science.gov (United States)

Yang, Kui; Dang, Xiaoqun; Baines, Joel D

2017-10-15

Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by U L 15, U L 28, and U L 33. The U L 33-encoded protein (pU L 33) interacts with pU L 28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pU L 33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of U L 33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pU L 33 C terminus did not affect viral replication or the interaction of pU L 33 with pU L 28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pU L 33 mutant interacted with pU L 28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pU L 33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA. IMPORTANCE This paper shows a role for pU L 33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components. Copyright © 2017 Yang et al.

The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

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Kenrick A Vassall

Full Text Available The classic isoforms of myelin basic protein (MBP are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99- containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90 upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107 with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012. Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the

Generation and Characterization of an IgG4 Monomeric Fc Platform.

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Lu Shan

Full Text Available The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

Science.gov (United States)

Case, Heather B; Dickenson, Nicholas E

2018-04-17

Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

International Nuclear Information System (INIS)

Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

1988-01-01

Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125 I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule

47 CFR 90.723 - Selection and assignment of frequencies.

Science.gov (United States)

2010-10-01

... 220-222 MHz Band § 90.723 Selection and assignment of frequencies. (a) Phase II applications for frequencies in the 220-222 MHz band shall specify whether their intended use is for 10-channel nationwide... medical use. Phase II applicants for frequencies for public safety/mutual aid use or emergency medical use...

40 CFR 141.723 - Requirements to respond to significant deficiencies identified in sanitary surveys performed by EPA.

Science.gov (United States)

2010-07-01

... deficiencies identified in sanitary surveys performed by EPA. 141.723 Section 141.723 Protection of Environment... performed by EPA, systems must respond in writing to significant deficiencies identified in sanitary survey... will address significant deficiencies noted in the survey. (d) Systems must correct significant...

Isothermal Bainite Processing of ASTM A723 Components

Science.gov (United States)

1993-09-01

4 List of Illustrations 1. Photograph of typical cylinders used in the present study ................................. 7 2. CCT diagram for... CCT ) diagram obtained by thermal and magnetic analyses for the high nickel (-3 percent) ASTM A723 steel. The bainite knee for the 2 percent nickel...block. Also shown is the slope giving the average cooling rate in the critical region of the CCT diagram . This average rate happens to approximately

Direct labelling of monomeric antibody fragments Fab' with 99mTc

International Nuclear Information System (INIS)

Li Jun; Wang Shizhen; Yang Ziyi

1994-01-01

Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG

Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

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Rodrigo Jácome

Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

Analytical verification and quality assessment of the Tosoh HLC-723GX HbA1c analyzer

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Marko Ris

2017-04-01

Full Text Available Objectives: Ion-exchange high-performance liquid chromatography (IE-HPLC has long been used as a reproducible and versatile analytical tool for HbA1c measurement.In this study, we performed analytical verification and quality assessment of the recently introduced small IE-HPLC Tosoh HLC-723GX HbA1c analyzer, and a comparison of results to immunoassay (IA and capillary electrophoresis (CE. Design and methods: The total imprecision of Tosoh HLC-723GX was verified according to CLSI EP15-A2 protocol using commercial control materials (C-QC and pooled human whole blood samples (HWB. The Sigma metric was used for the evaluation of quality targets. HbA1c results were compared to automated CE (MiniCap Flex Piercing, Sebia, France and IA (Tina-quant HbA1c Gen 2, Cobas Integra 400+, Roche Diagnostics, USA procedures. Results: The total imprecision of Tosoh HLC-723GX-HbA1c for IFCC(mmol/mol and NGSP(% units was: 1.91/1.25% (HbA1c=31 mmol/mol/5.0% and 0.51/0.63% (HbA1c=84 mmol/mol/9.8% for C-QC, and 0.39/0.2% (HbA1c=47 mmol/mol/6.5% and 0.77/0.46% (HbA1c=94 mmol/mol/10.8% in HWB samples, respectively. Bland-Altman analysis did not reveal any deviation of the results between Tosoh HLC-723GX and CE: mean difference 0.0% (95%CI: −0.02927 to 0.02653%, while the mean HbA1c difference against IA was −0.07% (95%CI: −0.1039 to −0.02765. At the selected HbA1c clinical decision level (48 mmol/mol/6,5%, six sigma analysis gave σ value of 3.91, within a desirable classification of performance. Conclusion: The analytical performance of the Tosoh HLC-723GX complies with the rigorous quality criteria for clinical use of HbA1c, with the results comparable to the CE procedure. Tosoh HLC-723GX provides a plausible analytical choice for reliable HbA1c measurement in low-volume laboratories. Keywords: HbA1c, Quality targets, Six sigma, Tosoh HLC-723GX analyzer

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

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DeVita Anne

2002-04-01

Full Text Available Abstract Background Proteorhodopsin (pR is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic γ-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. Results A pR purification procedure is described that utilizes Phenylsepharose™ and hydroxylapatite columns and yields 85% (w/w purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like photoproduct. Both of these occur with a similar rise time (4–10 μs as reported for monomeric bR in detergent. Conclusions The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved; or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly.

Monomeric insulins and their experimental and clinical implications.

Science.gov (United States)

Brange, J; Owens, D R; Kang, S; Vølund, A

1990-09-01

Due to the inherent pharmacokinetic properties of available insulins, normoglycemia is rarely, if ever, achieved in insulin-dependent diabetic patients without compromising their quality of life. Subcutaneous insulin absorption is influenced by many factors, among which the associated state of insulin (hexameric) in pharmaceutical formulation may be of importance. This review describes the development of a series of human insulin analogues with reduced tendency to self-association that, because of more rapid absorption, are better suited to meal-related therapy. DNA technology has made it possible to prepare insulins that remain dimeric or even monomeric at high concentration by introducing one or a few amino acid substitutions into human insulin. These analogues were characterized and used for elucidating the mechanisms involved in subcutaneous absorption and were investigated in preliminary clinical studies. Their relative receptor binding and in vitro potency (free-fat cell assay), ranging from 0.05 to 600% relative to human insulin, were strongly correlated (r = 0.97). In vivo, most of the analogues exhibited approximately 100% activity, explainable by a dominating receptor-mediated clearance. This was confirmed by clamp studies in which correlation between receptor binding and clearance was observed. Thus, an analogue with reduced binding and clearance gives higher circulating concentrations, counterbalancing the reduced potency at the cellular level. Absorption studies in pigs revealed a strong inverse correlation (r = 0.96) between the rate of subcutaneous absorption and the mean association state of the insulin analogues. These studies also demonstrated that monomeric insulins were absorbed three times faster than human insulin. In healthy subjects, rates of disappearance from subcutis were two to three times faster for dimeric and monomeric analogues than for human insulin. Concomitantly, a more rapid rise in plasma insulin concentration and an earlier

20 CFR 404.723 - When evidence of marriage is required.

Science.gov (United States)

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false When evidence of marriage is required. 404... DISABILITY INSURANCE (1950- ) Evidence Evidence of Age, Marriage, and Death § 404.723 When evidence of marriage is required. If you apply for benefits as the insured person's husband or wife, widow or widower...

Glucose(xylose isomerase production by Streptomyces sp. CH7 grown on agricultural residues

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Kankiya Chanitnun

2012-09-01

Full Text Available Streptomyces sp. CH7 was found to efficiently produce glucose(xylose isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

Diagnostic potential of IS6110, 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples

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Negi S

2007-01-01

Full Text Available Purpose: The correlation between the presence of specific gene sequence of M. tuberculosis and specific diagnosis of clinical tuberculosis is not known. This study compared the results of polymerase chain reaction (PCR amplification of M . tuberculosis specific DNA sequences (IS6110, 65kDa, 38kDa and mRNA coding for 85 B protein from different clinical samples of pulmonary and extrapulmonary tuberculosis. Methods: One hundred and seventy-two clinical samples from suspected tuberculosis patients were tested for smear examination, culture (LJ and rapid BACTEC 460 TB system and PCR. PCR was performed with specific primers for the targets: IS6110, 65kDa, 38kDa and 85B. Results: Each PCR test was found to have a much higher positivity than conventional test and BACTEC culture ( P < 0.05. Smear positive samples (56 and the samples (36 showing positive results by conventional methods (smear and LJ medium culture and BACTEC were found to be positive by all PCR protocols. No significant difference was found between the four PCR protocols ( P >0.05. The primer specific for amplifying the 123bp IS6110 fragment gave the highest positivity (83%, followed by 65kDa, 38kDa and 85B RT-PCR in descending order. Conclusions: These data suggest that the presence of IS6110 correlates more closely with the diagnosis of clinical tuberculosis than that of 65kDa, 38kDa and 85B proteins.

Anthocyanins and Their Variation in Red Wines I. Monomeric Anthocyanins and Their Color Expression

OpenAIRE

Chang-Qing Duan; Malcolm J. Reeves; Qiu-Hong Pan; Lin Mu; Na-Na Liang; Fei He; Jun Wang

2012-01-01

Originating in the grapes, monomeric anthocyanins in young red wines contribute the majority of color and the supposed beneficial health effects related to their consumption, and as such they are recognized as one of the most important groups of phenolic metabolites in red wines. In recent years, our increasing knowledge of the chemical complexity of the monomeric anthocyanins, their stability, together with the phenomena such as self-association and copigmentation that can stabilize and enha...

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

Science.gov (United States)

Rosen, G; Shoshani, M; Naor, R; Sela, M N

2001-12-01

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

Anthocyanins and Their Variation in Red Wines I. Monomeric Anthocyanins and Their Color Expression

Directory of Open Access Journals (Sweden)

Chang-Qing Duan

2012-02-01

Full Text Available Originating in the grapes, monomeric anthocyanins in young red wines contribute the majority of color and the supposed beneficial health effects related to their consumption, and as such they are recognized as one of the most important groups of phenolic metabolites in red wines. In recent years, our increasing knowledge of the chemical complexity of the monomeric anthocyanins, their stability, together with the phenomena such as self-association and copigmentation that can stabilize and enhance their color has helped to explain their color representation in red wine making and aging. A series of new enological practices were developed to improve the anthocyanin extraction, as well as their color expression and maintenance. This paper summarizes the most recent advances in the studies of the monomeric anthocyanins in red wines, emphasizing their origin, occurrence, color enhancing effects, their degradation and the effect of various enological practices on them.

31 CFR 501.723 - Prehearing disclosures; methods to discover additional matter.

Science.gov (United States)

2010-07-01

... (9)) and the Privacy Act (5 U.S.C. 552a). Parties may obtain discovery regarding any matter, not... Information Act (5 U.S.C. 552(b)(1) through (9)). (g) Updating discovery. A party who has made an initial..., PROCEDURES AND PENALTIES REGULATIONS Trading With the Enemy Act (TWEA) Penalties § 501.723 Prehearing...

Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer

Energy Technology Data Exchange (ETDEWEB)

Wei, Jia; Tong, Liang

2015-10-12

Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases1, 2, 3, 4, 5, 6. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope7, 8. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known1, 9, 10, 11, 12, 13, 14, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A15, 16 and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis.

Brain-specific interaction of a 91-kDa membrane-bound protein with the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Issinger, O G; Braulke, T

1996-01-01

The cytoplasmic tail of the 300 kDa mannose 6-phosphate receptor (MPR 300-CT) is thought to play an important role in sorting and targeting of lysosomal enzymes and the insulin-like growth factor II along the biosynthetic and endocytic pathway. In this study a brain specific 91 kDa protein and a ...... in neuronal cells....

Radioimmunoguided surgery using the monoclonal antibody B72.3 in colorectal tumors

International Nuclear Information System (INIS)

Sickle-Santanello, B.J.; O'Dwyer, P.J.; Mojzisik, C.

1987-01-01

The authors have developed a hand-held gamma-detecting probe (GDP) for intraoperative use that improves the sensitivity of external radioimmunodetection. Radiolabeled monoclonal antibody (MAb) B72.3 was injected in six patients with primary colorectal cancer and 31 patients with recurrent colorectal cancer an average of 16 days preoperatively. The GDP localized the MAb B72.3 in 83 percent of sites. The technique, known as a radioimmunoguided surgery (RIGS) system did not alter the surgical procedure in patients with primary colorectal cancer but did alter the approach in 26 percent (8/31) of patients with recurrent colorectal cancer. Two patients avoided unnecessary liver resections and two underwent extraabdominal approaches to document their disease. The RIGS system may influence the short-term morbidity and mortality of surgery for colorectal cancer. Larger series and longer follow-up are needed to determine whether the RIGS system confers a survival advantage to the patient with colorectal cancer

Multistage modeling of protein dynamics with monomeric Myc oncoprotein as an example.

Science.gov (United States)

Liu, Jiaojiao; Dai, Jin; He, Jianfeng; Niemi, Antti J; Ilieva, Nevena

2017-03-01

We propose to combine a mean-field approach with all-atom molecular dynamics (MD) into a multistage algorithm that can model protein folding and dynamics over very long time periods yet with atomic-level precision. As an example, we investigate an isolated monomeric Myc oncoprotein that has been implicated in carcinomas including those in colon, breast, and lungs. Under physiological conditions a monomeric Myc is presumed to be an example of intrinsically disordered proteins that pose a serious challenge to existing modeling techniques. We argue that a room-temperature monomeric Myc is in a dynamical state, it oscillates between different conformations that we identify. For this we adopt the Cα backbone of Myc in a crystallographic heteromer as an initial ansatz for the monomeric structure. We construct a multisoliton of the pertinent Landau free energy to describe the Cα profile with ultrahigh precision. We use Glauber dynamics to resolve how the multisoliton responds to repeated increases and decreases in ambient temperature. We confirm that the initial structure is unstable in isolation. We reveal a highly degenerate ground-state landscape, an attractive set towards which Glauber dynamics converges in the limit of vanishing ambient temperature. We analyze the thermal stability of this Glauber attractor using room-temperature molecular dynamics. We identify and scrutinize a particularly stable subset in which the two helical segments of the original multisoliton align in parallel next to each other. During the MD time evolution of a representative structure from this subset, we observe intermittent quasiparticle oscillations along the C-terminal α helix, some of which resemble a translating Davydov's Amide-I soliton. We propose that the presence of oscillatory motion is in line with the expected intrinsically disordered character of Myc.

In vitro degradation of the 32kDa PS II reaction centre protein

International Nuclear Information System (INIS)

Eckenswiller, L.C.; Greenberg, B.M.

1989-01-01

The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components

A 19-kDa C-terminal tryptic fragment of the α chain of Na/K-ATPase is essential for occlusion and transport of cations

International Nuclear Information System (INIS)

Karlish, S.J.D.; Goldshleger, R.; Stein, W.D.

1990-01-01

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the α chain, a largely intact β chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. The authors conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding sites, located on the cytoplasmic domain of the α chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect - mediated, presumably, by conformational changes of the protein

A 19-kDa C-terminal tryptic fragment of the. alpha. chain of Na/K-ATPase is essential for occlusion and transport of cations

Energy Technology Data Exchange (ETDEWEB)

Karlish, S.J.D.; Goldshleger, R. (Weizmann Institute of Science, Rehovot (Israel)); Stein, W.D. (Hebrew Univ. Jerusalem (Israel))

1990-06-01

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the {alpha} chain, a largely intact {beta} chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. The authors conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding sites, located on the cytoplasmic domain of the {alpha} chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect - mediated, presumably, by conformational changes of the protein.

Strontium-82/rubidium-82 generator

International Nuclear Information System (INIS)

Gennaro, G.P.; Haney, P.S.

1986-01-01

Hydroxylapatite, a compound having the formula: M 10 (PO 4 ) 6 (OH) 2 wherein M is calcium, strontium, barium, lead, iron, sodium, potassium, zinc, cadmium, magnesium, aluminium or a rare earth metal, is provided as a support medium for strontium-82 in a strontium-82/rubidium-82 parent-daughter radionuclide generator

Diagnostic potential of Fasciola gigantica-derived 14.5 kDa fatty acid binding protein in the immunodiagnosis of bubaline fascioliasis.

Science.gov (United States)

Allam, G; Bauomy, I R; Hemyeda, Z M; Diab, T M; Sakran, T F

2013-06-01

The 14.5 kDa fatty acid binding protein (FABP) was isolated from the crude extract of adult Fasciola gigantica worms. Polyclonal anti-FABP IgG was generated in rabbits immunized with prepared FABP antigen. Sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect coproantigen in stools and circulating Fasciola antigen (CA) in sera of 126 water buffaloes by using purified and horseradish peroxidase (HRP)-conjugated anti-FABP IgG. Sandwich ELISA sensitivity was 96.97% and 94.95%; while specificity was 94.12% and 82.35% for coproantigen and CA detection, respectively. However, sensitivity and specificity of the Kato-Katz technique was 73.74% and 100%, respectively. The diagnostic efficacy of sandwich ELISA was 96.55% and 93.1% for coproantigen and CA detection, respectively. In contrast, the diagnostic efficacy of the Kato-Katz technique was 77.59%. In conclusion, these results demonstrate that the purified 14.5 kDa FABP provides a more suitable antigen for immunodiagnosis of early and current bubaline fascioliasis by using sandwich ELISA.

Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

Science.gov (United States)

Langkjaer, L; Brange, J; Grodsky, G M; Guy, R H

1998-01-23

The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

Monomeric Aβ(1-40) and Aβ(1-42) Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil.

Science.gov (United States)

Roche, Julien; Shen, Yang; Lee, Jung Ho; Ying, Jinfa; Bax, Ad

2016-02-09

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ(1-40) and Aβ(1-42) into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ(1-42) compared to that of Aβ(1-40) are poorly understood. To explore in detail the structural propensity of the monomeric Aβ(1-40) and Aβ(1-42) peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ(1-40) and Aβ(1-42) peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JNCα, and (1)JNCα) recorded for Aβ(1-40) were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ(1-42), suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process.

Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

Energy Technology Data Exchange (ETDEWEB)

Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

2016-07-04

Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high-value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) including 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

Energy Technology Data Exchange (ETDEWEB)

Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

2016-07-04

Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer as well as its complex side chain structures, it has been a challenge to effectively depolymerize lignin and produce high value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) inclduing 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPCs yields obtained were 18% and 22% based on the initial weight of the lignin in SESPL and DACSL respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47%. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions.

Science.gov (United States)

Grossini, Elena; Farruggio, Serena; Qoqaiche, Fatima; Raina, Giulia; Camillo, Lara; Sigaudo, Lorenzo; Mary, David; Surico, Nicola; Surico, Daniela

2016-09-15

Perivascular adipose tissue can be involved in the process of cardiovascular pathology through the release of adipokines, namely adiponectins. Monomeric adiponectin has been shown to increase coronary blood flow in anesthetized pigs through increased nitric oxide (NO) release and the involvement of adiponectin receptor 1 (AdipoR1). The present study was therefore planned to examine the effects of monomeric adiponectin on NO release and Ca(2+) transients in porcine aortic endothelial cells (PAEs) in normal/high glucose conditions and the related mechanisms. PAEs were treated with monomeric adiponectin alone or in the presence of intracellular kinases blocker, AdipoR1 and Ca(2+)-ATPase pump inhibitors. The role of Na(+)/Ca(2+) exchanger was examined in experiments performed in zero Na(+) medium. NO release and intracellular Ca(2+) were measured through specific probes. In PAE cultured in normal glucose conditions, monomeric adiponectin elevated NO production and [Ca(2+)]c. Similar effects were observed in high glucose conditions, although the response was lower and not transient. The Ca(2+) mobilized by monomeric adiponectin originated from an intracellular pool thapsigargin- and ATP-sensitive and from the extracellular space. Moreover, the effects of monomeric adiponectin were prevented by kinase blockers and AdipoR1 inhibitor. Finally, in normal glucose condition, a role for Na(+)/Ca(2+) exchanger and Ca(2+)-ATPase pump in restoring Ca(2+) was found. Our results add new information about the control of endothelial function elicited by monomeric adiponectin, which would be achieved by modulation of NO release and Ca(2+) transients. A signalling related to Akt, ERK1/2 and p38MAPK downstream AdipoR1 would be involved. Copyright © 2016 Elsevier Inc. All rights reserved.

Cerebral microdialysis methodology--evaluation of 20 kDa and 100 kDa catheters.

Science.gov (United States)

Hutchinson, P J; O'Connell, M T; Nortje, J; Smith, P; Al-Rawi, P G; Gupta, A K; Menon, D K; Pickard, J D

2005-08-01

Microdialysis monitoring of cerebral metabolism is now performed in several neuro-intensive care units. Conventional microdialysis utilizes CMA70 catheters with 20 kDa molecular weight cut-off membranes enabling the measurement of small molecules such as glucose, lactate, pyruvate and glutamate. The CMA71 100 kDa molecular weight cut-off microdialysis catheter has recently been introduced to allow detection of larger molecules such as cytokines. The objective of this study was to perform in vitro and in vivo testing of the CMA71 microdialysis catheter, comparing its performance with the CMA70. In vitro comparison studies of three of each catheter using reference analyte solutions, demonstrated equivalent recovery for glucose, lactate, pyruvate and glutamate (range 94-97% for CMA70 and 88-103% for CMA71). In vivo comparison involved intracranial placement of paired CMA70 and CMA71 catheters (through the same cranial access device) in six patients with severe traumatic brain injury. Both catheters were perfused with CNS Perfusion Fluid without dextran at 0.3 microl min-1 with hourly sampling and bedside analysis on a CMA600 microdialysis analyser. The two catheters yielded equivalent results for glucose, lactate, pyruvate, glutamate and lactate/pyruvate ratio. CMA71 microdialysis catheters can, therefore, be used for routine clinical monitoring of extracellular substances, as well as for their intended research role of larger molecular weight protein sampling.

12 CFR 723.3 - What are the requirements for construction and development lending?

Science.gov (United States)

2010-01-01

... AFFECTING CREDIT UNIONS MEMBER BUSINESS LOANS § 723.3 What are the requirements for construction and... following additional requirements. (a) The aggregate of the net member business loan balances for all... associated member-borrowers, irrespective of the existence of a contractual commitment from a prospective...

Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation

Directory of Open Access Journals (Sweden)

Elena Grossini

2016-09-01

Full Text Available We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article “Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions” (Grossini et al., in press [1].

Distinct subcellular trafficking resulting from monomeric vs multimeric targeting to endothelial ICAM-1: implications for drug delivery.

Science.gov (United States)

Ghaffarian, Rasa; Muro, Silvia

2014-12-01

Ligand-targeted, receptor-mediated endocytosis is commonly exploited for intracellular drug delivery. However, cells-surface receptors may follow distinct endocytic fates when bound by monomeric vs multimeric ligands. Our purpose was to study this paradigm using ICAM-1, an endothelial receptor involved in inflammation, to better understand its regulation and potential for drug delivery. Our procedure involved fluorescence microscopy of human endothelial cells to determine the endocytic behavior of unbound ICAM-1 vs ICAM-1 bound by model ligands: monomeric (anti-ICAM) vs multimeric (anti-ICAM biotin-streptavidin conjugates or anti-ICAM coated onto 100 nm nanocarriers). Our findings suggest that both monomeric and multimeric ligands undergo a similar endocytic pathway sensitive to amiloride (∼50% inhibition), but not inhibitors of clathrin-pits or caveoli. After 30 min, ∼60-70% of both ligands colocalized with Rab11a-compartments. By 3-5 h, ∼65-80% of multimeric anti-ICAM colocalized with perinuclear lysosomes with ∼60-80% degradation, while 70% of monomeric anti-ICAM remained associated with Rab11a at the cell periphery and recycled to and from the cell-surface with minimal (drug delivery.

Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds.

Science.gov (United States)

Ma, Ruoshui; Guo, Mond; Lin, Kuan-Ting; Hebert, Vincent R; Zhang, Jinwen; Wolcott, Michael P; Quintero, Melissa; Ramasamy, Karthikeyan K; Chen, Xiaowen; Zhang, Xiao

2016-07-25

Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high-value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) including 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS.

Science.gov (United States)

Nelson, Bryant C; Sharpless, Katherine E

2003-01-29

Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.

Assessment of the PCDD/F fate from MSWI residue used in road construction in France.

Science.gov (United States)

Badreddine, R; François, D

2009-01-01

MSWI fly ash is susceptible to contain high amount of polychlorinated dibenzo-dioxins and polychlorinated dibenzo-furans. However, the use of MSWI residue for road construction started in France at a period when MSWI Bottom Ash and MSWI fly ash were not separated. From four old road sites, MSWI residue, road soils, reference soils and geo-textiles were sampled and their PCDD/F contents were analyzed. MSWI residue show a great heterogeneity but also high amounts of PCDD/F (14-2960 ng I-TEQ kg(-1)dm). Soils underlying the road show less heterogeneity and PCDD/F contents between 0.57 and 7.23 ng I-TEQ kg(-1)dm, lower than ordinary soils. Moreover, the specific analysis of the 17 toxic PCDD/F congeners (notably the 2,3,7,8-TetraCDD) indicates the very low harmfulness of road soils. The study also allows to assert the relation between the MSWI residue particle size and the PCDD/F content.

Modeling and Docking Studies on Novel Mutants (K71L and T204V of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

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Asita Elengoe

2014-04-01

Full Text Available The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO were mutated. 3D mutant models (K71L and T204V using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255, as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

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Song, Hyun-Ouk

2016-02-01

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Preparation and biological evaluation of 111In-, 177Lu- and 90Y-labeled DOTA analogues conjugated to B72.3

International Nuclear Information System (INIS)

Mohsin, Huma; Fitzsimmons, Jonathan; Shelton, Tiffani; Hoffman, Timothy J.; Cutler, Cathy S.; Lewis, Michael R.; Athey, Phillip S.; Gulyas, Gyongyi; Kiefer, Garry E.; Frank, R. Keith; Simon, Jaime; Lever, Susan Z.; Jurisson, Silvia S.

2007-01-01

Three 1,4,7,10-tetraazacyclododecane-N,N',N '' ,N '' '-tetraacetic acid (DOTA) analogues were evaluated for relative in vivo stability when radiolabeled with 111 In, 90 Y and 177 Lu and conjugated to the monoclonal antibody B72.3. The DOTA analogues evaluated were 'NHS-DOTA' [N-hydroxysuccinimdyl (NHS) group activating one carboxylate], 'Arm-DOTA' (also known as MeO-DOTA; with a p-NCS, o-MeO-benzyl moiety on the methylene group of one acetic acid arm) and 'Back-DOTA' (with a p-NCS-benzyl moiety on a backbone methylene group of the macrocycle). The B72.3 was conjugated to the DOTA analogues to increase the retention time of the radioloabeled conjugates in vivo in mice. The serum stability of the various radiometalated DOTA conjugates showed them to have good stability out to 168 h (all >95% except 111 In-NHS-DOTA-B72.3, which was 91% stable). Hydroxyapatite stability for the 111 In and 177 Lu DOTA-conjugates was >95% at 168 h, while the 90 Y DOTA-conjugates were somewhat less stable (between 90% and 95% at 168 h). The biodistribution studies of the radiometalated DOTA-conjugates showed that no significant differences were observed for the 111 In and 177 Lu analogues; however, the 90 Y analogues showed lower stabilities, as evidenced by their increased bone uptake relative to the other two [2-20% injected dose per gram (% ID/g) for 90 Y and 2-8% ID/g for 111 In and 177 Lu]. The lower stability of the 90 Y analogues could be due to the higher beta energy of 90 Y and/or to the larger ionic radius of Y 3+ . Based on the bone uptake observed, the 177 Lu-NHS-DOTA-B72.3 had slightly lower stability than the 177 Lu-Arm-DOTA-B72.3 and 177 Lu-Back-DOTA-B72.3, but not significantly at all time points. For 90 Y, the analogue showing the lowest stability based on bone uptake was 90 Y-Arm-DOTA-B72.3, perhaps because of the metal's larger ionic radius and potential steric interactions minimizing effective complexation. The 111 In analogues all showed similar biological

A Molecular Dynamics Study on RAGE-Aβ42 Interaction and the Influence of G82S RAGE Polymorphism on Aβ Interaction

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Sreeram Krishnan

2015-12-01

Full Text Available Interaction of amyloid peptides (Aβ with receptor for advanced glycation end products (RAGE elicits an inflammatory response and augments Alzheimer's disease (AD pathology. The present study was aimed to analyse the interactions of different forms of Aβ42 peptide with ligand binding domain of normal and G82S RAGE and their possible consequences in AD pathology. The structures of RAGE ectodomain (3CJJ, monomeric forms of Aβ42 - 1IYT (apolar and 1Z0Q (polar and fibrillar (2BEG were obtained from PDB. The structure of G82 and S82 RAGE was generated using SWISS MODEL. SIFT and PolyPhen analysis was performed to predict the phenotypic and functional effect of the amino acid substitution. The G82 and S82 variant structures were simulated in GROMACS and the 10 lowest energy structures were docked with different forms of Aβ42 using CLUSPRO in antibody mode. The lowest energy docked structure was further simulated for 5 ns. The structures corresponding to 0-5 ns were taken and the amino acid interactions were generated using PDBSUM. SIFT analysis indicated that G82S SNP had a tolerating effect on the structure of protein but polyphen predicted a probable damaging effect. Highest binding score was obtained with 2BEG docked with both G82 RAGE (-375.84 ± 7.425 Kcal/mol and G82S variant (-391.09 ± 13.391 Kcal/mol indicating that the fibrillar form showed better interaction. Compared to G82 RAGE, the S82 variant showed better interaction to all three forms of Aβ42. The results of study indicate that RAGE interacted better with fibrillar form of Aβ42 peptide and G82S mutation enhanced the binding affinity of RAGE towards amyloid peptides leading to enhanced inflammatory response.

Principal Component Regression Analysis of the Relation Between CIELAB Color and Monomeric Anthocyanins in Young Cabernet Sauvignon Wines

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Chang-Qing Duan

2008-11-01

Full Text Available Color is one of the key characteristics used to evaluate the sensory quality of red wine, and anthocyanins are the main contributors to color. Monomeric anthocyanins and CIELAB color values were investigated by HPLC-MS and spectrophotometry during fermentation of Cabernet Sauvignon red wine, and principal component regression (PCR, a statistical tool, was used to establish a linkage between the detected anthocyanins and wine coloring. The results showed that 14 monomeric anthocyanins could be identified in wine samples, and all of these anthocyanins were negatively correlated with the L*, b* and H*ab values, but positively correlated with a* and C*ab values. On an equal concentration basis for each detected anthocyanin, cyanidin-3-O-glucoside (Cy3-glu had the most influence on CIELAB color value, while malvidin 3-O-glucoside (Mv3-glu had the least. The color values of various monomeric anthocyanins were influenced by their structures, substituents on the B-ring, acyl groups on the glucoside and the molecular steric structure. This work develops a statistical method for evaluating correlation between wine color and monomeric anthocyanins, and also provides a basis for elucidating the effect of intramolecular copigmentation on wine coloring.

Monomeric insulins obtained by protein engineering and their medical implications.

Science.gov (United States)

Brange, J; Ribel, U; Hansen, J F; Dodson, G; Hansen, M T; Havelund, S; Melberg, S G; Norris, F; Norris, K; Snel, L

1988-06-16

The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

Estimation of residual stress distribution for pressurizer nozzle of Kori nuclear power plant considering safe end

Energy Technology Data Exchange (ETDEWEB)

Song, Tae Kwang; Bae, Hong Yeol; Chun, Yun Bae; Oh, Chang Young; Kim, Yun Jae [Korea University, Seoul (Korea, Republic of); Lee, Kyoung Soo; Park, Chi Yong [Korea Electric Power Research Institute, Daejeon (Korea, Republic of)

2008-08-15

In nuclear power plants, ferritic low alloy steel nozzle was connected with austenitic stainless steel piping system through alloy 82/182 butt weld. Accurate estimation of residual stress for weldment is important in the sense that alloy 82/182 is susceptible to stress corrosion cracking. There are many results which predict residual stress distribution for alloy 82/182 weld between nozzle and pipe. However, nozzle and piping system usually connected through safe end which has short length. In this paper, residual stress distribution for pressurizer nozzle of Kori nuclear power plant was predicted using FE analysis, which considered safe end. As a result, existing residual stress profile was redistributed and residual stress of inner surface was decreased specially. It means that safe end should be considered to reduce conservatism when estimating the piping system.

Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

Science.gov (United States)

Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

82Sr--82Rb radioisotope generator

International Nuclear Information System (INIS)

Grant, P.M.; Erdal, B.R.; O'Brien, H.A.

1976-01-01

An improved 82 Sr- 82 Rb radioisotope generator system, based upon the complexing ion exchange resin Chelex-100, has been developed. Columns of this material can be easily and rapidly milked, and the Rb-Sr separation factor for a fresh generator was found to be greater than 10 7 . Approximately 80 percent of the 82 Rb present was delivered in a 15-ml volume of aqueous 0.2 M NH 4 Cl solution. After more than 6 liters of eluant had been put through the generator, the Rb-Sr separation factor was still observed to be greater than 10 5 , and no unusual strontium breakthrough behavior was seen in the system over nearly three 82 Sr half lives. 2 claims, no drawings

Improved detection of a staphylococcal infection by monomeric and protein A-purified polyclonal human immunoglobulin

International Nuclear Information System (INIS)

Calame, W.

1993-01-01

The present study was undertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99m Tc-labelled monomeric human immunoglobulin (m-Ig), 99m Tc-labelled, protein A-purified, human immunoglobulin (A-IG) and 99m Tc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99m Tc-labelled Igs bound to bacteria in vitro: The percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-to-non-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purified Ig yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99m Tc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99m Tc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein A-purified monomeric Ig was the highest of all tracer agents tested. (orig.)

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Preclinical evaluation of intravenously administered 111In-and 90Y-labeled B72.3 immunoconjugate (GYK-DTPA) in beagle dogs

International Nuclear Information System (INIS)

Quadri, S.M.; Vriesendorp, H.M.; Yi Shao; Blum, J.E.; Leichner, P.K.; Williams, J.R.

1993-01-01

B72.3, a monoclonal antibody with reactivity against human adenocarcinomas was obtained from the Cytogen Corporation in the form of an immunoconjugate coupled with linker-chelator GYK-DTPA by using proprietary carbohydrate directed site specific chemistry. The immunoconjugate was radiolabeled with indium-111 or yttrium-90. A preclinical analysis was performed in 10 normal beagle dogs. The pharmacokinetics of intravenously administered indium- and yttrium-labeled immunoconjugates were compared serially in blood, bone marrow and urine samples. Compared to 90 Y less of the 111 In label ended up in urine and more was found in blood and bone marrow. Indium-labeled B72.3 GYK-DTPA had relatively higher uptake in most glandular tissues than 111 In-labeled antiferritin immunoconjugate. Bone marrow toxicity was the dose limiting side effect after intravenous infusion of 90 Y-labeled B72.3 GYK-DTPA. Toxicity was also observed in the liver but not in other organ systems. Recently other investigators obtained similar results with these immunoconjugates in human patients. A preclinical pharmacokinetic analysis of radioimmunoconjugates in beagle dogs provided useful information regarding bone marrow toxicity, liver toxicity and in vivo instability of the immunoconjugate. Data suggest that for future trials in human patients, a more stable chelated immunoconjugate for yttrium is needed to achieve less liver uptake and a better correlation with the 111 In-labeled product than the 90 Y-labeled B72.3 GYK-DTPA used in this investigation. (Author)

I222 crystal form of despentapeptide (B26-B30) insulin provides new insights into the properties of monomeric insulin.

Science.gov (United States)

Whittingham, Jean L; Youshang, Zhang; Záková, Lenka; Dodson, Eleanor J; Turkenburg, Johan P; Brange, Jens; Dodson, G Guy

2006-05-01

Despentapeptide (des-B26-B30) insulin (DPI), an active modified insulin, has been crystallized in the presence of 20% acetic acid pH 2. A crystal structure analysis to 1.8 A spacing (space group I222) revealed that the DPI molecule, which is unable to make beta-strand interactions for physiological dimer formation and is apparently monomeric in solution, formed an alternative lattice-generated dimer. The formation of this dimer involved interactions between surfaces which included the B9-B19 alpha-helices (usually buried by the dimer-dimer contacts within the native hexamer). The two crystallographically independent molecules within the dimer were essentially identical and were similar in conformation to T-state insulin as seen in the T(6) insulin hexamer. An unusual feature of each molecule in the dimer was the presence of two independent conformations at the B-chain C-terminus (residues B20-B25). Both conformations were different from that of native insulin, involving a 3.5 A displacement of the B20-B23 beta-turn and a repositioning of residue PheB25 such that it made close van der Waals contact with the main body of the molecule, appearing to stabilize the B-chain C-terminus.

Detection and differentiation of 22kDa and 20kDa Growth Hormone proteoforms in human plasma by LC-MS/MS

DEFF Research Database (Denmark)

Sanmartín, Gerard Such; Bache, N.; Bosch, J.

2015-01-01

Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20kDa GH proteoforms that demonstrate the administration of the synthetic hormone....... Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report...... a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH...

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

Science.gov (United States)

Kim, Kwangsig; Yang, Hyun Jong

2003-01-01

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis. PMID:12815325

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

Science.gov (United States)

Lüdi, H; Hasselbach, W

1985-11-21

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

Monomeric Aβ1–40 and Aβ1–42 Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil

Science.gov (United States)

2016-01-01

The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ1–40 and Aβ1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ1–42 compared to that of Aβ1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ1–40 and Aβ1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ1–40 and Aβ1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings (3JHNHα, 3JC′C′, 3JC′Hα, 1JHαCα, 2JNCα, and 1JNCα) recorded for Aβ1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process. PMID:26780756

Isoform composition and stoichiometry of the ∼ 90-kDa heat shock protein associated with glucocorticoid receptors

International Nuclear Information System (INIS)

Mendel, D.B.; Orti, E.

1988-01-01

The authors observed that the ∼ 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the ∼ 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the ∼ 90-kDa heat shock protein. The observation that TSTA and the ∼ 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested that the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the ∼ 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the ∼ 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free ∼ 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [ 35 S]methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two ∼ 90-kDa non-steroid-binding subunits. The consistency with which a ∼ 1:2 stoichiometric ratio of steroid binding to ∼ 90-kDa protein is observed supports the view that the ∼ 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

International Nuclear Information System (INIS)

Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

1989-01-01

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation

The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

Science.gov (United States)

A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

Tensile and low cycle fatigue properties of EUROFER97-steel after 16.3 dpa neutron irradiation at 523, 623 and 723 K

Energy Technology Data Exchange (ETDEWEB)

Materna-Morris, E., E-mail: edeltraud.materna-morris@kit.edu; Möslang, A., E-mail: anton.moeslang@kit.edu; Schneider, H.-C., E-mail: hans-christian.schneider@kit.edu

2013-11-15

Neutron-irradiated specimens of the reduced-activation tempered martensitic steel EUROFER97 were tested by tensile and low cycle conditions to detect the impact of irradiation on strength and lifetime. The irradiation temperature ranged from 523 to 723 K with an accumulated dose of up to 16.3 dpa. Tensile tests revealed a significant irradiation-induced hardening below 673 K with a peak of ∼430 MPa at 573 K but none was seen at 723 K, as expected. Despite the significant irradiation-induced reduction of uniform elongation, the total elongation is only reduced by about 50% below 673 K. Post-irradiation strain-controlled fatigue tests have been carried out at T{sub irrad} = T{sub test} = 523, 623 and 723 K. Pronounced cyclic softening was observed in all specimens. At 623 and 723 K, neutron irradiation had no effect on fatigue life within the data scatter. A significant lifetime increase has been observed at T{sub irrad} = T{sub test} = 523 K that advances with decreasing stress amplitude Δε (1% → 0.5%) up to a factor of ten. Scanning electron microscopy (SEM) analysis revealed ductile fracture and fatigue striations on the fracture surfaces. After push–pull fatigue testing, transmission electron microscopy (TEM) investigations showed the typical sub-cell formation, even at T{sub irrad} = T{sub test} = 523 K.

Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species.

Directory of Open Access Journals (Sweden)

Anna-Karin E Svensson

2010-04-01

Full Text Available Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1 can cause amyotrophic lateral sclerosis (ALS. Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.

The Evidence of Non n-glycan Linked Mannose in Exochitinase 42kDa, from Trichoderma harzianum BIO10671 Glycosylation

Directory of Open Access Journals (Sweden)

Muskhazli, M.

2006-01-01

Full Text Available Chitinase 42 kDa produced by Trichoderma harzianum has been proven as a prime compound to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction. Later it will initiate the process of the host cell becomes empty of cytoplasm, disintegrates and shows a rapid collapse. This study investigates the existence of N-glycan linked mannose in chitinase 42 kDa produced by the Malaysian T. harzianum strain BIO10671. The chitinase 42 kDa from T. harzianum BIO10671 was initially purified using anion exchange chromatography prior to a series of experiments such as immunoblotting against the chitinase 42 kDa antibody, lectin staining for detecting any terminal linked mannose, and galactofuranose detection to determine the presence of galatofuranase components in glycoproteins. The enzyme purification harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671 with strong indication of the chitinase 42 kDa presence on SDS-Page. This was confirmed by immunoblotting with a strong response around 42 kDa after overnight incubation in chitinase 42 kDa antibody suggesting that the gene for chitinase 42 kDa was greatly expressed in this strain. There are no intervation of galatofuranose on any of the terminal mannose in chitinase 42 kDa as shown by negative results on samples treated with or without endoglycosidase-H and lectin staining. Therefore, it can be concludeed that glycosylation occurred in the chitinase 42 kDa from T. harzianum 42 kDa was not in the form of N-glycan linked mannose as expected.

Two mechanisms for dissipation of excess light in monomeric and trimeric light-harvesting complexes

Energy Technology Data Exchange (ETDEWEB)

Dall' Osto, Luca [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Cazzaniga, Stefano [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Bressan, Mauro [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Paleček, David [Lund Univ. (Sweden). Dept. of Chemical Physics; Židek, Karel [Lund Univ. (Sweden). Dept. of Chemical Physics; Niyogi, Krishna K. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst., Dept. of Plant and Microbial Biology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Fleming, Graham R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry, Graduate Group in Applied Science and Technology; Zigmantas, Donatas [Lund Univ. (Sweden). Dept. of Chemical Physics; Bassi, Roberto [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Consiglio Nazionale delle Ricerche (CNR), Firenze (Italy). Istituto per la Protezione delle Piante (IPP)

2017-04-10

Oxygenic photoautotrophs require mechanisms for rapidly matching the level of chlorophyll excited states from light harvesting with the rate of electron transport from water to carbon dioxide. These photoprotective reactions prevent formation of reactive excited states and photoinhibition. The fastest response to excess illumination is the so-called non-photochemical quenching which, in higher plants, requires the luminal pH sensor PsbS and other yet unidentified components of the photosystem II antenna. Both trimeric light-harvesting complex II (LHCII) and monomeric LHC proteins have been indicated as site(s) of the heat-dissipative reactions. Different mechanisms have been proposed: Energy transfer to a lutein quencher in trimers, formation of a zeaxanthin radical cation in monomers. Here, we report on the construction of a mutant lacking all monomeric LHC proteins but retaining LHCII trimers. Its non-photochemical quenching induction rate was substantially slower with respect to the wild type. A carotenoid radical cation signal was detected in the wild type, although it was lost in the mutant. Here, we conclude that non-photochemical quenching is catalysed by two independent mechanisms, with the fastest activated response catalysed within monomeric LHC proteins depending on both zeaxanthin and lutein and on the formation of a radical cation. Trimeric LHCII was responsible for the slowly activated quenching component whereas inclusion in supercomplexes was not required. Finally, this latter activity does not depend on lutein nor on charge transfer events, whereas zeaxanthin was essential.

Formation of the 67-kDa laminin receptor by acylation of the precursor.

Science.gov (United States)

Butò, S; Tagliabue, E; Ardini, E; Magnifico, A; Ghirelli, C; van den Brûle, F; Castronovo, V; Colnaghi, M I; Sobel, M E; Ménard, S

1998-06-01

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.

Inhibition of Clostridium difficile toxin A and B by 1,2-cyclohexanedione modification of an arginine residue.

Science.gov (United States)

Balfanz, J; Rautenberg, P

1989-12-29

Toxin A (enterotoxin) and toxin B (cytotoxin) of Clostridium difficile were both inactivated by the arginine specific reagent 1,2-cyclohexanedione. Molecular stability during the inactivation process was demonstrated by SDS-PAGE analysis showing the same migration rates for modified and unmodified forms of the 230 kDa toxin A and of the 250 kDa toxin B. Cytotoxicity of both toxins as well as mouse lethality of the enterotoxin were drastically decreased as a result of the arginine modification. The reaction followed pseudo-first-order kinetics. Analysis of the data suggested that modification of a single arginine residue was sufficient to abolish the activity of both toxins.

Radioimmunoguided surgery using iodine 125 B72.3 in patients with colorectal cancer

International Nuclear Information System (INIS)

Cohen, A.M.; Martin, E.W. Jr.; Lavery, I.; Daly, J.; Sardi, A.; Aitken, D.; Bland, K.; Mojzisik, C.; Hinkle, G.

1991-01-01

Preliminary data using B72.3 murine monoclonal antibody labeled with iodine 125 suggested that both clinically apparent as well as occult sites of colorectal cancer could be identified intraoperatively using a hand-held gamma detecting probe. We report the preliminary data of a multicenter trial of this approach in patients with primary or recurrent colorectal cancer. One hundred four patients with primary, suspected, or known recurrent colorectal cancer received an intravenous infusion of 1 mg of B72.3 monoclonal antibody radiolabeled with 7.4 x 10 Bq of iodine 125. Twenty-six patients with primary colorectal cancer and 72 patients with recurrent colorectal cancer were examined. Using the gamma detecting probe, 78% of the patients had localization of the antibody in their tumor; this included 75% of primary tumor sites and 63% of all recurrent tumor sites; 9.2% of all tumor sites identified represented occult sites detected only with the gamma detecting probe. The overall sensitivity was 77% and a predictive value of a positive detection was 78%. A total of 30 occult sites in 26 patients were identified. In patients with recurrent cancer, the antibody study provided unique data that precluded resection in 10 patients, and in another eight patients it extended the potentially curative procedure

Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.

Science.gov (United States)

Mukherjee, T; Basu, D; Mahapatra, S; Goffin, C; van Beeumen, J; Basu, J

1996-01-01

The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin. PMID:8947487

Crystallization and preliminary crystallographic analysis of decameric and monomeric forms of C49S mutant thioredoxin-dependent AhpC from Helicobacter pylori

International Nuclear Information System (INIS)

Supangat; Seo, Kyung Hye; Furqoni, Ahmad; Kwon, Young-Chul; Cho, Myung-Je; Rhee, Kwang-Ho; Lee, Sang Yeol; Lee, Kon Ho

2008-01-01

Decameric and monomeric forms of recombinant C49S mutant AhpC from H. pylori have been crystallized. Diffraction data were collected to 2.8 and 2.25 Å, respectively. Cys49Ser mutant Helicobacter pylori alkyl hydroperoxide reductase (C49S HpAhpC) was purified under reducing conditions in monomeric and decameric forms. The monomeric form was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.25 Å resolution and belonged to space group C2, with unit-cell parameters a = 245.8, b = 140.7, c = 189.5 Å, β = 127°, and contained 20 molecules in the asymmetric unit. A crystal of the decameric form was obtained by the microbatch crystallization method and diffracted to 2.8 Å resolution. It belonged to space group C222, with unit-cell parameters a = 257.5, b = 417.5, c = 95.6 Å. The structure of the monomeric form of C49S HpAhpC has been solved by the molecular-replacement method

Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

International Nuclear Information System (INIS)

Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz; Burger, Gertraud; Hori, Manabu; Fujishima, Masahiro

2005-01-01

The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

Science.gov (United States)

Djaffar, I; Chen, Y P; Creminon, C; Maclouf, J; Cieutat, A M; Gayet, O; Rosa, J P

1994-05-15

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

International Nuclear Information System (INIS)

Chen, J.W.; Cunningham, M.D.; Galton, N.; Michaelis, E.K.

1988-01-01

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an ∼ 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 + M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L[ 3 H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes

Neurodevelopmental Expression Profile of Dimeric and Monomeric Group 1 mGluRs: Relevance to Schizophrenia Pathogenesis and Treatment.

Science.gov (United States)

Lum, Jeremy S; Fernandez, Francesca; Matosin, Natalie; Andrews, Jessica L; Huang, Xu-Feng; Ooi, Lezanne; Newell, Kelly A

2016-10-10

Group 1 metabotropic glutamate receptors (mGluR1/mGluR5) play an integral role in neurodevelopment and are implicated in psychiatric disorders, such as schizophrenia. mGluR1 and mGluR5 are expressed as homodimers, which is important for their functionality and pharmacology. We examined the protein expression of dimeric and monomeric mGluR1α and mGluR5 in the prefrontal cortex (PFC) and hippocampus throughout development (juvenile/adolescence/adulthood) and in the perinatal phencyclidine (PCP) model of schizophrenia. Under control conditions, mGluR1α dimer expression increased between juvenile and adolescence (209-328%), while monomeric levels remained consistent. Dimeric mGluR5 was steadily expressed across all time points; monomeric mGluR5 was present in juveniles, dramatically declining at adolescence and adulthood (-97-99%). The mGluR regulators, Homer 1b/c and Norbin, significantly increased with age in the PFC and hippocampus. Perinatal PCP treatment significantly increased juvenile dimeric mGluR5 levels in the PFC and hippocampus (37-50%) but decreased hippocampal mGluR1α (-50-56%). Perinatal PCP treatment also reduced mGluR1α dimer levels in the PFC at adulthood (-31%). These results suggest that Group 1 mGluRs have distinct dimeric and monomeric neurodevelopmental patterns, which may impact their pharmacological profiles at specific ages. Perinatal PCP treatment disrupted the early expression of Group 1 mGluRs which may underlie neurodevelopmental alterations observed in this model.

Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

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Ben Buelow

2009-07-01

Full Text Available Poly adenosine diphosphate-ribose polymerase-1 (PARP-1 is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR, and formation of the soluble 2(nd messenger monomeric adenosine diphosphate-ribose (mADPR. Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

Science.gov (United States)

Vora, J P; Owens, D R; Dolben, J; Atiea, J A; Dean, J D; Kang, S; Burch, A; Brange, J

1988-11-12

To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. Study in normal people at a diabetes research unit and a university department of medical physics. Seven healthy male volunteers aged 20-39 not receiving any other drugs. After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles

Science.gov (United States)

Mercer, Robert C. C.; Fu, Ze-Lin; Mays, Charles E.; Gapeshina, Hristina; Wohlgemuth, Serene L.; Acevedo-Morantes, Claudia Y.; Cashman, Neil R.; Coulthart, Michael B.; Jansen, Gerard H.; Stepanova, Maria

2018-01-01

To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting de novo formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie. PMID:29338055

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

Science.gov (United States)

Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

2011-09-01

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

Science.gov (United States)

Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

2015-04-01

Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

International Nuclear Information System (INIS)

Stasia, M.J.; Dianoux, A.C.; Vignais, P.V.

1989-01-01

In 32 P i -loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. The apparent molecular mass of the purified protein range between 20 and 23 kDa. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of our discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [γ- 32 P]ATP in the presence of bovine neutrophil PKC supplemented with Ca 2+ , phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. IEF of the 32 P-labeled 23-kDa protein followed by autoradiography revealed for discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein. The bands of the 23-kDa protein resolved by IEF and transfered to nitrocellulose showed ability to bind [ 35 S]GTP-γ-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDa protein was demonstrated on immunoblots after SDS-PAGE. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely, calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinum toxin

MONOMERIC ß-AMYLOID INTERACTS WITH TYPE-1 INSULIN-LIKE GROWTH FACTOR RECEPTORS TO PROVIDE ENERGY SUPPLY TO NEURONS

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Maria Laura eGiuffrida

2015-08-01

Full Text Available ß-amyloid (Aß1-42 is produced by proteolytic cleavage of the transmembrane type-1 protein, amyloid precursor protein. Under pathological conditions, Aß1-42 self-aggregates into oligomers, which cause synaptic dysfunction and neuronal loss, and are considered the culprit of Alzheimer’s disease (AD. However, Aß1-42 is mainly monomeric at physiological concentrations, and the precise role of monomeric Aß1-42 in neuronal function is largely unknown. We report that the monomer of Aß1-42 activates type-1 insulin-like growth factor receptors and enhances glucose uptake in neurons and peripheral cells by promoting the translocation of the Glut3 glucose transporter from the cytosol to the plasma membrane. In neurons, activity-dependent glucose uptake was blunted after blocking endogenous Aß production, and re-established in the presence of cerebrospinal fluid Aß. APP-null neurons failed to enhance depolarization-stimulated glucose uptake unless exogenous monomeric Aß1-42 was added. These data suggest that Aß1-42 monomers were critical for maintaining neuronal glucose homeostasis. Accordingly, exogenous Aß1-42 monomers were able to rescue the low levels of glucose consumption observed in brain slices from AD mutant mice.

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

Science.gov (United States)

Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

2008-02-01

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

DEFF Research Database (Denmark)

Schønning, Kristian; Bolmstedt, A; Novotny, J

1998-01-01

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation....

Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

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Worrall, Liam; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [School of Biological Sciences, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom)

2006-09-01

Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

Chemical shift changes provide evidence for overlapping single-stranded DNA and XPA binding sites on the 70 kDa subunit of human replication protein A

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Daughdrill, Gary W.; Buchko, Garry W.; Botuyan, Maria V.; Arrowsmith, Cheryl H.; Wold, Marc S.; Kennedy, Michael A.; Lowry, David F.

2003-07-15

Replication protein A (RPA) is a heterotrimeric single-stranded DNA (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, NMR spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA701-326), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H-15N correlation spectrum of uniformly 15N-labeled hRPA701-326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA binding domain that is between residues 183 and 296 of the hRPA701-326 fragment. The hRPA701-326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA binding domain to identify the binding surface with XPA-MBD. The XPA-MBD binding surface showed significant overlap with an ssDNA binding surface that was previously identified using NMR spectroscopy and X-ray crystallography.

Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.

Science.gov (United States)

Verma, Alok Kumar; Sharma, Akanksha; Kumar, Sandeep; Gupta, Rinkesh Kumar; Kumar, Dinesh; Gupta, Kriti; Giridhar, B H; Das, Mukul; Dwivedi, Premendra D

2016-06-01

Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to

Detection of calmodulin binding protein at 170 KDA in BALB, AKR, DON and chicken granulosa cells

International Nuclear Information System (INIS)

Selinfreund, R.; Lin, P.H.; Marrone, B.; Wharton, W.

1987-01-01

Calmodulin (CAM) has been shown to bind to the epidermal growth factor (EGF) receptor (170 kDa) and is phosphorylated in a EGF dependent manner in the A431 human epidermoid carcinoma cells. In the present study, they report 125 I-CAM binding to a 170 kDa protein detected in cell membrane vesicles of Balb/3T3, AKR, DON and chicken granulosa cells. Purified plasma membranes from these cells were resolved via electrophoresis (without heat denaturation) and electroblotted onto nictrocellulose paper. Upon hybridizing against 125 I-CAM, a distinct autoradiographic band occurred at 170 kDa for all the cells lines under study. The binding of CAM is specific and can be displaced with the addition of excess unlabeled CAM. The result suggest that 125 I-CAM may bind to the 170 kDa EGF receptor in BALB, AKR, DON and chicken granulosa cells

Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

International Nuclear Information System (INIS)

Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry

2005-01-01

Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure

Biosynthesis of 10 kDa and 7.5 kDa insulin-like growth factor II in a human rhabdomyosarcoma cell line

DEFF Research Database (Denmark)

Nielsen, F C; Haselbacher, G; Christiansen, Jan

1993-01-01

In the present study we have analysed the expression of insulin-like growth factor II (IGF-II) in the human rhabdomyosarcoma cell line IN157.IN157 cells express high levels of three IGF-II mRNAs of 6.0 kb, 4.8 kb and 4.2 kb. In contrast, normal skeletal muscle expresses a negligible amount of IGF......-II mRNA. Two forms of IGF-II with molecular masses of 7.5 kDa and 10 kDa, corresponding to the mature IGF-II and IGF-II with a C-terminal extension of 21 amino acids (IGF-IIE21), were secreted into the culture medium at amounts of 17 ng/ml (2.3 nM) and 15 ng/ml (1.5 nM), respectively. IN157 cells also......-II and IGF-IIE21 with Kd values of 0.5 nM and 2 nM, respectively, and IGF-I with about 500 times lower affinity. IGF-II and IGF-IIE21 stimulated DNA synthesis via the IGF-I receptor, whereas the IGF-II/Man 6-P receptor mediated their rapid internalization and inactivation. During culture of IN157 cells about...

Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

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Françoise Paquet

Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

Monomeric, Oligomeric and Polymeric Proteins in Huntington Disease and Other Diseases of Polyglutamine Expansion

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Guylaine Hoffner

2014-03-01

Full Text Available Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective.

Nanogapped impedimetric immunosensor for the detection of 16 kDa heat shock protein against Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Gopinath, Subash C. B.; Perumal, Veeradasan; Lakshmipriya, Thangavel; Rajintraprasad, Haarindraprasad; Rao, Balakrishnan S.; Arshad, M. K. Md; Hashim, Uda; Kumaresan, Ramanujam; Kotani, Norito; Chen, Yeng

2016-01-01

The 16 kDa heat shock protein (16 kDa HSP) against Mycobacterium tuberculosis (MT), expressed during the growth phase of MT, is a potential target in diagnostic tests for tuberculosis (TB). We describe here a method for impedimetric determination of the antigen by using a nanogapped dielectric surface consisting of a silver support coated with a thin finger-shaped coating made from zinc oxide and gold and patterned through a lift-off process. The electrode was characterized by scanning electron microscopy, field emission scanning electron microscopy, atomic force microscopy, and energy-dispersive X-ray spectroscopy. Surface chemical functionalization and immobilization of antibody against the 16 kDa HSP was evidenced by FTIR. In order to improve the detection limit, the antigen was conjugated to 10 nm gold nanoparticles. The resulting biosensor is capable of detecting the 16 kDa HSP in concentrations as low as 100 fM. The method covers a wide analytical range that extends from 100 fM to 1 nM. (author)

Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

Science.gov (United States)

Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

2016-11-01

Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features. Copyright © 2016. Published by Elsevier Inc.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Science.gov (United States)

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-03-19

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

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Javed M Khan

Full Text Available Banana lectin (BL is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS binding, size exclusion chromatography (SEC and dynamic light scattering (DLS. During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml at pH 2.0 while single peak (61.45 ml at pH 7.4. The hydrodynamic radii (R h of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.

mKikGR, a monomeric photoswitchable fluorescent protein.

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Satoshi Habuchi

Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

Recombinant DNA specifying the human amyloid. beta. precursor protein (ABPP) encodes a 95-kDa polypeptide

Energy Technology Data Exchange (ETDEWEB)

Mita, S; Sadlock, J; Herbert, J; Schon, E A

1988-10-11

Although the ABPP gene give rise to multiple mRNAs, the primary translation product of this gene is unknown. The longest published cDNA sequences predict a 770-aa polypeptide of 87 kDa. However, in immunoblots, ABPP migrated as a single species of >92 kDa in rat brain, and in human, as a species of 95-100 kDa in non-membrane bound form, as multiple species of 110-135 kDa in membrane-associated form and as a 130-kDa species in fibroblasts. The sizes of these larger species relative to the MW of ABPP predicted from the cDNA sequences have been attributed to postranslational modification. However, the authors have isolated a cDNA (lambdaHAP2) from a human fetal muscle lambdagt11 cDNA library encoding an 843-aa polypeptide with a deduced MW of 94,642. This cDNA contains both exons encoding an 843-aa polypeptide with a deduced MW of 94.642. This cDNA contains both exons encoding the protease inhibitor domains. Primer extension analysis indicates that the 5' terminus of this cDNA is 14 nt from a transcriptional start site. This cDNA, encoding the longest ABPP described to date, may explain some of the observations on the sizes of tissue-derived ABPP.

Sensitivity analyses of finite element method for estimating residual stress of dissimilar metal multi-pass weldment in nuclear power plant

Energy Technology Data Exchange (ETDEWEB)

Song, Tae Kwang; Bae, Hong Yeol; Kim, Yun Jae [Korea Unviersity, Seoul (Korea, Republic of); Lee, Kyoung Soo; Park, Chi Yong [Korea Electric Power Research Institute, Daejeon (Korea, Republic of)

2008-09-15

In nuclear power plants, ferritic low alloy steel components were connected with austenitic stainless steel piping system through alloy 82/182 butt weld. There have been incidents recently where cracking has been observed in the dissimilar metal weld. Alloy 82/182 is susceptible to primary water stress corrosion cracking. Weld-induced residual stress is main factor for crack growth. Therefore exact estimation of residual stress is important for reliable operating. This paper presents residual stress computation performed by 6'' safety and relief nozzle. Based on 2 dimensional and 3 dimensional finite element analyses, effect of welding variables on residual stress variation is estimated for sensitivity analysis.

Functional analysis of the human cytomegalovirus immune evasion protein, pUS322kDa

International Nuclear Information System (INIS)

Zhao Yiqiang; Biegalke, Bonita J.

2003-01-01

Human cytomegalovirus (HCMV) is an important opportunistic pathogen that infrequently causes disease in individuals with mature immune systems. The HCMV US3 gene encodes a 22-kDa protein that interferes with immune recognition of virally infected cells. The 22-kDa US3 protein binds to major histocompatibility complex (MHC) class I complexes, retaining them in the endoplasmic reticulum (ER), thereby decreasing the presentation of viral antigen to cytotoxic T cells. Our studies demonstrate that correct folding of the ER lumenal domain of the US3 protein is essential, but insufficient for interactions with MHC class I complexes. We demonstrate a requirement for the transmembrane domain of the 22-kDa US3 protein, confirming the results of others, and also show that the cytosolic carboxyl-terminal tail influences the function of the protein. Anchoring of the ER-lumenal immunoglobulin-like fold of the US3 protein to the membrane of the endoplasmic reticulum is critical for the binding and retention of MHC class I complexes

Selective defunctionalization by TiO2 of monomeric phenolics from lignin pyrolysis into simple phenols.

Science.gov (United States)

Mante, Ofei D; Rodriguez, Jose A; Babu, Suresh P

2013-11-01

This study is focused on defunctionalizing monomeric phenolics from lignin into simple phenols for applications such as phenol/formaldehyde resins, epoxidized novolacs, adhesives and binders. Towards this goal, Titanium dioxide (TiO2) was used to selectively remove hydroxyl, methoxy, carbonyl and carboxyl functionalities from the monomeric phenolic compounds from lignin to produce mainly phenol, cresols and xylenols. The results showed that anatase TiO2 was more selective and active compared to rutile TiO2. Catechols were found to be the most reactive phenolics and 4-ethylguaiacol the least reactive with anatase TiO2. An overall conversion of about 87% of the phenolics was achieved at 550°C with a catalyst-to-feed ratio of 5 w/w. Over 97% conversion of phenolics is achievable at moderate temperatures (550°C or ≤ 600°C) and a moderate catalyst-to-feed ratio of 6.5:1. The reactivity of catechols on TiO2 suggests that titania is a promising catalyst in the removal of hydroxyl moiety. Published by Elsevier Ltd.

Preparation and properties of a monomeric Mn(IV)-oxo complex.

Science.gov (United States)

Parsell, Trenton H; Behan, Rachel K; Green, Michael T; Hendrich, Michael P; Borovik, A S

2006-07-12

Manganese-oxo complexes have long been investigated because of their proposed roles in biological and chemical catalysis. However, there are few examples of monomeric complexes with terminal oxo ligands, especially those with oxomanganese(IV) units. A oxomanganese(IV) complex has been prepared from [MnIIIH3buea(O)]2- ([H3buea]3-, tris[(N'-tert-butylureaylato)-N-ethylene]aminato), a monomeric MnIII-O complex in which the oxo ligand arises from cleavage of dioxygen. Treating [MnIIIH3buea(O)]2- with [Cp2Fe]BF4 in either DMF at -45 degrees C or DMSO at room temperature produces [MnIVH3buea(O)]-: lambdamax = 635 nm; nu(Mn-16O) = 737 cm-1; nu(Mn-18O) = 709 cm-1; g = 5.15, 2.44, 1.63, D = 3.0 cm-1, E/D = 0.26, aMn = 66 G (A = 190 MHz). These spectroscopic properties support the assignment of a mononuclear MnIV-oxo complex with an S = 3/2 ground state. Density functional theory supports this assignment and the Jahn-Teller distortion around the high-spin MnIV center that would alter the molecular structure of [MnIVH3buea(O)]- from trigonal symmetry (as indicated by the highly rhombic EPR signal). [MnIVH3buea(O)]- is relatively unstable in DMSO, converting to [MnIIIH3buea(OH)]- via a proposed X-H bond cleavage. [MnIVH3buea(O)]- reacts with 1,2-diphenylhydrazine to from azobenzene (95% yield) and [MnIIIH3buea(OH)]-. The MnIV-oxo does not react with triphenyl- or tricyclohexylphosphine. However, O-atom transfer is observed with methyldiphenylphosphine and dimethylphenylphosphine, producing the corresponding phosphine oxides. These results illustrate the diverse reactivity of the MnIV-oxo unit.

Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

NARCIS (Netherlands)

Vlugt-Bergmans, van der C.J.B.; Werf, van der M.J.

2001-01-01

A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14

NARCIS (Netherlands)

Vlugt-Bergmans, C.J.B. van der; Werf, M.J. van der

2001-01-01

A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

Polyimide resin composites via in situ polymerization of monomeric reactants

Science.gov (United States)

Cavano, P. J.

1974-01-01

Thermo-oxidatively stable polyimide/graphite-fiber composites were prepared using a unique in situ polymerization of monomeric reactants directly on reinforcing fibers. This was accomplished by using an aromatic diamine and two ester-acids in a methyl alcohol solvent, rather than a previously synthesized prepolymer varnish, as with other A-type polyimides. A die molding procedure was developed and a composite property characterization conducted with high modulus graphite fiber tow. Flexure, tensile, compressive, and shear tests were conducted at temperatures from 72 to 650 F on laminates before and after exposures at the given temperatures in an air environment for times up to 1000 hours. The composite material was determined to be oxidatively, thermally, and hydrolytically stable.

Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements.

Science.gov (United States)

Mollah, A K M M; Stennis, Rhonda L; Mossing, Michael C

2003-05-01

The thermodynamic stabilities of three monomeric variants of the bacteriophage lambda Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.

Bioethanol production from forestry residues: A comparative techno-economic analysis

International Nuclear Information System (INIS)

Frankó, Balázs; Galbe, Mats; Wallberg, Ola

2016-01-01

Highlights: • A proposed cellulosic ethanol biorefinery in Sweden was simulated with Aspen Plus. • Forestry residues with different bark contents were evaluated as raw materials. • The bark content negatively influenced the minimum ethanol selling price. • Sensitivity analyses were performed to assess the influence of raw material cost. - Abstract: A techno-economic analysis was conducted to assess the feasibility of using forestry residues with different bark contents for bioethanol production. A proposed cellulosic ethanol biorefinery in Sweden was simulated with Aspen Plus. The plant was assumed to convert different forestry assortments (sawdust and shavings, fuel logs, early thinnings, tops and branches, hog fuel and pulpwood) to ethanol, pellets, biogas and electricity. The intention was not to obtain absolute ethanol production costs for future facilities, but to assess and compare the future potential of utilizing different forestry residues for bioethanol production. The same plant design and operating conditions were assumed in all cases, and the effect of including bark on the whole conversion process, especially how it influenced the ethanol production cost, was studied. While the energy efficiency (not including district heating) obtained for the whole process was between 67 and 69% regardless of the raw material used, the ethanol production cost differed considerably; the minimum ethanol selling price ranging from 0.77 to 1.52 USD/L. Under the basic assumptions, all the forestry residues apart from sawdust and shavings exhibited a negative net present value at current market prices. The profitability decreased with increasing bark content of the raw material. Sensitivity analyses showed that, at current market prices, the utilization of bark-containing forestry residues will not provide significant cost improvement compared with pulpwood unless the conversion of cellulose and hemicellulose to monomeric sugars is improved.

Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in ''Saccharomyces cerevisiae''

International Nuclear Information System (INIS)

Lenart, U.; Palamarczyk, G.

1995-01-01

The membrane-bound sterolglucoside synthase from the yeast ''Saccharomyces cerevisiae'' has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di- 125 I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDPglucose, which is a substrate for this enzyme. Upon photolysis the 125 I-labelled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlated with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast. (author). 10 refs, 5 figs, 1 tab

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

Science.gov (United States)

Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

2014-01-29

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

18 kDa translocator protein – implications in cell’s functions

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Agnieszka Kołodziejczyk

2015-01-01

Full Text Available The mitochondrial 18kDa Translocation Protein (TSPO was first identified in 1977 by its capability to bind benzodiazepines in peripheral tissues. It is more commonly known after its previous name – peripheral benzodiazepine receptor (PBR as opposed to the central benzodiazepine receptor (CBR, from which it differs by location, structure and function. It is ubiquitous with highest expression in steroid-producing tissues, like adrenal cortex, ovaries, testicles, and placenta. The role of TSPO is crucial for living; its inactivation results in early embryonic-lethal phenotype in mice. TSPO has been implicated in various functions of cell, including steroidogenesis, cellular respiration, reactive oxygen species production, heme biosynthesis, immunomodulation, apoptosis, and cellular proliferation. TSPO has been shown to interact with other cellular proteins: 32 kDa voltage-dependent anion channel (VDAC, 30 kDa adenine nucleotide translocase (ANT, cyclophilin D, hexokinase, creatinine kinase, diazepam binding inhibitor (DBI, phosphate carrier and Bcl-2 family. They are – involved in the formation and regulation of mitochondrial permeability transition pore (mPTP at the junction of the inner and outer mitochondrial membranes. While the function and characteristics of the mPTP are known, its well defined, but its structure remains speculative. Changes in TSPO expression are associated with multiple disorders, including cancer, ischaemia-reperfusion injury, neurological diseases and psychiatric disorders, atheromatosis, and others. – TSPO is able to bind cholesterol, porphyrins and other ligands with different affinity. The current knowledge of TSPO implicates its potential use as a diagnostic marker and therapeutic target in different diseases and their therapies.

18 kDa translocator protein – implications in cell’s functions

Directory of Open Access Journals (Sweden)

Agnieszka Kołodziejczyk

2015-06-01

Full Text Available The mitochondrial 18kDa Translocation Protein (TSPO was first identified in 1977 by its capability to bind benzodiazepines in peripheral tissues. It is more commonly known after its previous name – peripheral benzodiazepine receptor (PBR as opposed to the central benzodiazepine receptor (CBR, from which it differs by location, structure and function. It is ubiquitous with highest expression in steroid-producing tissues, like adrenal cortex, ovaries, testicles, and placenta. The role of TSPO is crucial for living; its inactivation results in early embryonic-lethal phenotype in mice. TSPO has been implicated in various functions of cell, including steroidogenesis, cellular respiration, reactive oxygen species production, heme biosynthesis, immunomodulation, apoptosis, and cellular proliferation. TSPO has been shown to interact with other cellular proteins: 32 kDa voltage-dependent anion channel (VDAC, 30 kDa adenine nucleotide translocase (ANT, cyclophilin D, hexokinase, creatinine kinase, diazepam binding inhibitor (DBI, phosphate carrier and Bcl-2 family. They are – involved in the formation and regulation of mitochondrial permeability transition pore (mPTP at the junction of the inner and outer mitochondrial membranes. While the function and characteristics of the mPTP are known, its well defined, but its structure remains speculative. Changes in TSPO expression are associated with multiple disorders, including cancer, ischaemia-reperfusion injury, neurological diseases and psychiatric disorders, atheromatosis, and others. – TSPO is able to bind cholesterol, porphyrins and other ligands with different affinity. The current knowledge of TSPO implicates its potential use as a diagnostic marker and therapeutic target in different diseases and their therapies.

Discovery of Radioiodinated Monomeric Anthraquinones as a Novel Class of Necrosis Avid Agents for Early Imaging of Necrotic Myocardium.

Science.gov (United States)

Wang, Qin; Yang, Shengwei; Jiang, Cuihua; Li, Jindian; Wang, Cong; Chen, Linwei; Jin, Qiaomei; Song, Shaoli; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Yin, Zhiqi

2016-02-16

Assessment of myocardial viability is deemed necessary to aid in clinical decision making whether to recommend revascularization therapy for patients with myocardial infarction (MI). Dianthraquinones such as hypericin (Hyp) selectively accumulate in necrotic myocardium, but were unsuitable for early imaging after administration to assess myocardial viability. Since dianthraquinones can be composed by coupling two molecules of monomeric anthraquinone and the active center can be found by splitting chemical structure, we propose that monomeric anthraquinones may be effective functional groups for necrosis targetability. In this study, eight radioiodinated monomeric anthraquinones were evaluated as novel necrosis avid agents (NAAs) for imaging of necrotic myocardium. All (131)I-anthraquinones showed high affinity to necrotic tissues and (131)I-rhein emerged as the most promising compound. Infarcts were visualized on SPECT/CT images at 6 h after injection of (131)I-rhein, which was earlier than that with (131)I-Hyp. Moreover, (131)I-rhein showed satisfactory heart-to-blood, heart-to-liver and heart-to-lung ratios for obtaining images of good diagnostic quality. (131)I-rhein was a more promising "hot spot imaging" tracer for earlier visualization of necrotic myocardium than (131)I-Hyp, which supported further development of radiopharmaceuticals based on rhein for SPECT/CT ((123)I and (99m)Tc) or PET/CT imaging ((18)F and (124)I) of myocardial necrosis.

Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

International Nuclear Information System (INIS)

Kelly, Mark J.S.; Krieger, Cornelia; Ball, Linda J.; Yu Yihua; Richter, Gerald; Schmieder, Peter; Bacher, Adelbert; Oschkinat, Hartmut

1999-01-01

NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins

Structural Characterization of Monomeric/Dimeric State of p59fyn SH2 Domain.

Science.gov (United States)

Huculeci, Radu; Kieken, Fabien; Garcia-Pino, Abel; Buts, Lieven; van Nuland, Nico; Lenaerts, Tom

2017-01-01

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59 fyn SH2 domain.

Comparison of excretory urographic contrast effects of dimeric and monomeric non-ionic iodinated contrast media in dogs

International Nuclear Information System (INIS)

Kishimoto, M.; Yamada, K.; Watanabe, A.; Miyamoto, K.; Iwasaki, T.; Miyake, Y.

2007-01-01

In excretory urography, the osmolarity of contrast media has rarely been treated as important in veterinary medicine. In this study, the contrast effect of two contrast media (monomeric iohexol and dimeric iodixanol) in the renal cortex and aorta were compared using computed tomography (CT). Five beagle dogs were used and the study employed a cross-over method for each contrast media. The results showed that there was no difference between the media in the aorta, but iodixanol showed higher CT value and a longer contrast effect than iohexol in the renal cortex, in spite of having the same iodine dosage. It is believed that iodixanol, with its low osmolarity, is diluted less by osmotic diuresis than monomeric iohexol. It is important to consider the osmolarity of the contrast media when evaluating the contrast effect, and it is essential to use the same contrast media for each examination, or the renal excretory speed will be under/overestimated

Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11.5-kDa peptide containing the putative 25-hydroxyvitamin D3 binding site

International Nuclear Information System (INIS)

Ray, R.; Holick, M.F.; Bouillon, R.; Baelen, H.V.

1991-01-01

In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D 3 for the binding site of the latter in hDBP and (2) 3 H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with 3 H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D 3

SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra

Science.gov (United States)

Khaliq, Binish; Falke, Sven; Negm, Amr; Buck, Friedrich; Munawar, Aisha; Saqib, Maria; Mahmood, Seema; Ahmad, Malik Shoaib; Betzel, Christian; Akrem, Ahmed

2017-06-01

Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50-55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 Å). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins.

α-SNAP prevents docking of the acrosome during sperm exocytosis because it sequesters monomeric syntaxin.

Directory of Open Access Journals (Sweden)

Facundo Rodríguez

Full Text Available α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs--wild type and M105I--inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160-295, unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans

78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

International Nuclear Information System (INIS)

Gonzalez-Noriega, Alfonso; Ortega Cuellar, Daniel D.; Michalak, Colette

2006-01-01

Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH 4 Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor

Effect of preemptive weld overlay on residual stress of repaired weldment in surge nozzle

Energy Technology Data Exchange (ETDEWEB)

Oh, Chang Young; Song, Tae Kwang; Bae, Hong Yeol; Chun, Yun Bae; Kim, Yun Jae [Korea Univ., Seoul (Korea, Republic of); Lee, Kyoung Soo; Park, Chi Yong [Korea Electric Power Research Institute, Daejeon (Korea, Republic of)

2008-07-01

In the welding process, weldments usually include repair weld during the manufacturing process. Repair welds is supposed to cause strong tensile residual stress. Moreover weldments, usually made by Alloy 82/182, is susceptible to PWSCC. Therefore, mitigation of welding residual stress in weldments is important for reliable operating. PWOL is one of the methods for mitigation and verified for over twenty years. In this paper, residual stress distribution of repaired weldments and the effect of PWOL on mitigation is examined for surge nozzle.

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.

Science.gov (United States)

Roy, M; Lee, R W; Kaarsholm, N C; Thøgersen, H; Brange, J; Dunn, M F

1990-06-12

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain*

Science.gov (United States)

Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J.; Ploplis, Victoria A.; Law, Ruby; Castellino, Francis J.

2014-01-01

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1–2 nm). However, addition of two PAM residues (Arg126-His127) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg113, His114, Glu116, Arg126, His127), mutation of which reduced PAM binding affinity for K2hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. PMID:24962580

Dimerization is not a determining factor for functional high affinity human plasminogen binding by the group A streptococcal virulence factor PAM and is mediated by specific residues within the PAM a1a2 domain.

Science.gov (United States)

Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J; Ploplis, Victoria A; Law, Ruby; Castellino, Francis J

2014-08-01

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Formation of amyloid fibers by monomeric light chain variable domains.

Science.gov (United States)

Brumshtein, Boris; Esswein, Shannon R; Landau, Meytal; Ryan, Christopher M; Whitelegge, Julian P; Phillips, Martin L; Cascio, Duilio; Sawaya, Michael R; Eisenberg, David S

2014-10-03

Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Discovery, characterization, and kinetic analysis of an alditol oxidase from streptomyces coelicolor

NARCIS (Netherlands)

Heuts, Dominic P. H. M.; van Hellemond, Erik W.; Janssen, Dick B.; Fraaije, Marco W.

2007-01-01

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each

The Beckman DxI 800 prolactin assay demonstrates superior specificity for monomeric prolactin.

LENUS (Irish Health Repository)

Byrne, Brendan

2010-02-01

Commercially available prolactin immunoassays detect macroprolactin to variable degrees. Best practice requires laboratories to assess the cross-reactivity of their prolactin assay with macroprolactin, and where appropriate, introduce a screen for the presence of macroprolactin. Our policy has been to reanalyse hyperprolactinaemic samples following polyethylene glycol (PEG) precipitation and to report the resultant value as the monomeric prolactin content of the sample. The goal of this study was to determine the need to continue PEG precipitation when prolactin measurements with the Wallac AutoDELFIA were replaced by the Beckman DxI 800.

Perfusion imaging using rubidium-82 ((82)Rb) PET in rats with myocardial infarction

DEFF Research Database (Denmark)

Clemmensen, Andreas Ettrup; Ghotbi, Adam Ali; Bodholdt, Rasmus Poul

2017-01-01

Assessing myocardial perfusion using 82Rb-PET is emerging as a valuable clinical tool.1,2 The rapid decay (T½ = 76 s) allows for absolute quantification of both rest and stress perfusion within 30 minutes. In addition to evaluation of epicardial disease with perfusion defects, also evaluation...... of balanced coronary and small vessel disease is possible. For further evaluation of how 82Rb-PET can be used clinically, pre-clinical application of the method would be valuable. However, so far no data on the use of 82Rb-PET in small animals have been published nor has the use of 82Rb-PET, to the best...

Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions

International Nuclear Information System (INIS)

Greenberg, B.M.; Gaba, V.; Canaani, O.; Malkin, S.; Mattoo, A.K.; Edelman, M.

1989-01-01

A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance

Endobronchial administration of iodine-131 B72.3 monoclonal antibody in patients with lung cancer

International Nuclear Information System (INIS)

Del Vecchio, S.; Mansi, L.; Petrillo, A.; Camera, L.; Salvatore, M.; Sofia, M.; Marra, A.; Carratu, L.

1991-01-01

We tested the feasibility of endobronchial administration of radiolabelled monoclonal antibodies (MoAbs) and the biodistribution of the radiotracer. Ten patients with histological confirmed adenocarcinoma or squamous cell carcinoma were studied. Nine received 470 μCi (103 μg) of 131 I-B72.3, a monoclonal antibody reacting against TAG 72 antigen, while one patient received 502 μCi (291 μg) of 131 I-4C4, an indifferent antibody used for comparison in a negative control study. The radiolabelled antibody was administered through a flexible fiberoptic bronchoscope and placed on the tumour mass under visual monitoring. Scans with a large field-of-view gamma-camera showed retention of 131 I-B72.3 at the tumour site up to 6-9 days in six of eight patients. No other organs were visualized with the exception of faint activity in the gastrointestinal tract, bladder and thyroid. On the contrary, the indifferent antibody 131 I-4C4 was not retained at the tumour site 6 days after MoAbs administration, and more prominent activity was found in the gastrointestinal tract. In one patient the study was not technically adequate because of failure of the delivery system. The vascular compartment contained less than 3% of the administered dose. We conclude that endobronchial administration is a feasible technique and allows stable and specific targetting of bronchial tumours. Furthermore, the low activity found in the plasma and other organs suggests that this approach may be used to deliver therapeutic doses of MoAbs to lung cancers. (orig.)

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways

Directory of Open Access Journals (Sweden)

Lewis D. Evans

2018-03-01

Full Text Available Summary: In Alzheimer’s disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon. : In contrast with predictions that transfer of the microtubule-associated protein tau between neurons is a toxic gain-of-function process in dementia, Evans et al. show that healthy human neurons efficiently take up both normal and aggregated tau, by distinct but overlapping uptake mechanisms. Keywords: Alzheimer’s disease, frontotemporal dementia, Tau, MAPT, iPSC, endocytosis, human neurons, intracellular transport

Evaluation of Anti-Trichinella spiralis Obtained by Sublingual and Conventional Immunizations with the 45kDa Protein

Directory of Open Access Journals (Sweden)

Francisca Chávez Ruvalcaba

2017-05-01

Full Text Available Trichinellosis is a cosmopolitan zoonotic disease produced mainly by the consumption of poorly cooked swine meat. Several studies have probed the efficiency of immunotherapy as a method for the treatment of trichinellosis. In this work, a 45 kDa immunodominant antigen was characterized, and the presence of IgA, IgM and IgG anti-Trichinella spiralis antibodies was evaluated during the course of the infection. In addition, the differences between sublingual and parenteral administration of the 45 kDa T. spiralis antigen were determined. Long Evans rats were used both to purify the 45 kDa antigen and to evaluate the immune response produced in six different groups: healthy and infected controls; two groups of immunized murines (sublingually and parenterally with 4 doses of the 45 kDa T. spiralis immunogen administered at days 0, 7, 14 and 21 and challenged with 500 T. spiralis infective larvae (IL 7 days after the last immunization; and finally, two groups of murines infected with 500 IL of T. spiralis, immunized at week 4 post infection by the same two routes. The humoral response was evaluated by indirect immunofluorescence by confocal microscopyin order to determine the presence of IgA, IgM and IgG antibodies.

11 Efficacy and Tolerability of HDM Injective Immunotherapy With Monomeric Allergoid

Science.gov (United States)

Compalati, Enrico; Atzeni, Isabella; Cabras, Sergio; Fancello, Paolo; Gaspardini, Giulio; Longo, Rocco; Patella, Vincenzo; Tore, Giorgio

2012-01-01

Background Subcutaneous immunotherapy (SCIT) is an effective treatment of respiratory allergy and carbamylated monomeric allergoids (monoids), by virtue of their reduced IgE-binding activity, resulted clinically safe by sublingual administration. Purpose of this study was to investigate the efficacy and tolerability of immunotherapy with house dust mites (HDM) monoid administered by injective route in patients with allergic rhinoconjunctivitis (AR). Methods A preparation of 0.70 mL of 10 BU/mL containing modified extract with 50% Dermatophagoides pteronyssinus and 50% Dermatophagoides farinae (amount of major allergen: 4 μg of group 1 per milliliter) was delivered monthly for 12 months, following a 5-week build-up induction phase (0.10–0.20–0.30–0.50–0.70 mL), to 58 patients (60% males, mean age 25.1 ± 12.7) suffering from AR due to mites for at least 2 years, whereas 60 patients with similar baseline characteristics were observed as controls. All patients were allowed to assume traditional drug therapy for their condition. At the end of the study changes from baseline in symptoms scores, in number of days with drug assumption, in severity of AR (according to ARIA classification) were compared between the 2 groups; moreover an overall assessment of clinical efficacy and tolerability was based on patients' and physicians' judgements (unsatisfactory, mild, good, optimal). Results In respect to baseline both groups showed, after 1 year, an improvement in symptoms score (P allergoid was associated with a significant clinical benefit observed through objective and subjective outcomes; the traditional safety of monomeric allergoids was confirmed by the subjective judgements of tolerability.

Relationship of molecular weight distribution profile of unreduced gluten protein extracts with quality characteristics of bread.

Science.gov (United States)

Chaudhary, Nisha; Dangi, Priya; Khatkar, B S

2016-11-01

A statistical correlation was established among the molecular weight distribution patterns of unreduced gluten proteins and physicochemical, rheological and bread-making quality characteristics of wheat varieties. Size exclusion chromatography fractionated the gluten proteins apparently into five peaks. Peak I signified glutenins (30-130kDa), peak II as gliadins (20-55kDa), peak III as very low molecular weight monomeric gliadins (10-28kDa), peak IV and V, collectively, as albumins and globulins (bread loaf volume (r=0.848(∗∗)); however, peak II had negative (r=-0.818(∗∗)) impact. Bread firmness increased with increment in peak II (r=0.625(∗∗)), and decreased with accretion in peak I (r=-0.623(∗∗)). Copyright © 2016 Elsevier Ltd. All rights reserved.

The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

International Nuclear Information System (INIS)

Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

2010-01-01

The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

77 FR 65834 - Residues of Fatty Acids, Tall-Oil, Ethoxylated Propoxylated; Tolerance Exemption

Science.gov (United States)

2012-10-31

... information in support of this action and considered its validity, completeness and reliability and the... definition of a polymer is given in 40 CFR 723.250(b) and the exclusion criteria for identifying these low... conforms to the definition of a polymer given in 40 CFR 723.250(b) and meets the following criteria that...

A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

Science.gov (United States)

Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

2015-07-24

An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides

Directory of Open Access Journals (Sweden)

Xueran Geng

2015-07-01

Full Text Available An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS, indicating the importance of tryptophan residue(s at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

Science.gov (United States)

Zhang, Guo-Qing; Wu, Ying-Ying; Ng, Tzi-Bun; Chen, Qing-Jun; Wang, He-Xiang

2013-01-01

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. PMID:23586045

A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

Directory of Open Access Journals (Sweden)

Guo-Qing Zhang

2013-01-01

Full Text Available A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.

7 CFR 82.2 - Administration.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Administration. 82.2 Section 82.2 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... PROGRAMS CLINGSTONE PEACH DIVERSION PROGRAM § 82.2 Administration. The program will be administered under...

40 CFR 82.260 - Definitions.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 17 2010-07-01 2010-07-01 false Definitions. 82.260 Section 82.260... STRATOSPHERIC OZONE Halon Emissions Reduction § 82.260 Definitions. Halon-containing equipment means equipment.... This group consists of the three halogenated hydrocarbons known as Halon 1211, Halon 1301, and Halon...

Effect of laser beam position on mechanical properties of F82H/SUS316L butt-joint welded by fiber laser

Energy Technology Data Exchange (ETDEWEB)

Serizawa, Hisashi, E-mail: serizawa@jwri.osaka-u.ac.jp [Joining and Welding Research Institute, Osaka University, 11-1 Mihogaoka, Ibaraki, Osaka 567-0047 (Japan); Mori, Daiki; Ogiwara, Hiroyuki; Mori, Hiroaki [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

2014-10-15

Highlights: • The micro hardness of weld metal in F82H/SUS316L joint partially decreases after PWHT by shifting beam position to SUS316L. • Charpy impact energy of F82H/SUS316L joint obviously increases after PWHT due to the release of residual stress. • The tensile strength of weld metal in F82H/SUS316L joint is higher than that of SUS316L. • The fiber laser welding seems to be one of the most candidate methods to join between F82H and SUS316L pipes practically. - Abstract: A dissimilar butt-joint between reduced activation ferritic/martensitic steel F82H and SUS316L austenitic stainless steel was made by 4 kW fiber laser and the influence of laser beam position on its mechanical properties before and after post-weld heat treatment (PWHT) was examined at room temperature. From the nano-indentation measurements and the microstructural observations, it is found that the micro hardness of weld metal partially decreases after PWHT by shifting beam position to SUS316L because its phase seems to move from only the martensitic phase to the mixture of austenitic and martensitic phases. In addition, Charpy impact test suggests that the impact energy slightly increases by shifting beam position before PWHT and obviously increases after PWHT due to the release of residual stress. Moreover, the tensile test indicates that the tensile strength of weld metal is higher than that of SUS316L and the fracture occurs at the base metal of SUS316L regardless of laser beam position.

42 CFR 460.82 - Marketing.

Science.gov (United States)

2010-10-01

... 42 Public Health 4 2010-10-01 2010-10-01 false Marketing. 460.82 Section 460.82 Public Health... Administrative Requirements § 460.82 Marketing. (a) Information that a PACE organization must include in its marketing materials. (1) A PACE organization must inform the public about its program and give prospective...

9 CFR 3.82 - Feeding.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Feeding. 3.82 Section 3.82 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE... Animal Health and Husbandry Standards § 3.82 Feeding. (a) The diet for nonhuman primates must be...

Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

DEFF Research Database (Denmark)

Andreasen, M.F.; Landbo, Anne-Katrine Regel; Christensen, L.P.

2001-01-01

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were...

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

NARCIS (Netherlands)

de Wit, M. Y.; Klatser, P. R.

1988-01-01

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M.

Residue levels and risk assessment of pesticides in nuts of China.

Science.gov (United States)

Liu, Yihua; Shen, Danyu; Li, Shiliang; Ni, Zhanglin; Ding, Ming; Ye, Caifen; Tang, Fubin

2016-02-01

The pesticide residue levels of three nuts (chestnut, walnut, pinenut) collected from seven main producing areas of China were investigated. Twenty-nine pesticides, including organophosphates (OPs), organochlorines (OCs), pyrethroids (PYs) and two fungicides (triadimefon and buprofezin) were analyzed by gas chromatography (GC). Four OPs (acephate, dimethoate, chlorpyrifos and parathion-methyl) were found in 11.4% samples, with the concentrations of 19.0 µg kg(-1) to 74.0 µg kg(-1). Six OCs (DDT, HCH, endosulfan, quintozene, aldrin and dieldrin) were found in 18.2% samples, with the concentrations of 2.0 µg kg(-1) to 65.7 µg kg(-1). Among OCs, p,p-DDE and α-HCH were the dominant isomer for DDT and HCH. Five PYs (fenpropathrin, fenvalerate, cypermethrin, bifenthrin and cyhalothrin) were found in 15.9% samples, with the concentrations of 2.5 µg kg(-1) to 433.0 µg kg(-1). Fenpropathrin was the most frequently detected pesticide. In addition, triadimefon and buprofezin were detected only in two samples. For the tested nuts, 25.0% samples with multiple residues (containing more than two pesticides) were noted, even up to 9.1% samples with five pesticide residues. The residue of 15.9% samples was higher than the maximum residue limits (MRLs) of China. The short-term risks for the tested nuts were below 1.2%, and the highest long-term risk was 12.58%. The cumulative risk (cHI) for the tested pesticides were 8.43% (OPs), 0.42% (OCs), 12.82% (PYs) and 0.15% (fungicides), respectively. The total cHI was 21.82%. There was no significant health risk for consumers via nuts consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

40 CFR 82.62 - Definitions.

Science.gov (United States)

2010-07-01

... Containing or Manufactured With Class II Substances § 82.62 Definitions. For purposes of this subpart... to subpart A. Class II Substance means any substance designated as class II in 40 CFR part 82.... Hydrochlorofluorocarbon means any substance listed as class II in 40 CFR part 82, appendix B to subpart A. Initial...

50 CFR 82.4 - Authority.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Authority. 82.4 Section 82.4 Wildlife and... PROTECTION ACT OF 1972) Introduction § 82.4 Authority. The Secretary of the Interior has delegated to the Director, Fish and Wildlife Service, his authority under the Marine Mammal Protection Act to enter into...

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

International Nuclear Information System (INIS)

Chen, J.; Varner, J.E.

1985-01-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) + RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) + RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) + RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

A de novo designed monomeric, compact three helix bundle protein on a carbohydrate template

DEFF Research Database (Denmark)

Malik, Leila; Nygård, Jesper; Christensen, Niels Johan

2015-01-01

De novo design and chemical synthesis of proteins and of other artificial structures, which mimic them, is a central strategy for understanding protein folding and for accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial...... the template could facilitate protein folding. Here we report the design and synthesis of 3-helix bundle carboproteins on deoxy-hexopyranosides. The carboproteins were analyzed by CD, AUC, SAXS, and NMR, which revealed the formation of the first compact, and folded monomeric carboprotein distinctly different...

Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

International Nuclear Information System (INIS)

Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

1986-01-01

The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-[ 14 C]-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed

Pharmacological Targeting Of Neuronal Kv7.2/3 Channels: A Focus On Chemotypes And Receptor Sites.

Science.gov (United States)

Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; Manocchio, Laura; Medoro, Alessandro; Mosca, Ilaria; Taglialatela, Maurizio

2017-10-12

The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing a characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for development of new drugs for neuronal, cardiac and metabolic diseases. In the present manuscript, we focus on describing the pharmacological relevance and the potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different modulator chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and we express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4.

Science.gov (United States)

Sakai, Y; Yoshida, N; Isogai, A; Tani, Y; Kato, N

1995-03-01

Fructosyl lysine oxidase (FLOD) was examined for its use in the enzymatic measurement of the level of glycated albumin in blood serum. To isolate microorganisms having such an enzyme activity, we used N epsilon-fructosyl N alpha-Z-lysine (epsilon-FL) as a sole nitrogen source in the enrichment culture medium. The isolated fungus, strain S-1F4, showed a high FLOD activity in the cell-free extract and was identified as Fusarium oxysporum. FLOD was purified to an apparent homogeneity on SDS-PAGE. The molecular mass of the subunit was 50 kDa on SDS-PAGE and seemed to exist in a monomeric form. The enzyme had an absorption spectrum characteristic of a flavoprotein and the flavin was found to be covalently bound to the enzyme. The enzyme acted against N epsilon-fructosyl N alpha-Z-lysine and N alpha-fructosyl N epsilon-Z-lysine and showed specificity for fructosyl lysine residues.

Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

DEFF Research Database (Denmark)

Andreasen, Mette Findal; Landbo, A K; Christensen, L P

2001-01-01

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were...... investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid......, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts...

Evaluation and application of alumina-based Rb-82 generators charged with high levels of Sr-82/85

International Nuclear Information System (INIS)

Yano, Y.; Budinger, T.F.; Chiang, G.; O'Brien, H.A.; Grant, P.M.

1979-01-01

Generator-produced Rb-82, a 75-sec positron emitter with potential for myocardial blood-flow imaging, was studied with various ion-exchange columns to evaluate the characteristics of alumina as an adsorber for the 25-day Sr-82 parent. Test columns of alumina, Bio Rex 70, and Chelex 100 were loaded with multimillicurie amounts of no-carrier-added Sr-82/Sr-85 (Sr-85 is a production contaminant). The breakthrough of Sr-82/Sr-85, and the yield of Rb-82, were determined for long-term elutions from each column with up to 4 liter of 2% NaCl solution at pH 8 to 9. The breakthrough of Sr-82/85 was 10 -6 to 10 -5 from alumina and 10 -6 to 10 -4 from Chelex 100 and Bio Rex 70. The effects of eluent flow rate and concentration, and of alumina volume, on the breakthrough and yield were also studied. An improved and automated Rb-82 generator was used for myocardial and brain blood-flow studies in experimental animals and in man; it was equipped with solenoid flow-control valves and five in. of lead shielding for the alumina columns, which were charged with 25 to 50 mCi Sr-82 (100 to 150 mCi Sr-85). The Rb-82 generator with alumina column provided up to 20 to 40 mCi of Rb-82 as often as every 5 to 10 min with -5 breakthrough of Sr-82/85 over the 2- to 3-mo, useful life of the generator

Novel DDR Processing of Corn Stover Achieves High Monomeric Sugar Concentrations from Enzymatic Hydrolysis (230 g/L) and High Ethanol Concentration (10% v/v) During Fermentation

Energy Technology Data Exchange (ETDEWEB)

Chen, Xiaowen; Jennings, Ed; Shekiro, Joe; Kuhn, Erik M.; O' Brien, Marykate; Wang, Wei; Schell, Daniel J.; Himmel, Mike; Elander, Richard T.; Tucker, Melvin P.

2015-04-03

Distilling and purifying ethanol, butanol, and other products from second and later generation lignocellulosic biorefineries adds significant capital and operating cost for biofuels production. The energy costs associated with distillation affects plant gate and life cycle analysis costs. Lower titers in fermentation due to lower sugar concentrations from pretreatment increase both energy and production costs. In addition, higher titers decrease the volumes required for enzymatic hydrolysis and fermentation vessels. Therefore, increasing biofuels titers has been a research focus in renewable biofuels production for several decades. In this work, we achieved over 200 g/L of monomeric sugars after high solids enzymatic hydrolysis using the novel deacetylation and disc refining (DDR) process on corn stover. The high sugar concentrations and low chemical inhibitor concentrations from the DDR process allowed ethanol titers as high as 82 g/L in 22 hours, which translates into approximately 10 vol% ethanol. To our knowledge, this is the first time that 10 vol% ethanol in fermentation derived from corn stover without any sugar concentration or purification steps has been reported. Techno-economic analysis shows the higher titer ethanol achieved from the DDR process could significantly reduce the minimum ethanol selling price from cellulosic biomass.

The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

NARCIS (Netherlands)

Wimmer, Bernhard; Lottspeich, Friedrich; Klei, Ida van der; Veenhuis, Marten; Gietl, Christine

1997-01-01

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons with molecular masses of 70 and 72 KDa, Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1987-01-01

We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H 2 O 2 , and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H 2 O 2 , the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

Science.gov (United States)

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

Directory of Open Access Journals (Sweden)

Anton R Dluzewski

2008-08-01

Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

Ultratight crystal packing of a 10 kDa protein

Energy Technology Data Exchange (ETDEWEB)

Trillo-Muyo, Sergio [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain); Jasilionis, Andrius [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Domagalski, Marcin J. [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Chruszcz, Maksymilian [University of South Carolina, 631 Sumter Street, Columbia, SC 29208 (United States); Minor, Wladek [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Kuisiene, Nomeda [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Arolas, Joan L.; Solà, Maria; Gomis-Rüth, F. Xavier, E-mail: xgrcri@ibmb.csic.es [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain)

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

A 43-kDa TDP-43 species is present in aggregates associated with frontotemporal lobar degeneration.

Directory of Open Access Journals (Sweden)

Patrick J Bosque

Full Text Available The transactive response DNA-binding protein (TDP-43 is a major component of the abnormal intracellular inclusions that occur in two common neurodegenerative diseases of humans: (1 a subtype of frontotemporal lobar degeneration and (2 amyotrophic lateral sclerosis. Genetics, experiments in cultured cells and animals, and analogy with other neurodegenerative diseases indicate that the process of TDP-43 aggregation is fundamental to the pathogenesis of these 2 diseases, but the process by which this aggregation occurs is not understood. Biochemical fractionation has revealed truncated, phosphorylated and ubiquitinated forms of TDP-43 in a detergent-insoluble fraction from diseased CNS tissue, while these forms are absent from controls. However, a large amount of the normally predominant 43-kDa form of TDP-43 is present in the detergent-insoluble fraction even from control brains, so it has not been possible to determine if this form of TDP-43 is part of pathological aggregates in frontotemporal lobe degeneration. We used semi-denaturing detergent-agarose gel electrophoresis to isolate high molecular weight aggregates containing TDP-43 that are present in the cerebral cortex of individuals with frontotemporal lobar degeneration but not that of controls. These aggregates include the same covalently modified forms of TDP-43 seen in detergent-insoluble extracts. In addition, aggregates include a 43-kDa TDP-43 species. This aggregated 43-kDa form of TDP-43 is absent or present only at low levels in controls. The presence of 43-kDa TDP-43 in aggregates raises the possibility that covalent modification is not a primary step in the pathogenic aggregation of TDP-43 associated with frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Interaction of chemokine receptor CXCR4 in monomeric and dimeric state with its endogenous ligand CXCL12: coarse-grained simulations identify differences.

Science.gov (United States)

Cutolo, Pasquale; Basdevant, Nathalie; Bernadat, Guillaume; Bachelerie, Françoise; Ha-Duong, Tâp

2017-02-01

Despite the recent resolutions of the crystal structure of the chemokine receptor CXCR4 in complex with small antagonists or viral chemokine, a description at the molecular level of the interactions between the full-length CXCR4 and its endogenous ligand, the chemokine CXCL12, in relationship with the receptor recognition and activation, is not yet completely elucidated. Moreover, since CXCR4 is able to form dimers, the question of whether the CXCR4-CXCL12 complex has a 1:1 or 2:1 preferential stoichiometry is still an open question. We present here results of coarse-grained protein-protein docking and molecular dynamics simulations of CXCL12 in association with CXCR4 in monomeric and dimeric states. Our proposed models for the 1:1 and 2:1 CXCR4-CXCL12 quaternary structures are consistent with recognition and activation motifs of both partners provided by the available site-directed mutagenesis data. Notably, we observed that in the 2:1 complex, the chemokine N-terminus makes more steady contacts with the receptor residues critical for binding and activation than in the 1:1 structure, suggesting that the 2:1 stoichiometry would favor the receptor signaling activity with respect to the 1:1 association.

Line-Shape Code Comparison through Modeling and Fitting of Experimental Spectra of the C ii 723-nm Line Emitted by the Ablation Cloud of a Carbon Pellet

Directory of Open Access Journals (Sweden)

Mohammed Koubiti

2014-07-01

Full Text Available Various codes of line-shape modeling are compared to each other through the profile of the C ii 723-nm line for typical plasma conditions encountered in the ablation clouds of carbon pellets, injected in magnetic fusion devices. Calculations were performed for a single electron density of 1017 cm−3 and two plasma temperatures (T = 2 and 4 eV. Ion and electron temperatures were assumed to be equal (Te = Ti = T. The magnetic field, B, was set equal to either to zero or 4 T. Comparisons between the line-shape modeling codes and two experimental spectra of the C ii 723-nm line, measured perpendicularly to the B-field in the Large Helical Device (LHD using linear polarizers, are also discussed.

Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2'-S-cycloadenosine.

Science.gov (United States)

Ikehara, M; Tezuka, T

1975-01-01

A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9. PMID:170595

Unique N-Glycan Moieties of the 66-kDa Cell Wall Glycoprotein from the Red Microalga Porphyridium sp.

Science.gov (United States)

Levy-Ontman, Oshrat; Arad, Shoshana (Malis); Harvey, David J.; Parsons, Thomas B.; Fairbanks, Antony; Tekoah, Yoram

2011-01-01

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man8–9Xyl1–2Me3GlcNAc2. The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins. PMID:21515680

Mapping monomeric threading to protein-protein structure prediction.

Science.gov (United States)

Guerler, Aysam; Govindarajoo, Brandon; Zhang, Yang

2013-03-25

The key step of template-based protein-protein structure prediction is the recognition of complexes from experimental structure libraries that have similar quaternary fold. Maintaining two monomer and dimer structure libraries is however laborious, and inappropriate library construction can degrade template recognition coverage. We propose a novel strategy SPRING to identify complexes by mapping monomeric threading alignments to protein-protein interactions based on the original oligomer entries in the PDB, which does not rely on library construction and increases the efficiency and quality of complex template recognitions. SPRING is tested on 1838 nonhomologous protein complexes which can recognize correct quaternary template structures with a TM score >0.5 in 1115 cases after excluding homologous proteins. The average TM score of the first model is 60% and 17% higher than that by HHsearch and COTH, respectively, while the number of targets with an interface RMSD benchmark proteins. Although the relative performance of SPRING and ZDOCK depends on the level of homology filters, a combination of the two methods can result in a significantly higher model quality than ZDOCK at all homology thresholds. These data demonstrate a new efficient approach to quaternary structure recognition that is ready to use for genome-scale modeling of protein-protein interactions due to the high speed and accuracy.

Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.

Science.gov (United States)

Fahie, Monifa; Chisholm, Christina; Chen, Min

2015-02-24

Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop's movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity, even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin-binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design, while OmpG's monomeric structure greatly simplifies nanopore production.

7 CFR 905.82 - Effective time.

Science.gov (United States)

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Effective time. 905.82 Section 905.82 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... TANGELOS GROWN IN FLORIDA Order Regulating Handling Miscellaneous Provisions § 905.82 Effective time. The...

Isolation of a 60 kDa protein with in vitro anticancer activity against ...

African Journals Online (AJOL)

Sea hares have greatly attracted the interest of all those investigating chemical defense substances. Most of these substances are low molecular weight compounds derived from algal diets. In vitro anticancer effect of a 60 kDa protein isolated from the purple fluid of Aplysia dactylomela on four human cancer cell lines was ...

30 CFR 256.82 - Environmental studies.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 2 2010-07-01 2010-07-01 false Environmental studies. 256.82 Section 256.82... OIL AND GAS IN THE OUTER CONTINENTAL SHELF Studies § 256.82 Environmental studies. (a) The Director... practicable, be designed to predict environmental impacts of pollutants introduced into the environments and...

7 CFR 945.82 - Effective time.

Science.gov (United States)

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Effective time. 945.82 Section 945.82 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... § 945.82 Effective time. The provisions of this subpart shall become effective at such time as the...

Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

Science.gov (United States)

Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

2018-04-01

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

Science.gov (United States)

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

Directory of Open Access Journals (Sweden)

Davidson Michael W

2008-03-01

Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

45 CFR 74.82 - Program income.

Science.gov (United States)

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Program income. 74.82 Section 74.82 Public Welfare... COMMERCIAL ORGANIZATIONS Special Provisions for Awards to Commercial Organizations § 74.82 Program income. The additional costs alternative described in § 74.24(b)(1) may not be applied to program income...

7 CFR 930.82 - Effective time.

Science.gov (United States)

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Effective time. 930.82 Section 930.82 Agriculture... Miscellaneous Provisions § 930.82 Effective time. The provisions of this part, and of any amendment thereto, shall become effective at such time as the Secretary may declare, and shall continue in force until...

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

Science.gov (United States)

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

Science.gov (United States)

Barkla, B J; Blumwald, E

1991-12-15

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

50 CFR 82.20 - Civil rights.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Civil rights. 82.20 Section 82.20 Wildlife... (MARINE MAMMAL PROTECTION ACT OF 1972) Administration § 82.20 Civil rights. Each cooperative agreement... Civil Rights Act of 1964, 42 U.S.C. 2000d-2000d-4, and with the Secretary's regulations promulgated...

50 CFR 216.82 - Dogs prohibited.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 7 2010-10-01 2010-10-01 false Dogs prohibited. 216.82 Section 216.82... Pribilof Islands Administration § 216.82 Dogs prohibited. In order to prevent molestation of fur seal herds, the landing of any dogs at Pribilof Islands is prohibited. [41 FR 49488, Nov. 9, 1976. Redesignated at...

11 CFR 100.82 - Bank loans.

Science.gov (United States)

2010-01-01

... 11 Federal Elections 1 2010-01-01 2010-01-01 false Bank loans. 100.82 Section 100.82 Federal... Contributions § 100.82 Bank loans. (a) General provisions. A loan of money to a political committee or a candidate by a State bank, a federally chartered depository institution (including a national bank) or a...

Comparative analysis of the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a using Mössbauer spectroscopy with a high velocity resolution

Science.gov (United States)

Alenkina, I. V.; Kumar, A.; Berkovsky, A. L.; Oshtrakh, M. I.

2018-02-01

A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and β-subunits. In contrast, the Mössbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mössbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

25 CFR 82.6 - Petition format.

Science.gov (United States)

2010-04-01

... 25 Indians 1 2010-04-01 2010-04-01 false Petition format. 82.6 Section 82.6 Indians BUREAU OF... REORGANIZED UNDER FEDERAL STATUTE AND OTHER ORGANIZED TRIBES § 82.6 Petition format. Petitions may consist of... of a petition must set forth at least a summary of the objectives of the petitioners and must show...

Residual fatigue lifetime estimation of railway axles for various loading spectra

Czech Academy of Sciences Publication Activity Database

Pokorný, Pavel; Hutař, Pavel; Náhlík, Luboš

2016-01-01

Roč. 82, APR (2016), s. 25-32 ISSN 0167-8442 R&D Projects: GA MŠk(CZ) EE2.3.20.0214; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081723 Keywords : Fatigue failure * Residual fatigue lifetime * Crack propagation * Railway axle * Load spectrum Subject RIV: JL - Materials Fatigue, Friction Mechanics Impact factor: 2.659, year: 2016

Fermentation of Arabinoxylan-Oligosaccharides, Oligofructose and their Monomeric Sugars by Hindgut Bacteria from Siberian Sturgeon and African Catfish in Batch Culture in vitro

NARCIS (Netherlands)

Geraylou, Z.; Rurangwa, E.; Wiele, van der T.; Courtin, C.M.; Delcour, J.A.; Buyse, J.; Ollevier, F.

2014-01-01

The in vitro fermentation of two Non-Digestible Oligosaccharide (NDO) preparations, Arabinoxylan- Oligosaccharides (AXOS) and Oligofructose (OF), and their respective monomeric sugars, xylose and fructose, were investigated by hindgut microbiota of two major aquaculture fish species, Siberian

64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells)

International Nuclear Information System (INIS)

Wright, M.; Hogset, A.; Alestrom, P.; Gautvik, K.M.

1988-01-01

A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [ 3 H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [ 3 H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [ 3 H]TRH, the 64 kDa protein cannot be demonstrated by [ 3 H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells

cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

International Nuclear Information System (INIS)

D'Arpa, P.; Machlin, P.S.; Ratrie, H. III; Rothfield, N.F.; Cleveland, D.W.; Earnshaw, W.C.

1988-01-01

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

Pirimiphos-methyl residues in stored wheat and barley, bread, burghul and parboiled wheat

International Nuclear Information System (INIS)

Hadjidemetriou, D.G.

1990-01-01

Residues of 14 C-pirimiphos-methyl in stored grain declined to 88% in wheat and 82% in barley after 12 months. Corresponding percentages with the unlabelled insecticide were 78% and 59% since only the parent chemical was determined. Surface residues, removed by washing the grain with water, decreased from 3.3 to 0.2 mg/kg for wheat and from 2.0 to 0.2 mg/kg for barley. Bound residues increased gradually with time and reached a maximum of 2.2% for wheat and 3.0% for barley in 12 months. Pirimiphos-methyl residues in flour increased from 1.1 at 0 time to 2.2 mg/kg after one year. The mean values of residues contained in the unwashed wheat grain were 81% for bran and 19% for flour. The loss in milling during preparation of wholemeal flour from prewashed grain was 7% for wheat and 6% for barley. Processed products from wheat showed residue losses ranging from 24 to 45%. (author). 16 refs, 2 figs, 2 tabs

Complexes of 3.6 kDa Maltodextrin with Some Metals

Directory of Open Access Journals (Sweden)

Christopher H. Schilling

2004-06-01

Full Text Available Preparation of magnesium, lanthanum, and bismuth(III complexes of 3.6 kDa maltodextrin and some properties of the resulting materials are presented. The metal derivatives contain metals bound to the oxygen atoms of the hydroxyl groups of maltodextrin. Additionally, the metal atoms are coordinated to the hydroxyl groups of the D-glucose units of the macroligand. Such coordination stabilized the metal – oxygen bond against hydrolysis, even in boiling water. The presence of magnesium and lanthanum atoms increased the thermal stability of maltodextrin, whereas bismuth atoms decreased it.

The mitochondrial 60-kDa heat shock protein in marine invertebrates: biochemical purification and molecular characterization.

Science.gov (United States)

Choresh, Omer; Loya, Yossi; Müller, Werner E G; Wiedenmann, Jörg; Azem, Abdussalam

2004-03-01

Sessile marine invertebrates undergo constant direct exposure to the surrounding environmental conditions, including local and global environmental fluctuations that may lead to fatal protein damage. Induction of heat shock proteins (Hsps) constitutes an important defense mechanism that protects these organisms from deleterious stress conditions. In a previous study, we reported the immunological detection of a 60-kDa Hsp (Hsp60) in the sea anemone Anemonia viridis (formerly called Anemonia sulcata) and studied its expression under a variety of stress conditions. In the present study, we show that the sponge Tetilla sp. from tidal habitats with a highly variable temperature regime is characterized by an increased level of Hsp60. Moreover, we show the expression of Hsp60 in various species among Porifera and Cnidaria, suggesting a general importance of this protein among marine invertebrates. We further cloned the hsp60 gene from A viridis, using a combination of conventional protein isolation methods and screening of a complementary deoxyribonucleic acid library by polymerase chain reaction. The cloned sequence (1764 bp) encodes for a protein of 62.8 kDa (588 amino acids). The 62.8-kDa protein, which contains an amino terminal extension that may serve as a mitochondrial targeting signal, shares a significant identity with mitochondrial Hsp60s from several animals but less identity with Hsp60s from either bacteria or plants.

21 CFR 500.82 - Definitions.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Definitions. 500.82 Section 500.82 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS GENERAL Regulation of Carcinogenic Compounds Used in Food-Producing Animals § 500...

22 CFR 8.2 - Policy.

Science.gov (United States)

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Policy. 8.2 Section 8.2 Foreign Relations... solely for advisory functions and any decision taken pursuant to the advice or recommendation of an... advisory committees will be open to the public unless there is a compelling reason which requires...

Calmyonemin: a 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate).

Science.gov (United States)

David, C; Viguès, B

1994-01-01

Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calcium-binding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein cross-reacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myoneme-mediated retraction of the ciliature.

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State

Directory of Open Access Journals (Sweden)

Claus U. Pietrzik

2017-04-01

Full Text Available The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aβ secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin β and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin β, with the latter exhibiting a preference for APP in its dimeric state.

Method for the isolation of biologically active monomeric immunoglobulin A from a plasma fraction.

Science.gov (United States)

Leibl, H; Tomasits, R; Wolf, H M; Eibl, M M; Mannhalter, J W

1996-04-12

A purification method for immunoglobulin A (IgA) yielding monomeric IgA with a purity of over 97% has been developed. This procedure uses ethanol-precipitated plasma (Cohn fraction III precipitate) as the starting material and includes heparin-Sepharose adsorption, dextran sulfate and ammonium sulfate precipitation, hydroxyapatite chromatography, batch adsorption by an anion-exchange matrix and gel permeation. Additional protein G Sepharose treatment leads to an IgA preparation of greater than 99% purity. The isolated IgA presented with an IgA subclass distribution, equivalent to IgA in unfractionated plasma, and was biologically active, as was shown by its ability to down-modulate Haemophilus influenzae-b-induced IL-6 secretion of human monocytes.

27 CFR 31.82 - Hotels.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Hotels. 31.82 Section 31... Same Premises § 31.82 Hotels. The proprietor of a hotel who conducts the sale of liquors throughout the hotel premises is only required to register under this part for one place. For example, different areas...

27 CFR 24.82 - Samples.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Samples. 24.82 Section 24.82 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... vinegar and salted wine as defined in § 24.215, the proprietor shall submit under separate cover at the...

Isolation of 62 kda protein with antioxidant properties from natural honey

International Nuclear Information System (INIS)

Mohammed, S.E.A.R.

2014-01-01

Fourteen natural honey samples from Libya, Sudan and Pakistan were evaluated for their antioxidant activity by employing 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay. The scavenging activity of honey samples were in the range of 18-32% when compared to control. A 62 kDa protein was isolated from honey by gel filtration chromatography followed by reverse phase HPLC showed significant radical scavenging activity. The research pointed out the antioxidative role of honey proteins and possibility of their contribution to the therapeutic value of the natural honey. (author)

21 CFR 82.3 - Definitions.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Definitions. 82.3 Section 82.3 Food and Drugs FOOD... gloss white means a suspension in water of co-precipitated aluminum hydroxide and barium sulfate. (j... interpretations of terms contained in section 201 of the Federal Food, Drug, and Cosmetic Act shall be applicable...

7 CFR 3015.82 - Financial status report.

Science.gov (United States)

2010-01-01

... 7 Agriculture 15 2010-01-01 2010-01-01 false Financial status report. 3015.82 Section 3015.82 Agriculture Regulations of the Department of Agriculture (Continued) OFFICE OF THE CHIEF FINANCIAL OFFICER, DEPARTMENT OF AGRICULTURE UNIFORM FEDERAL ASSISTANCE REGULATIONS Financial Reporting Requirements § 3015.82...

Holistic versus monomeric strategies for hydrological modelling of human-modified hydrosystems

Directory of Open Access Journals (Sweden)

I. Nalbantis

2011-03-01

Full Text Available The modelling of human-modified basins that are inadequately measured constitutes a challenge for hydrological science. Often, models for such systems are detailed and hydraulics-based for only one part of the system while for other parts oversimplified models or rough assumptions are used. This is typically a bottom-up approach, which seeks to exploit knowledge of hydrological processes at the micro-scale at some components of the system. Also, it is a monomeric approach in two ways: first, essential interactions among system components may be poorly represented or even omitted; second, differences in the level of detail of process representation can lead to uncontrolled errors. Additionally, the calibration procedure merely accounts for the reproduction of the observed responses using typical fitting criteria. The paper aims to raise some critical issues, regarding the entire modelling approach for such hydrosystems. For this, two alternative modelling strategies are examined that reflect two modelling approaches or philosophies: a dominant bottom-up approach, which is also monomeric and, very often, based on output information, and a top-down and holistic approach based on generalized information. Critical options are examined, which codify the differences between the two strategies: the representation of surface, groundwater and water management processes, the schematization and parameterization concepts and the parameter estimation methodology. The first strategy is based on stand-alone models for surface and groundwater processes and for water management, which are employed sequentially. For each model, a different (detailed or coarse parameterization is used, which is dictated by the hydrosystem schematization. The second strategy involves model integration for all processes, parsimonious parameterization and hybrid manual-automatic parameter optimization based on multiple objectives. A test case is examined in a hydrosystem in Greece

Mycobacterium tuberculosis 38 kDa Antigen Purification and Potential Diagnostic Use by Piezoelectric Immunosensors

Directory of Open Access Journals (Sweden)

Paula A. MARÍN

2015-01-01

Un paso crucial en el desarrollo de un inmunosensor piezoeléctrico para la detección de tuberculosis (TB, es la selección y obtención de los inmunoreactivos empleados en el inmunoensayo y la estrategia para la biofuncionalización del transductor. Diversos estudios han reportado el uso del antígeno proteico 38kDa (Ag38kDa de Mycobacterium tuberculosis (Mtb como un buen biomarcador de la enfermedad y el cumplimiento de las características físicas y bioquímicas para ser inmovilizado por monocapas autoensambladas (SAMs, en la superficie del electrodo de oro de cristales piezoeléctricos. Un inmunosensor piezoeléctrico desarrollado a partir de un antígeno nativo purificado de Mtb podría ser un método alternativo simple para la detección de Mtb con ventajas de rapidez y reusabilidad, contribuyendo al control y el tratamiento oportuno de la enfermedad. En este estudio se presenta el proceso de purificación del Ag38kDa a partir de proteínas de secreción filtradas de cultivo (CFP de Mtb para ser usado como inmunoreactivo con potencial aplicación en la detección de Mtb con inmunosensores piezoeléctricos. Se obtuvieron cristales funcionalizados mediante la técnica modificada de monocapas autoensambladas (SAMs, con el antígeno nativo purificado y CFP. Las superficies biofuncionalizadas fueron caracterizadas cualitativamente con microscopía de fuerza atómica (AFM para validar las condiciones de optimización del protocolo de inmovilización con antígenos de secreción de Mtb. Estos cristales modificados pueden ser acoplados a un sistema de caracterización de un inmunosensor piezoeléctrico para la detección de Mtb mediante un inmunoensayo competitivo directo.

Energy research program 82

International Nuclear Information System (INIS)

1982-01-01

The energy research program 82 (EFP-82) is prepared by the Danish ministry of energy in order to continue the extension of the Danish energy research and development started through the former trade ministry's programs EM-1 (1976) and EM-2 (1978), and the energy ministry's programs EFP-80 and EFP-81. The new program is a continuation of the activities in the period 1982-84 with a total budget of 100 mio.Dkr. The program gives a brief description of background, principles, organization and financing, and a detailed description of each research area. (BP)

Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events

DEFF Research Database (Denmark)

Jackers, P; Clausse, N; Fernandez, M

1996-01-01

A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed...

Microstructural evolution of HFIR-irradiated low activation F82H and F82H-10B steels

International Nuclear Information System (INIS)

Wakai, E.; Shiba, K.; Sawai, T.; Hashimoto, N.; Robertson, J.P.; Klueh, R.L.

1998-01-01

Microstructures of reduced-activation F82H (8Cr-2W-0.2V-0.04Ta) and the F82H steels doped with 10 B, irradiated at 250 and 300 C to 3 and 57 dpa in the High Flux Isotope Reactor (HFIR), were examined by TEM. In the F82H irradiated at 250 C to 3 dpa, dislocation loops, small unidentified defect clusters with a high number density, and a few MC precipitates were observed in the matrix. The defect microstructure after 300 C irradiation to 57 dpa is dominated by the loops, and the number density of loops was lower than that of the F82H- 10 B steel. Cavities were observed in the F82H- 10 B steels, but the swelling value is insignificant. Small particles of M 6 C formed on the M 23 C 6 carbides that were present in both steels before the irradiation at 300 C to 57 dpa. A low number density of MC precipitate particles formed in the matrix during irradiation at 300 C to 57 dpa

50 CFR 82.7 - Coordination with States.

Science.gov (United States)

2010-10-01

...-IN-AID (MARINE MAMMAL PROTECTION ACT OF 1972) Application for Grants § 82.7 Coordination with States... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Coordination with States. 82.7 Section 82.7 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR...

Biosynthesis of inulin from sucrose using inulosucrase from Lactobacillus gasseri DSM 20604.

Science.gov (United States)

Ni, Dawei; Zhu, Yingying; Xu, Wei; Bai, Yuxiang; Zhang, Tao; Mu, Wanmeng

2018-04-01

Inulin is composed of fructose residues connected by β-(2, 1) glycosidic linkages with many promising physiochemical and physiological properties. In this study, an inulin-producing inulosucrase gene from Lactobacillus gasseri DSM 20604 was cloned, expressed and purified. SDS-PAGE and gel filtration found that the recombinant inulosucrase is a monomeric protein with a molecular weight of 63KDa. The optimal pH for its sucrose hydrolysis and transfructosylation activities was pH 5.5. The optimal temperatures were measured to be 45, 25, and 35°C for sucrose hydrolysis, transfructosylation, and total activity, respectively. Biosynthesis studies showed that the optimal enzyme dosage was 4.5U/g sucrose. Higher sucrose concentrations immensely contributed to inulin biosynthesis; the inulin yield reached its maximum after 1.5h of reaction. Structural analyses of the polysaccharide produced by the recombinant enzyme from sucrose revealed that it is an inulin-type fructan with a molecular weight of 5.858×10 6 Da. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification and characterization of tenecin 4, a new anti-Gram-negative bacterial peptide, from the beetle Tenebrio molitor.

Science.gov (United States)

Chae, Jun-Ho; Kurokawa, Kenji; So, Young-In; Hwang, Hyun Ok; Kim, Min-Su; Park, Ji-Won; Jo, Yong-Hun; Lee, Yong Seok; Lee, Bok Luel

2012-03-01

The biochemical characterization of novel antimicrobial peptides (AMPs) and the determination of ligand molecules that induce AMP production are essential for understanding the host innate immune response in insects. Here, we purified a new 14-kDa AMP, named tenecin 4, from the larval hemolymph of the beetle Tenebrio molitor. Tenecin 4 contains 14% glycine residues and has moderate similarities both to the C-terminal region of Drosophila attacin and to silk-moth gloverin proteins. Purified tenecin 4 showed bactericidal activity against Gram-negative Escherichia coli but not against Gram-positive Bacillus subtilis or the fungus Candida albicans. Tenecin 4 production was induced by Toll cascade-activating ligands, such as β-1,3-glucan, lysine-type peptidoglycan and active Spätzle, and by the probable Imd pathway-activating ligand monomeric meso-diaminopimelic acid-type peptidoglycan. Taken together, these data show that tenecin 4 is a defense protein against Gram-negative pathogens and is induced by multiple ligands in Tenebrio larvae. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

Science.gov (United States)

Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

2013-01-01

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

40 CFR 82.150 - Purpose and scope.

Science.gov (United States)

2010-07-01

...) PROTECTION OF STRATOSPHERIC OZONE Recycling and Emissions Reduction § 82.150 Purpose and scope. (a) The purpose of this subpart is to reduce emissions of class I and class II refrigerants and their substitutes... 40 Protection of Environment 17 2010-07-01 2010-07-01 false Purpose and scope. 82.150 Section 82...

Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells.

Science.gov (United States)

Lin, Jianfei; Chen, He; Luo, Ling; Lai, Yongrong; Xie, Wei; Kee, Kehkooi

2015-01-01

To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

21 CFR 1250.82 - Potable water systems.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Potable water systems. 1250.82 Section 1250.82... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.82 Potable water systems. The following conditions must be met by vessel water systems used for the storage and distribution of water which has met...

Evaluation of the hemoglobin A1c-analyzer TOSOH HLC-723 G7.

Science.gov (United States)

Gremmels, Heinz-Detlef; Richter, Anja; Watzke, Ivonne

2003-01-01

The TOSOH HLC-723 G7 is a compact analyzer designed for the measurement of HbA1c under routine laboratory conditions. The analyzer has an automatic blood tube supply and positive sample identification. Samples are transported automatically via racks in a continuous-load mode, cap piercing is optional. Tests devoted to the assessment of reproducibility and accuracy of analytical results indicated that over a test period of 17 days, the intra-assay variation (CV) was 1.79%, and the inter-assay variation 2.60%, respectively. A comparison with the predecessor model G5 showed a very good correlation (r = 0.997, y = 1.0041x - 0.00172; n = 149). The presence of high triglyceride, bilirubin or urea concentrations in patient samples did not influence the analytical precision. The labile HbA1c fraction (L-A1c) is clearly separated during chromatography and thus does not compromise HbA1c analysis. With a protocol of 1.2 minutes, the TOSOH G7 is a very fast analyzer, designed for laboratories with a high throughput of samples.

Stability of the anthocyanins extracted from residues of the wine industry

Directory of Open Access Journals (Sweden)

Edmar Clemente

2011-09-01

Full Text Available Anthocyanins are highly important due to their antioxidant capacity. They are the most important among the phenolic compounds and one of the main natural dyes used in the food industry. In this research, residue of processed grapes was used to investigate the presence of anthocyanins, the possibility of their extraction from the residue, and their stability. The extraction solution consisted of 70 mL of ethanol 70% and 30 mL of HCl 0.1% at pH 2.0. The results found for the processed grapes residue was 26.20 mg.100 g-1. In order to evaluate stability, caffeic acid was added at 0.5:1 w/v; 0.8:1 w/v; and 1:1 w/v concentrations. Anthocyanins extract reached the greatest stability at 0.5:1 w/v concentration, with 82.47% color retention and a half-life period of 15 days. Therefore, the use of this organic acid as a stabilizer for anthocyanins is feasible.

7 CFR 8.2 - Delegation of authority.

Science.gov (United States)

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Delegation of authority. 8.2 Section 8.2 Agriculture Office of the Secretary of Agriculture 4-H CLUB NAME AND EMBLEM § 8.2 Delegation of authority. The... Agriculture, may authorize the use of the 4-H Club Name and Emblem in accordance with the regulations in this...

Fate and possible nutritional and toxicological significance of methylbromide residues in fumigated cocoa beans. Coordinated programme on isotopic tracer-aided studies on foreign chemical residues in food

International Nuclear Information System (INIS)

Adomako, D.

1975-03-01

Methyl bromide residues in cocoa as a result of fumigation under practical conditions have been studied. Cocoa beans were exposed to 14 C-labelled CH 3 Br for 24 hours at 20-32degC and dosage 23.5 to 28mg/1 of fumigants (moisture content of 6-7%). Whole unroasted beans and their shells and nibs as well as shells and nibs of roasted (105degC for 30 min) beans were extracted separately with toluene (for free CH 3 Br) and the dried residues wet combusted. 14 C-activities were determined by liquid scintillation counting. Total methyl bromide (bound and free) was equivalent 83-98ppm in whole unroasted beans 35 hours after treatment and aeration, and 31, 15 and 10ppm after 7, 42 and 70 days respectively in one set of experiments and 37, 53 and 42ppm after 76, 60 and 51 days in another set. Approximately 80% of the residue occurred in the shells which constitute only 12 to 13% of unroasted beans. 99% of the total residues appeared to be in chemically-bound form. Roasting reduced the total residues by 32 to 62% in nibs and 3.6 to 14% in shells. A striking effect of roasting was the occurrence in roasted nibs of residues as (extractable) carbon-14 (62-82% of the total residues compared to 16.5 - 27% in unroasted nibs). The bound residues behaved as methylated derivatives whilst the volatile fraction behaved as volatile aroma compounds formed by Maillard type reactions from the 14 C-labelled amino acides and sugars

Faradaurate nanomolecules: a superstable plasmonic 76.3 kDa cluster.

Science.gov (United States)

Dass, Amala

2011-12-07

Information on the emergence of the characteristic plasmonic optical properties of nanoscale noble-metal particles has been limited, due in part to the problem of preparing homogeneous material for ensemble measurements. Here, we report the identification, isolation, and mass spectrometric and optical characterization of a 76.3 kDa thiolate-protected gold nanoparticle. This giant molecule is far larger than any metal-cluster compound, those with direct metal-to-metal bonding, previously known as homogeneous molecular substances, and is the first to exhibit clear plasmonic properties. The observed plasmon emergence phenomena in nanomolecules are of great interest, and the availability of absolutely homogeneous and characterized samples is thus critical to establishing their origin. © 2011 American Chemical Society

Enzymatic properties and primary structures of two α-amylase isozymes from the Pacific abalone Haliotis discus hannai

OpenAIRE

Kumagai, Yuya; Satoh, Takuya; Inoue, Akira; Ojima, Takao

2013-01-01

Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58 kDa and 82 kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30℃ and 6.7, and 30℃ and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, ...

Efficiency of the refining processes in removing 14C-dichlorvos residues in soybean oil

International Nuclear Information System (INIS)

Soliman, S.M.

2006-01-01

Crude soybean oil extracted from grains treated with 14 C-dichlorvos at a dose 24 mg insecticide / kg seeds and stored for 30 weeks was subjected to different refining processes such as alkali treatment, bleaching, winterization and deodorization. The effect of the refining processes on the nature and magnitude of the originally present residues was investigated. The insecticide residues in crude oil and cake amounted to 9.5% and 55% , respectively, of original residues inside the seeds. Extraction of the seeds with hexane gave crude oil with 9.5 % of original residues in seeds. The l4 C-activity in the crude stored Soya beans oil could be reduced by about 82% of radioactivity originally present in crude oil eliminated by simulated commercial processes locally used for oil refining. A high percentage of the residues (50-55%) were eliminated during alkali treatment and bleaching. Refining of soybeans oil fortified with '1 4 C-dichlorovos. The final refined oil had only 13% of the radioactivity originally present, mainly in the form of dichlorvos, dimethyl and monomethyl phosphate in addition to desmethyl dichlorvos in oil with aged residues

Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.

Directory of Open Access Journals (Sweden)

Su Ma

Full Text Available ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.

TIMP-1 resistant matrix metalloproteinase-9 is the predominant serum active isoform associated with MRI activity in patients with multiple sclerosis.

Science.gov (United States)

Trentini, Alessandro; Manfrinato, Maria C; Castellazzi, Massimiliano; Tamborino, Carmine; Roversi, Gloria; Volta, Carlo A; Baldi, Eleonora; Tola, Maria R; Granieri, Enrico; Dallocchio, Franco; Bellini, Tiziana; Fainardi, Enrico

2015-08-01

The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown. To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis. We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing-remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls. Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively). Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity. © The Author(s), 2015.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

Science.gov (United States)

Horn, James V C; Ellena, Rachel A; Tran, Jesse J; Beck, Wendy H J; Narayanaswami, Vasanthy; Weers, Paul M M

2017-08-01

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystallization of a 79 kDa fragment of the hook protein FlgE from Campylobacter jejuni

International Nuclear Information System (INIS)

Kido, Yasuji; Yoon, Young-Ho; Samatey, Fadel A.

2011-01-01

A 79 kDa fragment of FlgE from C. jejuni has been crystallized. A 79 kDa fragment of the bacterial flagellar hook protein FlgE from Campylobacter jejuni was cloned, overexpressed, purified and crystallized. Two different crystal forms were obtained. Synchrotron X-ray diffraction data showed that the first crystal form, which diffracted to 4.9 Å resolution, belonged to the tetragonal crystal system, with space group I4 1 22 and unit-cell parameters a = b = 186.2, c = 386.6 Å, α = β = γ = 90°. The second crystal form diffracted to 2.5 Å resolution and belonged to the monoclinic crystal system, with space group P2 1 and unit-cell parameters a = 75.7, b = 173.8, c = 150.8 Å, α = γ = 90, β = 106.5°. SeMet protein was also overexpressed, purified and crystallized, and a 2.6 Å resolution MAD data set was collected

40 CFR 82.36 - Approved refrigerant handling equipment.

Science.gov (United States)

2010-07-01

... equipment. 82.36 Section 82.36 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PROTECTION OF STRATOSPHERIC OZONE Servicing of Motor Vehicle Air Conditioners § 82.36...-12, Extraction and Recycle Equipment for Mobile Automotive Air-Conditioning Systems, and Standard of...

Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

Directory of Open Access Journals (Sweden)

Edward C Twomey

Full Text Available Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15 protein in the complex with a mitogen-activated protein (MAP kinase, extracellular regulated kinase 2 (ERK2, which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

Interaction of Classical Platinum Agents with the Monomeric and Dimeric Atox1 Proteins: A Molecular Dynamics Simulation Study

Directory of Open Access Journals (Sweden)

Xiaolei Wang

2013-12-01

Full Text Available We carried out molecular dynamics simulations and free energy calculations for a series of binary and ternary models of the cisplatin, transplatin and oxaliplatin agents binding to a monomeric Atox1 protein and a dimeric Atox1 protein to investigate their interaction mechanisms. All three platinum agents could respectively combine with the monomeric Atox1 protein and the dimeric Atox1 protein to form a stable binary and ternary complex due to the covalent interaction of the platinum center with the Atox1 protein. The results suggested that the extra interaction from the oxaliplatin ligand–Atox1 protein interface increases its affinity only for the OxaliPt + Atox1 model. The binding of the oxaliplatin agent to the Atox1 protein might cause larger deformation of the protein than those of the cisplatin and transplatin agents due to the larger size of the oxaliplatin ligand. However, the extra interactions to facilitate the stabilities of the ternary CisPt + 2Atox1 and OxaliPt + 2Atox1 models come from the α1 helices and α2-β4 loops of the Atox1 protein–Atox1 protein interface due to the cis conformation of the platinum agents. The combinations of two Atox1 proteins in an asymmetric way in the three ternary models were analyzed. These investigations might provide detailed information for understanding the interaction mechanism of the platinum agents binding to the Atox1 protein in the cytoplasm.

Widespread occurrence of chemical residues in beehive matrices from apiaries located in different landscapes of Western France.

Directory of Open Access Journals (Sweden)

Olivier Lambert

Full Text Available The honey bee, Apis mellifera, is frequently used as a sentinel to monitor environmental pollution. In parallel, general weakening and unprecedented colony losses have been reported in Europe and the USA, and many factors are suspected to play a central role in these problems, including infection by pathogens, nutritional stress and pesticide poisoning. Honey bee, honey and pollen samples collected from eighteen apiaries of western France from four different landscape contexts during four different periods in 2008 and in 2009 were analyzed to evaluate the presence of pesticides and veterinary drug residues.A multi-residue analysis of 80 compounds was performed using a modified QuEChERS method, followed by GC-ToF and LC-MS/MS. The analysis revealed that 95.7%, 72.3% and 58.6% of the honey, honey bee and pollen samples, respectively, were contaminated by at least one compound. The frequency of detection was higher in the honey samples (n = 28 than in the pollen (n = 23 or honey bee (n = 20 samples, but the highest concentrations were found in pollen. Although most compounds were rarely found, some of the contaminants reached high concentrations that might lead to adverse effects on bee health. The three most frequent residues were the widely used fungicide carbendazim and two acaricides, amitraz and coumaphos, that are used by beekeepers to control Varroa destructor. Apiaries in rural-cultivated landscapes were more contaminated than those in other landscape contexts, but the differences were not significant. The contamination of the different matrices was shown to be higher in early spring than in all other periods.Honey bees, honeys and pollens are appropriate sentinels for monitoring pesticide and veterinary drug environmental pollution. This study revealed the widespread occurrence of multiple residues in beehive matrices and suggests a potential issue with the effects of these residues alone or in combination on honey bee health.

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.

Science.gov (United States)

Yamamoto, K; Ichinose, Y; Shinagawa, H; Makino, K; Nakata, A; Iwanaga, M; Honda, T; Miwatani, T

1990-12-01

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Dicty_cDB: CHE818 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 8.1 Habrotrocha constricta 82 kDa heat shock protein 2 (hsp82-2) gene, partial cds. 88 3e-48 4 AF143854 |AF143854.1 Sinantherina soci...alis 82 kD heat shock protein (hsp82) gene, partial cds.

Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

International Nuclear Information System (INIS)

Hristov, K.; Mitev, V.; Knox, K.

2006-01-01

Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

Dose-finding study of carbamylated monomeric allergoid tablets in grass-allergic rhinoconjunctivitis patients.

Science.gov (United States)

Mösges, Ralph; Rohdenburg, Christina; Eichel, Andrea; Zadoyan, Gregor; Kasche, Elena-Manja; Shah-Hosseini, Kija; Lehmacher, Walter; Schmalz, Petra; Compalati, Enrico

2017-11-01

To determine the optimal effective and safe dose of sublingual immunotherapy tablets containing carbamylated monomeric allergoids in patients with grass pollen-induced allergic rhinoconjunctivitis. In this prospective, randomized, double-blind, active-controlled, multicenter, Phase II study, four different daily doses were applied preseasonally for 12 weeks. Of 158 randomized adults, 155 subjects (safety population) received 300 units of allergy (UA)/day (n = 36), 600 UA/day (n = 43), 1000 UA/day (n = 39), or 2000 UA/day (n = 37). After treatment, 54.3, 47.6, 59.0 and 51.4% of patients, respectively, ceased to react to the highest allergen concentration in a conjunctival provocation test. Furthermore, the response threshold improved in 70.4, 62.9, 76.7 and 66.7% of patients, respectively. No serious adverse events occurred. This study found 1000 UA/day to be the optimal effective and safe dose.

The substructure of immunoglobulin G resolved to 25 kDa using amplitude modulation AFM in air

International Nuclear Information System (INIS)

Thomson, Neil H.

2005-01-01

Amplitude modulation (or tapping-mode) atomic force microscopy (AM AFM or TM AFM) in air can reveal sub-molecular details of isolated multi-subunit proteins, such as immunoglobulin G (IgG) antibodies, on atomically flat support surfaces such as mica [A. San Paulo, R. Garcia, Biophys. J. 78(3) (2000) 1599]. This is achieved by controlling the microscope imaging parameters (e.g. cantilever drive frequency and set-point amplitude) to keep the AFM tip predominantly in the attractive force regime. Under these conditions, the 50 kDa F c and F ab subunits can be resolved when the molecule has the appropriate orientation on the surface. The presence of a water layer on hydrophilic mica is an important factor affecting imaging contrast, a consequence of capillary neck formation between tip and surface [L. Zitzler, S. Herminghaus, F. Mugele, Phys. Rev. B 66(15) (2002) 155436]. Desiccation of samples to remove surface bound water layers can yield reproducible imaging of the IgG substructure [N.H. Thomson, J. Microsc. (Oxford) 217(3) (2004) 193]. This approach has also given higher resolution than previously achieved, down to about 25 kDa, and these data are detailed here. These subdomains are formed as two immunoglobulin folds from the light and heavy peptide chains of the IgG crossover. This result has been validated by comparing the AFM images with X-ray crystallography data from the protein data bank. These data show that the AFM can obtain 25 kDa resolution on isolated protein molecules with commercially available silicon tips, but, as expected for a local probe technique, resolution is highly dependent on the macromolecular orientation on the support surface

130 kDa phosphatase from the liver of labeo rohita: isolation: purification and some kinetic properties

International Nuclear Information System (INIS)

Siddiqua, A.; Sherazi, M.; Shah, A.H.; Khan, A.R.; Khan, H.U.

2009-01-01

An isoenzyme of high molecular weight acid phosphatase (HM-ACP) from the live of fish rohu (Labeo Rohita) was isolated and purified to homogeneity. The enzyme had specific activity of 14.96 U/mg and a recovery of about 4%. The purification procedure included ammonium sulphate precipitation and series of chromatographic separations on SP-Sephadex C-50, CM-Cellulose and Sephacryl HR-200 columns. Nealry 500-folds purification was achieved. The molecular weight was estimated to be 120-130 kDa by polyacrylamide gel electrophoresis (PAGE) of native enzyme and 130 kDa by gel filtration on calibrated Sephadex G-100 column. sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced and non-reduced condition showed a band corresponding to 66 kDa confirming the dimeric nature of enzyme. para nitrophenyl phosphate and flavin mononucleotide were hydrolyzed effectively by the enzyme and found to be good substrates. Optimum temperature for the enzyme was 50 degree C and temperature stability was 0 degree-50 degree C. Similarly optimum ph for the enzyme was 5.4 and ph stability was 4.8-6.0. The K/sub m/ for the p-nitrophenyl phosphate was estimated to be 0.15 mM. The enzyme was competitively inhibited by the phosphate, vanadate, molybdate, tartrate, fluoride and pyridoxal-5-PO/sub 4/ while pyridoxamine-5-PO/sub 4/ showed poor inhibition. Metal ions such as Ag/sup +/, Cu/sup ++/ Zn/sup ++/ showed strong inhibition on the enzyme activity while other divalent ions like Mg/sup ++/, Mn/sup ++/ and Co/sup ++/ were found to be poor inhibitors. Modifiers like EDTA, methanol, ethanol, acetone and glycerol had no effect on the enzyme's activity. (author)

40 CFR 82.34 - Prohibitions and required practices.

Science.gov (United States)

2010-07-01

.... 82.34 Section 82.34 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PROTECTION OF STRATOSPHERIC OZONE Servicing of Motor Vehicle Air Conditioners § 82.34..., any class I or class II substance that is suitable for use as a refrigerant in motor vehicle air...

Interplay of histidine residues of the Alzheimer’s disease Aβ peptide governs its Zn-induced oligomerization

Science.gov (United States)

Istrate, Andrey N.; Kozin, Sergey A.; Zhokhov, Sergey S.; Mantsyzov, Alexey B.; Kechko, Olga I.; Pastore, Annalisa; Makarov, Alexander A.; Polshakov, Vladimir I.

2016-02-01

Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1-16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation.

34 CFR 82.300 - Professional and technical services.

Science.gov (United States)

2010-07-01

... and analysis directly applying any professional or technical discipline. For example, drafting or a... 34 Education 1 2010-07-01 2010-07-01 false Professional and technical services. 82.300 Section 82... by Other Than Own Employees § 82.300 Professional and technical services. (a) The prohibition on the...

Residual nilpotence and residual solubility of groups

International Nuclear Information System (INIS)

Mikhailov, R V

2005-01-01

The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa

NARCIS (Netherlands)

Van Dorsselaer, A.; Bitsch, F.; Green, B.; Jarvis, S.; Lepage, P.; Bischoff, Rainer; Kolbe, V.J.; Roitsch, C.

1990-01-01

Mass measurement by electrospray mass spectrometry (ESMS) is used as a rapid preliminary verification of the identity of various recombinant proteins ranging from 7 to 44 kDa with an accuracy of 0.01-0.03%. ESMS not only improves the speed but also the reliability of the protein structure

MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

Science.gov (United States)

Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2013-04-01

Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

34 CFR 82.205 - Professional and technical services.

Science.gov (United States)

2010-07-01

..., drafting of a legal document accompanying a bid or proposal by a lawyer is allowable. Similarly, technical... 34 Education 1 2010-07-01 2010-07-01 false Professional and technical services. 82.205 Section 82... by Own Employees § 82.205 Professional and technical services. (a) The prohibition on the use of...

29 CFR 452.82 - Reprisal for exercising rights.

Science.gov (United States)

2010-07-01

... 29 Labor 2 2010-07-01 2010-07-01 false Reprisal for exercising rights. 452.82 Section 452.82 Labor Regulations Relating to Labor OFFICE OF LABOR-MANAGEMENT STANDARDS, DEPARTMENT OF LABOR LABOR-MANAGEMENT... DISCLOSURE ACT OF 1959 Campaign Safeguards § 452.82 Reprisal for exercising rights. A member has a right to...

Ftr82 Is Critical for Vascular Patterning during Zebrafish Development

Directory of Open Access Journals (Sweden)

Hsueh-Wei Chang

2017-01-01

Full Text Available Cellular components and signaling pathways are required for the proper growth of blood vessels. Here, we report for the first time that a teleost-specific gene ftr82 (finTRIM family, member 82 plays a critical role in vasculature during zebrafish development. To date, there has been no description of tripartite motif proteins (TRIM in vascular development, and the role of ftr82 is unknown. In this study, we found that ftr82 mRNA is expressed during the development of vessels, and loss of ftr82 by morpholino (MO knockdown impairs the growth of intersegmental vessels (ISV and caudal vein plexus (CVP, suggesting that ftr82 plays a critical role in promoting ISV and CVP growth. We showed the specificity of ftr82 MO by analyzing ftr82 expression products and expressing ftr82 mRNA to rescue ftr82 morphants. We further showed that the knockdown of ftr82 reduced ISV cell numbers, suggesting that the growth impairment of vessels is likely due to a decrease of cell proliferation and migration, but not cell death. In addition, loss of ftr82 affects the expression of vascular markers, which is consistent with the defect of vascular growth. Finally, we showed that ftr82 likely interacts with vascular endothelial growth factor (VEGF and Notch signaling. Together, we identify teleost-specific ftr82 as a vascular gene that plays an important role for vascular development in zebrafish.

Residues leaching from 'Factory of barren ores'

International Nuclear Information System (INIS)

Rakhmatov, N.; Khakimov, N.; Nazarov, Kh.M.; Barotov, A.M.

2012-01-01

The objective of the present work is safe management of residues from Factory of Barren Ores, their reprocessing, expenditures reduction for remediation of Istiklol city former uranium tailings. For this purpose, some experiences were adopted - Factory of Barren Ores tailing use for filling up the open pit where water with uranium content 3-5 mg/l is located. Factory of Barren Ores waste are passed through heap leaching and have some amount of uranium salts dissolved in water. Thus, we propose to dissolve uranium from Factory of Barren Ores wastes with uranium bearing water flowing out from gallery and filling up the open-pit by radioactive wastes. In so doing, uranium content flowing out from gallery will increase twice, and further, passing them through apricot's shell, as a sorbent, we will clean the water against radionuclides. Residue samples with uranium content 0,015% from Factory of Barren Ores and uranium bearing waters from gallery 1 with uranium content 0,0025 g/l were used for laboratory tests. After which, a slurry was prepared by means of residue mixing with water in ratio of solid and liquid phases (S:L) - 1:2 and 9,7 ml of sulfuric acid (Ρ=1,82) was added which corresponds consumption by H 2 SO 4 176,54 kg/t. For the first test, leaching was carried out during 4 hours at ph=1,6 at room temperature. For the second test, leaching was carried out at 60 d eg C a nd ph=1,6 during 4 hours. Slurry heating and mixing was carried out by means of magnetic mixer. The basic residue leaching indicators are provided in this article.

Usefulness of food chain information provided by Dutch finishing pig producers to control antibiotic residues in pork.

Science.gov (United States)

van Wagenberg, Coen P A; Backus, Gé B C; van der Vorst, Jack G A J; Urlings, Bert A P

2012-11-01

The EU prescribes that food business operators must use food chain information to assist in food safety control. This study analyses usefulness of food chain information about antibiotic usage covering the 60-day period prior to delivery of pigs to slaughter in the control of antibiotic residues in pork. A dataset with 479 test results for antibiotic residues in tissue samples of finishing pigs delivered to a Dutch slaughter company was linked to information provided by pig producers about antibiotic usage in these finishing pigs. Results show that twice as many producers reported using antibiotics in the group of 82 producers with antibiotic residues (11.0%) compared to the group without antibiotic residues (5.5%) (p=0.0686). For 89% of consignments with a finishing pig with antibiotic residues, the producer reported 'did not use antibiotics'. Food chain information about antibiotic usage provided by Dutch pig producers was no guarantee for absence of antibiotic residues in delivered finishing pigs. Copyright © 2012 Elsevier B.V. All rights reserved.

Residues of 14C-chlorpyrifos in coconut by radiotracer techniques

International Nuclear Information System (INIS)

Tejada, A.W.; Calumpang, S.M.F.; Magallona, E.D.

1992-01-01

A coconut tree was root-infused with 5 g a.i. chlorpyrifos (Lorsban plus 1.4 uCi of 14 C-chlorpyrifos. Coconut samples both young and mature, were taken 24 h up to 60 days after root infusion. Analysis of the meat and water was done. The maximum uptake of chlorpyrifos equivalents was on the 13th day after root infusion both in mature and young fruit. The residue in water reached the peak on the 20th day after infusion and declined thereafter. A preharvest interval (PHI) of more than 60 days maybe recommended for safe consumption. The usual practice of storing copra in treated warehouses until it is processed into oil was simulated in the laboratory to determine the residues absorbed on the meat. Forty kg of copra with moisture content of 7-12% were stored in an improvised warehouse, i.e. 1 m x 1.5 m x 1 m wooden box, which was treated with 100 ml 1% a.i. Lorsban 50 WP plus 100 uCi 14 C-chlorpyrifos for protection against insects. Analysis showed that 14 C-chlorpyrifos equivalents ranged from 22.6 mg/kg on the 30th day to 8.2 mg/kg on the 90th day with a 63.7% reduction in residues. Bound residues were detected on samples stored for 60-90 days, with levels of 1.0 to 0.2 ug/g 14 C-chlorpyrifos equivalents. This is within the Maximum Residue Limit of 2 mg/kg set by FAO/WHO for chlorpyrifos in most agricultural commodities. Chlorpyrifos residues were also determined at various stages of refining and processing of crude coconut oil. It was subjected to alkali refining, bleaching and finally steaming. The residues were reduced by as much as 32.7%. (author). 18 refs.; 2 figs.; 3 tabs

PURIFICACIÓN DEL ANTÍGENO 38 kDa DE Mycobacterium tuberculosis Y SU POTENCIAL USO EN DIAGNÓSTICO MEDIANTE INMUNOSENSORES PIEZOELÉCTRICOS

OpenAIRE

Paula A. Marín; Luz E. Botero; Jaime A. Robledo; Ana M. Murillo; Robinson A. Torres; Yeison J. Montagut; Elizabeth Pabón; Marisol Jaramillo

2015-01-01

Un paso crucial en el desarrollo de un inmunosensor piezoeléctrico para la detección de tuberculosis (TB), es la selección y obtención de los inmunoreactivos empleados en el inmunoensayo y la estrategia para la biofuncionalización del transductor. Diversos estudios han reportado el uso del antígeno proteico 38kDa (Ag38kDa) de Mycobacterium tuberculosis ( Mtb ) como un buen biomarcador de la enfermedad y el cumplimiento de las características físicas y bioquímicas para ser inmovilizado por mon...

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein

Directory of Open Access Journals (Sweden)

Coetzer Theresa L

2006-11-01

Full Text Available Abstract Background Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181 is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. Methods 4.1R structural domains (30, 16, 10 and 22 kDa domain and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. Results Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. Conclusion The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.

Annual report 1981-82

International Nuclear Information System (INIS)

The research and teaching programmes of the Universities Research Reactor, Risley, U.K. are reviewed giving outlines of the individual experiments undertaken during 1981-82 and the reactor operation and safety arrangements. (U.K.)

Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis

International Nuclear Information System (INIS)

Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

2009-01-01

A monomeric mutant of Azami-Green from G. fascicularis was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal belonged to space group P1 and diffracted X-rays to 2.20 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 Å, α = 90.96, β = 103.41, γ = 101.79°. The Matthews coefficient (V M = 2.10 Å 3 Da −1 ) indicated that the crystal contained two mAG molecules per asymmetric unit

Cooperative Interactions between 480 kDa Ankyrin-G and EB Proteins Assemble the Axon Initial Segment.

Science.gov (United States)

Fréal, Amélie; Fassier, Coralie; Le Bras, Barbara; Bullier, Erika; De Gois, Stéphanie; Hazan, Jamilé; Hoogenraad, Casper C; Couraud, François

2016-04-20

The axon initial segment (AIS) is required for generating action potentials and maintaining neuronal polarity. Significant progress has been made in deciphering the basic building blocks composing the AIS, but the underlying mechanisms required for AIS formation remains unclear. The scaffolding protein ankyrin-G is the master-organizer of the AIS. Microtubules and their interactors, particularly end-binding proteins (EBs), have emerged as potential key players in AIS formation. Here, we show that the longest isoform of ankyrin-G (480AnkG) selectively associates with EBs via its specific tail domain and that this interaction is crucial for AIS formation and neuronal polarity in cultured rodent hippocampal neurons. EBs are essential for 480AnkG localization and stabilization at the AIS, whereas 480AnkG is required for the specific accumulation of EBs in the proximal axon. Our findings thus provide a conceptual framework for understanding how the cooperative relationship between 480AnkG and EBs induces the assembly of microtubule-AIS structures in the proximal axon. Neuronal polarity is crucial for the proper function of neurons. The assembly of the axon initial segment (AIS), which is the hallmark of early neuronal polarization, relies on the longest 480 kDa ankyrin-G isoform. The microtubule cytoskeleton and its interacting proteins were suggested to be early key players in the process of AIS formation. In this study, we show that the crosstalk between 480 kDa ankyrin-G and the microtubule plus-end tracking proteins, EBs, at the proximal axon is decisive for AIS assembly and neuronal polarity. Our work thus provides insight into the functional mechanisms used by 480 kDa ankyrin-G to drive the AIS formation and thereby to establish neuronal polarity. Copyright © 2016 the authors 0270-6474/16/364421-13$15.00/0.

Two-stage catalytic up-grading of vacuum residue of a Wandoan coal liquid. [Vacuum residue of coal liquid

Energy Technology Data Exchange (ETDEWEB)

Mochida, I.; Sakanishi, K.; Korai, Y.; Fujitsu, H.

1986-08-01

A successive two-stage hydrotreatment using a commercial Ni-Mo/Al/sub 2/O/sub 3/ catalyst (HDN-30) was applied to the vacuum residue of a Wandoan coal liquid to achieve high levels of hydrocracking, hydrodenitrogenation and hydrodeoxygenation. Two-stage hydrotreatment in 1-methylnaphthalene containing 20wt% fluoranthene as a solvent at solvent/coal liquid ratio of unity removed 83% (overall) of nitrogen and 90% (overall) of oxygen in the asphaltene (benzene-soluble fraction) at 380/sup 0/C for 3 h and at 420/sup 0/C for 3h under hydrogen pressure of 15 MPa and 14 MPa, respectively, while the single stage treatment at 420/sup 0/C for 3 h removed only 41% and 46%, respectively. The same two-stage treatment allowed the overall denitrogenation of 51% and the overall deoxygenation of 67% from a mixture of asphaltene and preasphaltene (THF-soluble fraction). Addition of the catalyst prior to the second stage reaction increased the removal of nitrogen and oxygen to 75 and 82%, respectively, indicating significant catalyst deactivation by the preasphaltene fraction in the first stage. Increasing the solvent/coal liquid ratio to 2 or addition of tetrahydrofluoranthene as a component of the solvent increased the removal of nitrogen and oxygen to 70 and 80%, respectively. Such two-stage hydrotreatment was also effective in refining the whole residue, allowing denitrogenations and deoxygenations of 68 and 75% respectively using tetrahydrofluoranthene. The coke, unreacted coal and minerals in the residue may not cause acute catalyst deactivation. High dissolving ability of the reaction solvents is very effective to decrease catalyst deactivation by carbon deposition. The successive two-stage hydrotreatment also enhanced hydrocracking of polar and resin fractions in the residue into oils (conversion, 65%). (Abstract Truncated)

Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

Science.gov (United States)

Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard

2014-10-01

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.

PDX experimental results in FY82

International Nuclear Information System (INIS)

Kaye, S.M.; Bell, M.; Bol, K.

1983-08-01

This report presents a detailed summary of the major experimental results of PDX in FY82 and represents the efforts of the entire PDX group. Topics covered include β-scaling and fishbone studies, fluctuations, disruptions, impurities and impurity transport, power handling, limiter conditioning, edge studies, plasma fueling, counter-injection, and diagnostic development. A less detailed version will appear as the FY82 PDX contribution to the PPPL Annual Report

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-01-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

STS-82 Pilot Scott Horowitz at SLF

Science.gov (United States)

1997-01-01

STS-82 Pilot Scott J. 'Doc' Horowitz flashes a wide grin for photographers after he lands his T-38 jet at KSCs Shuttle Landing Facility. Horowitz and the other six members of the STS-82 crew came from their home base at Johnson Space Center in Houston, TX, to spend the last few days before launch at KSC. STS-82 is scheduled for liftoff on Feb. 11 during a 65-minute launch window which opens at 3:56 a.m. EST. The 10-day flight aboard the Space Shuttle Discovery will be the second Hubble Space Telescope servicing mission.

Secretion of 35SO4-labeled proteins from isolated rat hepatocytes

International Nuclear Information System (INIS)

von Wuertemberg, M.M.F.; Fries, E.

1989-01-01

Sulfation is a Golgi-specific modification of secretory proteins. We have characterized the proteins that are labeled with 35 SO 4 in cultures of rat hepatocytes and studied their transport to the medium. Analysis by polyacrylamide gel electrophoresis showed that of the five most heavily labeled proteins, four had well-defined mobilities--apparent molecular masses of 188, 142, 125, and 82 kDa--whereas one was electrophoretically heterogeneous--apparent molecular mass of 35-45 kDa. Judging by their relatively high resistance to acid treatment, the sulfate residues in the 125- and 35-45-kDa proteins were linked to carbohydrate. Some of the secreted proteins were sialylated. In samples of pulse-labeled cells, there appeared to be no unsialylated forms, indicating that sulfation occurred after sialylation, presumably in the trans Golgi. Kinetic experiments showed that the cellular half-life was the same for all the sulfated proteins--about 8 min--consistent with the idea that transport from the Golgi complex to the cell surface occurs by liquid bulk flow

The 18-kDa mitochondrial translocator protein in gliomas: from the bench to bedside.

OpenAIRE

Janczar, Karolina; Su, Zhangjie; Raccagni, Isabella; Anfosso, Andrea; Kelly, Charlotte; Durrenberger, Pascal F; Gerhard, Alexander; Roncaroli, Federic

2015-01-01

The 18-kDa mitochondrial translocator protein (TSPO) is known to be highly expressed in several types of cancer, including gliomas, whereas expression in normal brain is low. TSPO functions in glioma are still incompletely understood. The TSPO can be quantified pre-operatively with molecular imaging making it an ideal candidate for personalized treatment of patient with glioma. Studies have proposed to exploit the TSPO as a transporter of chemotherapics to selectively target tumour cells in t...

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein

International Nuclear Information System (INIS)

Ross, W.; Bertrand, W.; Morrison, A.

1990-01-01

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed. We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

International Nuclear Information System (INIS)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

1990-01-01

A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

Energy Technology Data Exchange (ETDEWEB)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

1990-08-01

A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

Isotopic yields and kinetic energies of primary residues in 1 A GeV 208Pb + p reactions

International Nuclear Information System (INIS)

Enqvist, T.; Wlazlo, W.; Armbruster, P.

2000-09-01

The production of primary residual nuclei in the reaction 1 A GeV 208 Pb on proton has been studied by measuring isotopic distributions for all elements from titanium (Z=22) to lead (Z=82). Kinematical properties of the residues were also determined and used to disentangle the relevant reaction mechanisms, spallation (projectile fragmentation) and fission. The fragment separator FRS at GSI, Darmstadt, was used to separate and identify the reaction products. The measured production cross sections are highly relevant for the design of accelerator-driven subcritical reactors and for the planning of future radioactive-beam facilities. (orig.)

40 CFR 8.2 - Applicability and effect.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Applicability and effect. 8.2 Section 8... OF NONGOVERNMENTAL ACTIVITIES IN ANTARCTICA § 8.2 Applicability and effect. (a) This part is intended to ensure that potential environmental effects of nongovernmental activities undertaken in Antarctica...

The iron uptake mechanisms of enteroinvasive Escherichia coli Mecanismos de captação de ferro de Escherichia coli enteroinvasora

Directory of Open Access Journals (Sweden)

Agda Andrade

2000-09-01

Full Text Available Enteroinvasive Escherichia coli strains (EIEC of different serotypes isolated from patients with acute diarrhea were examined for the ability to produce siderophores and iron-regulated outer membrane proteins (IROMP. For iron starvation cultures were grown at 37°C in LB supplied with 200 muM of a-alpha’dypirydil. All strains produced enterobactin and twelve (40% produced aerobactin. The strains showed IROMP varying from 67-82 kDa. Proteins were either induced or stimulated by the iron starvation. Differences were observed in the electrophoretic profile among the serotypes, originating 5 electrophoretic profiles. All serotypes expressed proteins of 82 kDa (FepA and 76 kDa (IutA (except serotype O28ac:H- that did not produce the 76 kDa protein. Several strains (O29:H-, O144:H-, O152:H-, and O167:H- expressed IutA in the outer membrane, in the absence of aerobactin production. Additionally to well characterized proteins (FepA and IutA, we found two IROMP of unknown function in some serotypes: a 71 kDa protein was detected in three profiles and a 67 kDa protein was present in serotype O152:H-. Moreover, two bands (39 and 43 kDa which were not iron-regulated bound specifically to human lactoferrin.Cepas de Escherichia coli enteroinvasora de diferentes sorotipos isoladas de pacientes com diarréia aguda foram examinadas quanto a capacidade de produzir sideróforos e proteínas de membrana externa reguladas pelo ferro (IROMP. O crescimento bacteriano em meio com deficiência em Fe foi obtido em caldo Lúria acrescido de 200 mM de alfa-a’dipiridil. Todas as cepas produziram enterobactina e 40% produziram aerobactina. As cepas produziram IROMPs com MM variando de 82-67 kDa. As proteínas foram induzidas ou estimuladas pela deficiência de ferro. Diferenças foram observadas no perfil eletroforético entre os sorotipos, originando 5 perfis eletroforéticos. Todos os sorotipos, com exceção do sorotipo O28ac:H- (onde a proteína de 76 kDa não foi

Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): first discovery of giant hexagonal-bilayer hemoglobin in the former "Pogonophora" group.

Science.gov (United States)

Meunier, Cédric; Andersen, Ann C; Bruneaux, Matthieu; Le Guen, Dominique; Terrier, Peran; Leize-Wagner, Emmanuelle; Zal, Franck

2010-01-01

Siboglinids are symbiotic polychete annelids having hemoglobins as essential oxygen- and sulfide-carriers for their endosymbiotic bacteria. We analyzed the structure of the hemoglobins from two species of siboglinids: the monilifera Sclerolinum contortum and the frenulata Oligobrachia webbi (i.e. haakonmosbiensis) from Norwegian cold seeps. Measured by Multi-Angle Laser Light Scattering (MALLS), Sclerolinum shows a 3190+/-50 kDa hexagonal bilayer hemoglobin (HBL-Hb) and a 461+/-46 kDa ring-Hb, just as vestimentifera, whereas Oligobrachia has a 409+/-3.7 kDa ring-Hb only. Electrospray Ionization-Mass Spectrometry (ESI-MS) showed Sclerolinum HBL-Hb composed of seven monomeric globins (15-16 kDa), three disulfide-bonded globin heterodimers and three linkers. The heterodimers always contain globin-b (15814.4+/-1.5 Da). Sclerolinum ring-Hb is composed of globins and dimers with identical masses as its HBL-Hb, but lacks linkers. Oligobrachia ring-Hb has three globin monomers (14-15 kDa) only, with no disulfide-bonded dimers. Comparison of Sclerolinum hemoglobins between Storegga and Haakon Mosby Mud Volcano, using the normalized height of deconvoluted ESI-MS peaks, shows differences in globin monomers abundances that could reflect genetic differences or differential gene expression between distinct seep populations. The discovery of HBL-Hb in Sclerolinum is a new element supporting the hypothesis of monilifera being phylogenetically more closely related to vestimentifera, than to frenulata.

Myocardial study with rubidium-82 using positron emission tomography

Energy Technology Data Exchange (ETDEWEB)

De Landsheere, C.; Brihaye, C.; Chevigne, M.; Guillaume, M.; Lamotte, D.; Larock, M.P.; Quaglia, L.; Rigo, P. (Universite de Liege (Belgium))

1982-01-01

The rubidium-82 is eluted from a strontium-82/rubidium-82 generator. Its short half-life (78 sec.) enables the sequential repetition of dynamic studies. Myocardial ischemia produced in the dog by a stenosis of the left anterior descending artery is investigated. In this case, intravenous administration of rubidium demonstrates a decrease in the cation uptake in the anterior wall of the left ventricle. Through ischemia studies various parameters which are liked to modify the rubidium distribution according to the equation describing the cation exchanges, are considered. The generator strontium-82/rubidium-82 delivers a reference tracer for myocardial flow evaluation without requiring a cyclotron which is then available for the direct production of metabolic tracers.

Quadratic residues and non-residues selected topics

CERN Document Server

Wright, Steve

2016-01-01

This book offers an account of the classical theory of quadratic residues and non-residues with the goal of using that theory as a lens through which to view the development of some of the fundamental methods employed in modern elementary, algebraic, and analytic number theory. The first three chapters present some basic facts and the history of quadratic residues and non-residues and discuss various proofs of the Law of Quadratic Reciprosity in depth, with an emphasis on the six proofs that Gauss published. The remaining seven chapters explore some interesting applications of the Law of Quadratic Reciprocity, prove some results concerning the distribution and arithmetic structure of quadratic residues and non-residues, provide a detailed proof of Dirichlet’s Class-Number Formula, and discuss the question of whether quadratic residues are randomly distributed. The text is a valuable resource for graduate and advanced undergraduate students as well as for mathematicians interested in number theory.

[E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction].

Science.gov (United States)

Huo, Yujia; Lu, Qiaonan; Zheng, Xiaowei; Ma, Yuanfang; Lu, Feng

2016-02-04

To explore effects of FtsZ mutants FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) on FtsZ self-assembly and interaction of FtsZ with MreB in Escherichia coli strains. METHODS) We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis methods, and purified targeted proteins by affinity chromatography. QN6(ftsZ::yfp-cat), QN7(tsZ::yfp-cat), QN8(ftsZ(R78G)::yfp-cat) and QN9 (ftsZ(D82A):.:yfp-cat) strains were constructed by linear DNA homologous recombination. We observed cellular localization pattern of FtsZ and its mutants in E. coli by living cell imaging experiments, examined interaction of FtsZ/FtsZ*-FtsZ* and FtsZ/FtsZ*-MreB by Coimmunoprecipitation and bacteria two hybrid, and analyzed assembly characteristics of FtsZ mutants by Light scattering. RESULTS) The Yfp-labeled FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) mutant proteins failed to assemble into functional Z-ring structure and localize correctly in E. coli strains. Interaction of FtsZ with its mutants, or FtsZ*-FtsZ* and FtsZ*-MreB interaction were weakened or completely disappeared. In addition, in vitro experiments show that E75A, R78G and D82A mutations decreased the polymerization efficiency of FtsZ monomer. FtsZ E75, R78 and D82 are critical amino acids in the assembly, function of FtsZ protein and FtsZ-MreB interaction in E. coli strains.

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

Science.gov (United States)

Calderon, L A; Teles, R C L; Leite, J R S A; Franco, O L; Grossi-de-Sá, M F; Medrano, F J; Bloch, C; Freitas, S M

2005-08-01

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

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Mohammad Matiur Rahman

2014-08-01

Full Text Available The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013. The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9 enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic -1,4-glucanase (EC 3.2.1.4. A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

Science.gov (United States)

Rahman, Mohammad; Inoue, Akira; Ojima, Takao

2014-08-01

The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic ?-1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

27 CFR 70.82 - Payment on notice and demand.

Science.gov (United States)

2010-04-01

... demand. 70.82 Section 70.82 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... Collection of Excise and Special (Occupational) Tax Notice and Demand § 70.82 Payment on notice and demand. Upon receipt of notice and demand from the appropriate TTB officer, there shall be paid at the place...

Electrophoretic and zymographic techniques for production monitoring of two lipase forms from Candida antarctica DSM 70725

Directory of Open Access Journals (Sweden)

Dimitrijević Aleksandra S.

2012-01-01

Full Text Available Yeast Candida antarctica produces two lipase forms, which are widely used as catalysts in variety of organic reactions, many of which are applied on a large scale. In this work, production of two forms of lipase from C. antarctica DSM 70725 (CAL A and CAL B was monitored during seven days of cultivation in the optimal medium using different electrophoretic and zymographic techniques. According to electrophoresis after silver staining, C. antarctica lipase A (molecular mass 45 kDa was produced starting from the second day of cultivation. C. antarctica lipase B (CAL B was also produced starting from the second day, but protein was present in the fermentation broth predominantly as dimer (molecular weight 66 kDa, while presence of monomeric form of CAL B (molecular weight of 33 kDa was observed starting from the fourth day of cultivation. Both types of zymograms (based on hydrolysis and synthesis reactions were used for detection of lipase activity in the fermentation broth. C. antarctica lipase A showed activity only in hydrolytic zymogram, when α-naphtyl butyrate was used as substrate. In the same zymogram, with α-naphtyl acetate as substrate no CAL A activity was detected. Similarly, CAL A showed no activity in synthesis based zymograms towards oleic acid and octanol as substrates, indicating that CAL A is not active towards very short or long-chain substrates. As opposite of CAL A, both monomeric and dimeric form of CAL B were detected in the all zymograms, suggesting that CAL B is active towards wide range of substrates, regardless to the chain length. Thus, zymogram based on hydrolysis of α-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics.

27 CFR 479.82 - Rate of tax.

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2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2010-04-01 2010-04-01 false Rate of tax. 479.82... OTHER FIREARMS Transfer Tax § 479.82 Rate of tax. The transfer tax imposed with respect to firearms... transfer tax on any firearm classified as “any other weapon” shall be at the rate of $5 for each such...

Characterization of monomeric DNA-binding protein Histone H1 in Leishmania braziliensis.

Science.gov (United States)

Carmelo, Emma; González, Gloria; Cruz, Teresa; Osuna, Antonio; Hernández, Mariano; Valladares, Basilio

2011-08-01

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

Science.gov (United States)

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

A Bayesian framework based on a Gaussian mixture model and radial-basis-function Fisher discriminant analysis (BayGmmKda V1.1) for spatial prediction of floods

Science.gov (United States)

Tien Bui, Dieu; Hoang, Nhat-Duc

2017-09-01

In this study, a probabilistic model, named as BayGmmKda, is proposed for flood susceptibility assessment in a study area in central Vietnam. The new model is a Bayesian framework constructed by a combination of a Gaussian mixture model (GMM), radial-basis-function Fisher discriminant analysis (RBFDA), and a geographic information system (GIS) database. In the Bayesian framework, GMM is used for modeling the data distribution of flood-influencing factors in the GIS database, whereas RBFDA is utilized to construct a latent variable that aims at enhancing the model performance. As a result, the posterior probabilistic output of the BayGmmKda model is used as flood susceptibility index. Experiment results showed that the proposed hybrid framework is superior to other benchmark models, including the adaptive neuro-fuzzy inference system and the support vector machine. To facilitate the model implementation, a software program of BayGmmKda has been developed in MATLAB. The BayGmmKda program can accurately establish a flood susceptibility map for the study region. Accordingly, local authorities can overlay this susceptibility map onto various land-use maps for the purpose of land-use planning or management.

A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin).

Science.gov (United States)

Brange, J; Dodson, G G; Edwards, D J; Holden, P H; Whittingham, J L

1997-04-01

The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 A spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer-monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy.

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein

International Nuclear Information System (INIS)

Lee, G.; Ronai, Z.A.; Pincus, M.R.; Brandt-Rauf, P.W.; Weinstein, I.B.; Murphy, R.B.; Delohery, T.M.; Nishimura, S.; Yamaizumi, Z.

1989-01-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with modified p21 protein, the cells were pulsed with [ 35 S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21 - protein complexes. By using this technique, the authors found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. They suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes

21 CFR 82.1260 - D&C Orange No. 10.

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2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false D&C Orange No. 10. 82.1260 Section 82.1260 Food... CERTIFIED PROVISIONALLY LISTED COLORS AND SPECIFICATIONS Drugs and Cosmetics § 82.1260 D&C Orange No. 10. The color additive D&C Orange No. 10 shall conform in identity and specifications to the requirements...

21 CFR 82.1261 - D&C Orange No. 11.

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2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false D&C Orange No. 11. 82.1261 Section 82.1261 Food... CERTIFIED PROVISIONALLY LISTED COLORS AND SPECIFICATIONS Drugs and Cosmetics § 82.1261 D&C Orange No. 11. The color additive D&C Orange No. 11 shall conform in identity and specifications to the requirements...

Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus

NARCIS (Netherlands)

You, Z.O.; Nadala, E.C.B.; Yang, J.S.; Hulten, van M.C.W.; Loh, P.C.

2002-01-01

A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum

Cardiac 82rubidium PET/CT: initial European experience

International Nuclear Information System (INIS)

Groves, Ashley M.; Dickson, John C.; Kayani, Irfan; Endozo, Raymondo; Blanchard, Patty; Shastry, Manu; Prvulovich, Elizabeth; Waddington, Wendy A.; Ben-Haim, Simona; Bomanji, Jamshed B.; Ell, Peter J.; Speechly-Dick, Marie-Elsya; McEwan, Jean R.

2007-01-01

Myocardial perfusion with PET/CT has advantages over conventional SPECT. We describe our initial European experience using 82 Rubidium-PET/CT, as part of a clinical myocardial perfusion service. We studied the first 100 patients (64 male; 36 female, mean age = 60: SD +/-12.5y, mean body mass index = 30: SD +/-6.9kg/m 2 ) who underwent 82 Rubidium cardiac PET/CT in our institution. Thirty patients had recently undergone coronary angiography. Patients underwent imaging during adenosine infusion and at rest. Images were acquired over 5 minutes using a GE-PET/CT instrument. Image quality was described as good, adequate or inadequate. Images were reported patient-by-patient by a minimum of five nuclear medicine physicians. A segment-by-segment analysis (17-segment model) was also performed. Image quality was good in 77%, adequate 23% and inadequate 0%. There was no statistical difference in image quality between obese and non-obese patients (Fisher's exact test, p = 0.2864). 59% had normal perfusion studies, 29% had inducible ischaemia, 12% had myocardial infarction (11% with super added ischaemia). There was reduced 82 Rubidium uptake in 132/1700 segments during stress. There was reduced 82 Rubidium uptake at rest in 42/1700 segments. The 82 Rubidium PET/CT findings were consistent with the angiographic findings in 28/30 cases. We show that, even from initial use of 82 Rubidium, it is possible to perform myocardial perfusion studies quickly with good image quality, even in the obese. The PET findings correlated well in the third of the cases where angiography was available. As such, 82 Rubidium cardiac PET/CT is likely to be an exciting addition to the European nuclear physician/ cardiologist's radionuclide imaging arsenal. (orig.)

Mucins CA 125, CA 19.9, CA 15.3 and TAG-72.3 as tumor markers in patients with lung cancer: comparison with CYFRA 21-1, CEA, SCC and NSE.

Science.gov (United States)

Molina, Rafael; Auge, Jose Maria; Escudero, Jose Miguel; Marrades, Ramon; Viñolas, Nuria; Carcereny, Emilio; Ramirez, Jose; Filella, Xavier

2008-01-01

Tumor marker serum levels were prospectively studied in 289 patients with suspected, but unconfirmed, lung cancer and in 513 patients with lung cancer [417 non-small cell lung cancer (NSCLC) patients and 96 small cell lung cancer (SCLC) patients]. In patients with benign disease, abnormal serum levels were found for the following tumor markers: CEA (in 6.6% of patients); CA 19.9 (6.2%); CA 125 (28.7%); NSE (0.7%); CYFRA (8.7%); TAG-72.3 (4.2%); SCC (3.5%), and CA 15.3 (3.5%). Excluding patients with renal failure or liver diseases, tumor marker specificity improved with abnormal levels in 0.5% for NSE, 0.9% for SCC, 2.8% for CEA, CA 15.3 and TAG-72.3, 3.8% for CA 19.9, 4.2% for CYFRA and 21.4% for CA 125. Excluding CA 125, one of the markers was abnormal in 15% of patients without malignancy. Tumor marker sensitivity was related to cancer histology and tumor extension. NSE had the highest sensitivity in SCLC and CYFRA and CEA in NSCLC. Significantly higher concentrations of CEA, SCC, CA 125, CA 15.3 and TAG-72.3 were found in NSCLC than in SCLC. Likewise, significantly higher CEA (p tumors. Using a combination of 3 tumor markers (CEA, CYFRA 21-1 in all histologies, SCC in squamous tumors and CA 15.3 in adenocarcinomas), a high sensitivity may be achieved in all histological types. Tumor markers may be useful in the histological differentiation of NSCLC and SCLC. Using specific criteria for the differentiation of SCLC and NSCLC, the sensitivity was 84.2 and 68.8%, the specificity was 93.8 and 99.7%, the positive predictive value was 98.3 and 98.5% and the negative predictive value was 57.7 and 93.3%, respectively. Copyright 2008 S. Karger AG, Basel.

Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

Science.gov (United States)

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

2008-12-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex.

Science.gov (United States)

Siritapetawee, J; Thammasirirak, S; Samosornsuk, W

2012-01-01

Artocarpus heterophyllus (jackfruit) is a latex producing plant. Plant latex is produced from secretory cells and contains many intergradients. It also has been used in folk medicine. This study aimed to purify and characterize the biological activities of a protease from jackfruit latex. A protease was isolated and purified from crude latex of a jackfruit tree by acid precipitation and ion exchange chromatography. The proteolytic activities of protein were tested using gelatin- and casein-zymography. The molecular weight and isoelectric point (pl) of protein were analysed by SDS/12.5% PAGE and 2D-PAGE, respectively. Antimicrobial activity of protein was analysed by broth microdilution method. In addition, the antibacterial activity of protein against Pseudomonas aeruginosa ATCC 27853 was observed and measured using atomic force microscopy (AFM) technique. The purified protein contained protease activity by digesting gelatin- and casein-substrates. The protease was designated as antimicrobial protease-48 kDa or AMP48 due to its molecular mass on SDS-PAGE was approximately 48 kDa. The isoelectric point (pl) of AMP48 was approximately 4.2. In addition, AMP48 contained antimicrobial activities by it could inhibit the growths of Pseudomonas aeruginosa ATCC 27853 and clinical isolated Candida albicans at minimum inhibitory concentration (MIC) 2.2 mg/ml and Minimum microbicidal concentration (MMC) 8.8 mg/ml. AFM image also supported the antimicrobial activities of AMP48 by the treated bacterial morphology and size were altered from normal.

Positron annihilation lifetime measurements of vanadium alloy and F82H irradiated with fission and fusion neutrons

International Nuclear Information System (INIS)

Sato, K.; Inoue, K.; Yoshiie, T.; Xu, Q.; Wakai, E.; Kutsukake, C.; Ochiai, K.

2009-01-01

V-4Cr-4Ti, F82H, Ni and Cu were irradiated with fission and fusion neutrons at room temperature and 473 K. Defect structures were analyzed and compared using positron annihilation lifetime measurement, and microstructural evolution was discussed. The mean lifetime of positrons (the total amount of residual defects) increased with the irradiation dose. The effect of cascade impact was detected in Ni at room temperature. The size and the number of vacancy clusters were not affected by the displacement rate in the fission neutron irradiation at 473 K for the metals studied. The vacancy clusters were not formed in V-4Cr-4Ti irradiated at 473 K in the range of 10 -6 -10 -3 dpa. In F82H irradiated at 473 K, the defect evolution was prevented by pre-existing defects. The mean lifetime of positrons in fission neutron irradiation was longer than that in fusion neutron irradiation in V-4Cr-4Ti at 473 K. It was interpreted that more closely situated subcascades were formed in the fusion neutron irradiation and subcascades interacted with each other, and consequently the vacancy clusters did not grow larger.

21 CFR 82.1255 - D&C Orange No. 5.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false D&C Orange No. 5. 82.1255 Section 82.1255 Food and... PROVISIONALLY LISTED COLORS AND SPECIFICATIONS Drugs and Cosmetics § 82.1255 D&C Orange No. 5. (a) The color additive D&C Orange No. 5 shall conform in identity and specifications to the requirements of § 74.1255(a...

21 CFR 82.1254 - D&C Orange No. 4.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false D&C Orange No. 4. 82.1254 Section 82.1254 Food and... PROVISIONALLY LISTED COLORS AND SPECIFICATIONS Drugs and Cosmetics § 82.1254 D&C Orange No. 4. The color additive D&C Orange No. 4 shall conform in identity and specifications to the requirements of § 74.1254(a...

Recommended activation detector cross sections (RNDL-82)

International Nuclear Information System (INIS)

Bondars, Kh.Ya.; Lapenas, A.A.

1984-01-01

The results of the comparison between measured and calculated average cross sections in 5 benchmark experiments are presented. Calculations have been based on the data from 10 libraries of evaluated cross sections. The recommended library (RNDL-82) of the activation detector cross sections has been created on the basis of the comparison. RNDL-82, including 26 reactions, and the basic characteristics of the detectors are presented. (author)

Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome

International Nuclear Information System (INIS)

Mimori, Tsuneyo; Ohosone, Yasuo; Hama, Nobuaki; Suwa, Akira; Akizuki, Masashi; Homma, Mitsuo; Griffith, A.J.; Hardin, J.A.

1990-01-01

Anti-Ku (p70/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA. In the present study, the authors isolated full-length cDNAs that encode the 80-kDa Ku subunit. Initial screening of a human spleen cDNA library with anti-Ku antibodies yielded a cDNA of 1.0 kilobase (kb) (termed K71) encoding a portion of the 80-kDa Ku polypeptide (identification based on immunological criteria). In RNA blots, this cDNA hybridized with two mRNAs of 3.4 and 2.6 kb. In vitro transcription and translation experiments produced an immunoprecipitable polypeptide which comigrated with the 80-kDa Ku subunit. The Ku80-6 cDNA proved to be 3304 nucleotides in length, with an additional poly(A) tail, closely approximating the size of the larger mRNA. It contains a single long open reading frame encoding 732 amino acids. The putative polypeptide has a high content of acidic amino acids and a region with periodic repeat of leucine in every seventh position which may form the leucine zipper structure. In genomic DNA blots, probes derived from the opposite ends of cDNA Ku80-6 hybridized with several nonoverlapping restriction fragments from human leukocyte DNA, indicating that the gene encoding the 80-kDa Ku polypeptide is divided into several exons by intervening sequences

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096.

Science.gov (United States)

Kumar, C Ganesh; Kamle, Avijeet; Kamal, Ahmed

2013-01-01

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE-1, AtFAE-2, and AtFAE-3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH-dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5-8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca(2+), K(+), and Mg(2+) stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k(cat) /K(m)) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE-1 belonged to type A while AtFAE-2 and AtFAE-3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers.

A Bayesian framework based on a Gaussian mixture model and radial-basis-function Fisher discriminant analysis (BayGmmKda V1.1 for spatial prediction of floods

Directory of Open Access Journals (Sweden)

D. Tien Bui

2017-09-01

Full Text Available In this study, a probabilistic model, named as BayGmmKda, is proposed for flood susceptibility assessment in a study area in central Vietnam. The new model is a Bayesian framework constructed by a combination of a Gaussian mixture model (GMM, radial-basis-function Fisher discriminant analysis (RBFDA, and a geographic information system (GIS database. In the Bayesian framework, GMM is used for modeling the data distribution of flood-influencing factors in the GIS database, whereas RBFDA is utilized to construct a latent variable that aims at enhancing the model performance. As a result, the posterior probabilistic output of the BayGmmKda model is used as flood susceptibility index. Experiment results showed that the proposed hybrid framework is superior to other benchmark models, including the adaptive neuro-fuzzy inference system and the support vector machine. To facilitate the model implementation, a software program of BayGmmKda has been developed in MATLAB. The BayGmmKda program can accurately establish a flood susceptibility map for the study region. Accordingly, local authorities can overlay this susceptibility map onto various land-use maps for the purpose of land-use planning or management.

Human biodistribution and radiation dosimetry of 82Rb.

Science.gov (United States)

Senthamizhchelvan, Srinivasan; Bravo, Paco E; Esaias, Caroline; Lodge, Martin A; Merrill, Jennifer; Hobbs, Robert F; Sgouros, George; Bengel, Frank M

2010-10-01

Prior estimates of radiation-absorbed doses from (82)Rb, a frequently used PET perfusion tracer, yielded discrepant results. We reevaluated (82)Rb dosimetry using human in vivo biokinetic measurements. Ten healthy volunteers underwent dynamic PET/CT (6 contiguous table positions, each with separate (82)Rb infusion). Source organ volumes of interest were delineated on the CT images and transferred to the PET images to obtain time-integrated activity coefficients. Radiation doses were estimated using OLINDA/EXM 1.0. The highest mean absorbed organ doses (μGy/MBq) were observed for the kidneys (5.81), heart wall (3.86), and lungs (2.96). Mean effective doses were 1.11 ± 0.22 and 1.26 ± 0.20 μSv/MBq using the tissue-weighting factors of the International Commission on Radiological Protection (ICRP), publications 60 and 103, respectively. Our current (82)Rb dosimetry suggests reasonably low radiation exposure. On the basis of this study, a clinical (82)Rb injection of 2 × 1,480 MBq (80 mCi) would result in a mean effective dose of 3.7 mSv using the weighting factors of the ICRP 103-only slightly above the average annual natural background exposure in the United States (3.1 mSv).

Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein.

Science.gov (United States)

Pan, Jingxi; Han, Jun; Borchers, Christoph H; Konermann, Lars

2009-09-09

Amide H/D exchange (HDX) mass spectrometry (MS) is widely used for protein structural studies. Traditionally, this technique involves protein labeling in D(2)O, followed by acid quenching, proteolytic digestion, and analysis of peptide deuteration levels by HPLC/MS. There is great interest in the development of alternative HDX approaches involving the top-down fragmentation of electrosprayed protein ions, instead of relying on enzymatic cleavage and solution-phase separations. A number of recent studies have demonstrated that electron capture dissociation (ECD) results in fragmentation of gaseous protein ions with little or no H/D scrambling. However, the successful application of this approach for in-depth protein conformational studies has not yet been demonstrated. The current work uses horse myoglobin as a model system for assessing the suitability of HDX-MS with top-down ECD for experiments of this kind. It is found that ECD can pinpoint the locations of protected amides with an average resolution of less than two residues for this 17 kDa protein. Native holo-myoglobin (hMb) shows considerable protection from exchange in all of its helices, whereas loops are extensively deuterated. Fraying is observable at some helix termini. Removal of the prosthetic heme group from hMb produces apo-myoglobin (aMb). Both hMb and aMb share virtually the same HDX protection pattern in helices A-E, whereas helix F is unfolded in aMb. In addition, destabilization is evident for some residues close to the beginning of helix G, the end of helix H, and the C-terminus of the protein. The structural changes reported herein are largely consistent with earlier NMR data for sperm whale myoglobin, although small differences between the two systems are evident. Our findings demonstrate that the level of structural information obtainable with top-down ECD for small to medium-sized proteins considerably surpasses that of traditional HDX-MS experiments, while at the same time greatly reducing

Differential Recognition of CD1d-[alpha]-Galactosyl Ceramide by the V[beta]8.2 and V[beta]7 Semi-invariant NKT T Cell Receptors

Energy Technology Data Exchange (ETDEWEB)

Pellicci, Daniel G.; Patel, Onisha; Kjer-Nielsen, Lars; Pang, Siew Siew; Sullivan, Lucy C.; Kyparissoudis, Konstantinos; Brooks, Andrew G.; Reid, Hugh H.; Gras, Stephanie; Lucet, Isabelle S.; Koh, Ruide; Smyth, Mark J.; Mallevaey, Thierry; Matsuda, Jennifer L.; Gapin, Laurent; McCluskey, James; Godfrey, Dale I.; Rossjohn, Jamie; PMCI-A; Monash; UCHSC; Melbourne

2009-09-02

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR{alpha} chain is typically invariant, the {beta} chain expression is more diverse, where three V{beta} chains are commonly expressed in mice. We report the structures of V{alpha}14-V{beta}8.2 and V{alpha}14-V{beta}7 NKT TCRs in complex with CD1d-{alpha}-galactosylceramide ({alpha}-GalCer) and the 2.5 {angstrom} structure of the human NKT TCR-CD1d-{alpha}-GalCer complex. Both V{beta}8.2 and V{beta}7 NKT TCRs and the human NKT TCR ligated CD1d-{alpha}-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V{beta} domains of the V{beta}8.2 and V{beta}7 NKT TCR-CD1d complexes resulted in altered TCR{beta}-CD1d-mediated contacts and modulated recognition mediated by the invariant {alpha} chain. Mutagenesis studies revealed the differing contributions of V{beta}8.2 and V{beta}7 residues within the CDR2{beta} loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V{beta} usage in NKT cells.

Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

DEFF Research Database (Denmark)

Rosorius, O; Issinger, O G; Braulke, T

1993-01-01

The cytoplasmic tail of the human 300-kDa mannose 6-phosphate receptor (MPR 300-CT) is an excellent substrate for casein kinase II in vitro. The phosphorylated MPR 300-CT was cross-linked by means of bis(sulfosuccinimidyl)suberate mainly to a cytosolic protein of 35 kDa (referred to as TIP 35...... with TIP 35 is phosphorylation-specific. Furthermore, TIP 35 was only cross-linked to the MPR 300-CT phosphorylated by casein kinase II whereas the MPR 300-CT phosphorylated by protein kinase A failed to cross-link to TIP 35. These results indicate that the cytoplasmic tail of the MPR 300 interacts...

Correlation between phosphorylation level of a hippocampal 86kDa protein and extinction of a behaviour in a model of Wernicke-Korsakoff syndrome.

Science.gov (United States)

Pires, Rita G W; Pereira, Sílvia R C; Carvalho, Fabiana M; Oliveira-Silva, Ieda F; Ferraz, Vany P; Ribeiro, Angela M

2007-06-04

The effects of chronic ethanol and thiamine deficiency, alone or associated, on hippocampal protein phosphorylation profiles ranging in molecular weight from 30 to 250kDa molecular weight, in stimulated (high K(+) concentration) and unstimulated (basal) conditions were investigated. These treatments significantly changed the phosphorylation level of an 86kDa phosphoprotein. Thiamine deficiency, but not chronic ethanol, induced a decrease in a behavioural extinction index, which is significantly correlated to the phosphorylation level of the p86 protein. These data add to and extend previous findings by our laboratory implicating the involvement of hippocampal neurotransmission components in extinction of a behaviour which involves learning of environmental spatial cues.

Hydrogen permeation behavior through F82H at high temperature

Energy Technology Data Exchange (ETDEWEB)

Matsuda, S.; Katayama, K.; Shimozori, M.; Fukada, S. [Interdisciplinary Graduate School of Engineering Science, Kyushu University, Kyushu (Japan); Ushida, H. [Energy Science and Engineering, Faculty of Engineering, Kyushu University, Kyushu (Japan); Nishikawa, M. [Malaysia-Japan International Institute of Technology, UTM, Kuala Lumpur (Malaysia)

2015-03-15

F82H is a primary candidate of structural material and coolant pipe material in a blanket of a fusion reactor. Understanding tritium permeation behavior through F82H is important. In a normal operation of a fusion reactor, the temperature of F82H will be controlled below 550 C. degrees because it is considered that F82H can be used up to 30,000 hours at 550 C. degrees. However, it is necessary to assume the situation where F82H is heated over 550 C. degrees in a severe accident. In this study, hydrogen permeation behavior through F82H was investigated in the temperature range from 500 to 800 C. degrees. In some cases, water vapor was added in a sample gas to investigate an effect of water vapor on hydrogen permeation. The permeability of hydrogen in the temperature range from 500 to 700 C. degrees agreed well with the permeability reported by E. Serra et al. The degradation of the permeability by water vapor was not observed. After the hydrogen permeation reached in a steady state at 700 C. degrees, the F82H sample was heated to 800 C. degrees. The permeability of hydrogen through F82H sample which was once heated up to 800 C. degrees was lower than that of the original one. (authors)

75 FR 80744 - Airworthiness Directives; McDonnell Douglas Corporation Model DC-9-81 (MD-81), DC-9-82 (MD-82...

Science.gov (United States)

2010-12-23

...-1203; Directorate Identifier 2010-NM-168-AD] RIN 2120-AA64 Airworthiness Directives; McDonnell Douglas... amends Sec. 39.13 by adding the following new airworthiness directive (AD): McDonnell Douglas Corporation... Douglas Corporation Model DC-9-81 (MD-81), DC-9-82 (MD-82), DC-9-83 (MD-83), DC-9-87 (MD-87) and MD-88...

9 CFR 82.15 - Replacement birds and poultry.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Replacement birds and poultry. 82.15... AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DIS- EASE (END) AND CHLAMYDIOSIS Exotic Newcastle Disease (END) § 82.15 Replacement birds and poultry. Birds...

Inhibition of Bifidobacterium Cell Wall 51.74 kDa Adhesin Isolated from Infants Feces Towards Adhesion of Enteric Phatogen E. coli on Enterocyte Balb/C Mice

Directory of Open Access Journals (Sweden)

I Sukrama

2012-01-01

Full Text Available Objectives: To determine 51.74 kDa adhesin of Bifidobacterium sp cell wall isolated from infants feces as an anti adhesion of E. coli on enterocyte mice. Methods: Randomized Posttest-Only Control Group Design was employed to investigate adherence ability of this adhesin towards E.coli adhesion on mice entherocyte. Results: In this research, it was obtained, that the 51.74 kDa adhesin cell wall of Bifidobacterium sp has an ability to inhibit adhesion of E. coli on mice enterocyte. The ability was increased as an increase of adhsein concentration. Conclusions: that can be drawn from this research is the finding of 51.74 kDa adhesin cell wall of Bifidobacterium sp isolated from infants feces that can inhibit adhseion of E. coli on mice enterocyte. Future work that can be carried out are further researches concerning whether these protein can be applied to inhibit adherence of other pathogen bacteria

Purification and characterization of a protease-resistant phytase of Aspergillus oryzae SBS50 whose properties make it exceptionally useful as a feed supplement.

Science.gov (United States)

Sapna; Singh, Bijender

2017-10-01

An extracellular phytase of Aspergillus oryzae SBS50 was purified to homogeneity using ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Purified phytase has a monomeric molecular mass of ∼80kDa exhibiting its optimal activity at pH 5.0 and 50°C with a T 1/2 of 300min at 50°C. Phytase of A. oryzae displayed broad substrate specificity with V max and K m values of 58.82μmol/ml/min and 1.14mM, respectively, for calcium phytate. Purity and homogeneity of the phytase was confirmed by high performance liquid chromatography and MALDI-TOF analysis revealed the identification of a peptide showing homology with acid phosphatase of Aspergillus oryzae RIB40. Among the inhibitors, 2,3-butanedione and sodium molybdate significantly inhibited the enzyme activity. Phytase of A. oryzae showed protease-resistance and was more stable during storage at 4°C and -20°C as compared to room temperature. Among all the feed samples, mustard oil cake was dephytinized more efficiently than other feed samples. These unique properties suggested that the phytase has the potential to be useful as an animal feed supplement. Copyright © 2017 Elsevier B.V. All rights reserved.

42 CFR 408.82 - Conditions for group billing.

Science.gov (United States)

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Conditions for group billing. 408.82 Section 408.82 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE... for group billing. CMS agrees to a group billing arrangement only if the following conditions are met...

42 CFR 102.82 - Calculation of death benefits.

Science.gov (United States)

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Calculation of death benefits. 102.82 Section 102... COMPENSATION PROGRAM Calculation and Payment of Benefits § 102.82 Calculation of death benefits. (a... paragraph (d) of this section for the death benefit available to dependents. (2) Deceased person means an...

Allergen-Specific Immunotherapy with Monomeric Allergoid in a Mouse Model of Atopic Dermatitis

Science.gov (United States)

Babakhin, Alexander; Andreev, Sergey; Nikonova, Alexandra; Shilovsky, Igor; Buzuk, Andrey; Elisyutina, Olga; Fedenko, Elena; Khaitov, Musa

2015-01-01

Atopic dermatitis (AD) is a widespread and difficult to treat allergic skin disease and is a tough challenge for healthcare. In this study, we investigated whether allergen-specific immunotherapy (ASIT) with a monomeric allergoid obtained by succinylation of ovalbumin (sOVA) is effective in a mouse model of atopic dermatitis. An experimental model of AD was reproduced by epicutaneous sensitization with ovalbumin (OVA). ASIT was performed with subcutaneous (SC) administration of increasing doses of OVA or sOVA. The levels of anti-OVA antibodies, as well as cytokines, were detected by ELISA. Skin samples from patch areas were taken for histologic examination. ASIT with either OVA or sOVA resulted in a reduction of both the anti-OVA IgE level and the IgG1/IgG2a ratio. Moreover, ASIT with sOVA increased the IFN-γ level in supernatants after splenocyte stimulation with OVA. Histologic analysis of skin samples from the sites of allergen application showed that ASIT improved the histologic picture by decreasing allergic inflammation in comparison with untreated mice. These data suggest that ASIT with a succinylated allergen represents promising approach for the treatment of AD. PMID:26275152

Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization.

Science.gov (United States)

Osterbye, T; Jørgensen, K H; Fredman, P; Tranum-Jensen, J; Kaas, A; Brange, J; Whittingham, J L; Buschard, K

2001-06-01

Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8--A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Directory of Open Access Journals (Sweden)

Nida Toufiq

Full Text Available Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L. cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form.

Science.gov (United States)

Martín, Mariana; Rius, Sebastián Pablo; Podestá, Florencio Esteban

2011-06-01

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

An economical method for production of (2H, (13CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome.

Directory of Open Access Journals (Sweden)

Algirdas Velyvis

Full Text Available NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13CH(3 methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2H; β-(2H;γ-(13C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr (13C,(1H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[(13C,(1H]-Thr.

Expression of 65-kDa oncofetal protein in experimental hepatoma after antivancer therapy

International Nuclear Information System (INIS)

Mirowski, M.; Rozalski, M.; Krajewska, U.; Wierbicki, R.; Hanausek, M.

1997-01-01

We have tested the expression of 65-kDa oncofetal protein (p65) after combined treatment with menadione and methotrexate in hamsters transplanted with Kirkman-Robbins hepatoma. The treatment of tumor-bearing animals with these compounds significantly inhibited both the tumor development and the expression of p65. This inhibition in tumor tissue was calculated from densitograms of Western blots. The inhibition of p65 was also confirmed in the serum of hepatoma bearing animals by using solid-phase radioimmunoassay (RIA) to quantify the specificity of polyclonal antibodies to fetal p65 molecules. Additionally, p65 was shown to localize both in cytoplasm an in the nuclear extracts prepared from hepatoma tissue. (author)

Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble

DEFF Research Database (Denmark)

Christgau, S; Schierbeck, H; Aanstoot, H J

1991-01-01

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic...... acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular...

Residual stresses

International Nuclear Information System (INIS)

Sahotra, I.M.

2006-01-01

The principal effect of unloading a material strained into the plastic range is to create a permanent set (plastic deformation), which if restricted somehow, gives rise to a system of self-balancing within the same member or reaction balanced by other members of the structure., known as residual stresses. These stresses stay there as locked-in stresses, in the body or a part of it in the absence of any external loading. Residual stresses are induced during hot-rolling and welding differential cooling, cold-forming and extruding: cold straightening and spot heating, fabrication and forced fitting of components constraining the structure to a particular geometry. The areas which cool more quickly develop residual compressive stresses, while the slower cooling areas develop residual tensile stresses, and a self-balancing or reaction balanced system of residual stresses is formed. The phenomenon of residual stresses is the most challenging in its application in surface modification techniques determining endurance mechanism against fracture and fatigue failures. This paper discusses the mechanism of residual stresses, that how the residual stresses are fanned and what their behavior is under the action of external forces. Such as in the case of a circular bar under limit torque, rectangular beam under limt moment, reclaiming of shafts welds and peening etc. (author)

Identification of phosphoproteins coupled to initiation of motility in live epididymal mouse sperm

Science.gov (United States)

Tash, J. S.; Bracho, G. E.

1998-01-01

A method for collecting live immotile cauda epididymal mouse sperm that initiate motility by dilution into an activation buffer is described. Sperm in collection buffer showed low percent motility (MOT) and population progression (PRG) that increased 10-fold and 9-fold, respectively, during the first 2 min after dilution into activation buffer. Western phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) analysis revealed a 120 kDa protein that markedly increased in pT content during initiation of motility and may be related to FP130, the motility-coupled axonemal protein of sea urchin sperm. A prominent 82 kDa protein that was pS and pT-phosphorylated in immotile and motile sperm is likely the fibrous sheath component AKAP82 that is phosphorylated during spermatogenesis. Analysis of live human sperm also identified a prominent 120 kDa pT protein. Thus it appears that phosphorylation of FP130 and related 120 kDa proteins in mouse, and perhaps human sperm, represent common targets during motility initiation in sperm. Copyright 1998 Academic Press.

Structure of the Putative 32 kDa Myrosinase Binding Protein from Arabidopsis (At3g16450.1) Determined by SAIL-NMR

Science.gov (United States)

Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira Mei; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, JunGoo; Güntert, Peter; Aceti, David J.; Markley, John L.; Kainosho, Masatsune

2009-01-01

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme, myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniformly 13C/15N labeling methods, we used our stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected with the SAIL-protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at the low concentration (0.2 mM) of the protein product. We collected additional NOESY data and solved the three-dimensional structure with the CYANA software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent, but similar, lectin-fold domains composed of three β-sheets. PMID:19021763

Development, validation and evaluation of an analytical method for the determination of monomeric and oligomeric procyanidins in apple extracts.

Science.gov (United States)

Hollands, Wendy J; Voorspoels, Stefan; Jacobs, Griet; Aaby, Kjersti; Meisland, Ane; Garcia-Villalba, Rocio; Tomas-Barberan, Francisco; Piskula, Mariusz K; Mawson, Deborah; Vovk, Irena; Needs, Paul W; Kroon, Paul A

2017-04-28

There is a lack of data for individual oligomeric procyanidins in apples and apple extracts. Our aim was to develop, validate and evaluate an analytical method for the separation, identification and quantification of monomeric and oligomeric flavanols in apple extracts. To achieve this, we prepared two types of flavanol extracts from freeze-dried apples; one was an epicatechin-rich extract containing ∼30% (w/w) monomeric (-)-epicatechin which also contained oligomeric procyanidins (Extract A), the second was an oligomeric procyanidin-rich extract depleted of epicatechin (Extract B). The parameters considered for method optimisation were HPLC columns and conditions, sample heating, mass of extract and dilution volumes. The performance characteristics considered for method validation included standard linearity, method sensitivity, precision and trueness. Eight laboratories participated in the method evaluation. Chromatographic separation of the analytes was best achieved utilizing a Hilic column with a binary mobile phase consisting of acidic acetonitrile and acidic aqueous methanol. The final method showed linearity for epicatechin in the range 5-100μg/mL with a correlation co-efficient >0.999. Intra-day and inter-day precision of the analytes ranged from 2 to 6% and 2 to 13% respectively. Up to dp3, trueness of the method was >95% but decreased with increasing dp. Within laboratory precision showed RSD values <5 and 10% for monomers and oligomers, respectively. Between laboratory precision was 4 and 15% (Extract A) and 7 and 30% (Extract B) for monomers and oligomers, respectively. An analytical method for the separation, identification and quantification of procyanidins in an apple extract was developed, validated and assessed. The results of the inter-laboratory evaluation indicate that the method is reliable and reproducible. Copyright © 2017. Published by Elsevier B.V.

40 CFR 82.152 - Definitions.

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2010-07-01

..., restaurants and other food service establishments. Cold storage includes the equipment used to store meat... apprentice in service, maintenance, repair, or disposal of appliances with the U.S. Department of Labor's... Administrator pursuant to § 82.160. Certified refrigerant recovery or recycling equipment means equipment...

40 CFR 82.32 - Definitions.

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2010-07-01

... Administrator pursuant to § 82.38. (b) Approved refrigerant recycling equipment means equipment certified by the... equipment extracts and recycles refrigerant or extracts refrigerant for recycling on-site or reclamation off... for reclamation. (2) Refrigerant from reclamation facilities that is used for the purpose of...

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

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José Andrés Morgado-Díaz

2005-07-01

Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

Science.gov (United States)

Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

1989-11-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts

Science.gov (United States)

Xu, Congfeng; Zhang, Yanhui H.; Thangavel, Muthusamy; Richardson, Mekel M.; Liu, Li; Zhou, Bin; Zheng, Yi; Ostrom, Rennolds S.; Zhang, Xin A.

2009-01-01

Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.—Xu, C., Zhang, Y. H., Thangavel, M., Richardson, M. M., Liu, L., Zhou, B., Zheng, Y., Ostrom, R. S., Zhang, X. A. CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts. PMID:19497983

Photomonomerization of pyrimidine dimers by indoles and proteins

Energy Technology Data Exchange (ETDEWEB)

Chen, J.; Huang, C.W.; Hinman, L.; Gordon, M.P.; Deranleau, D.A.

1976-01-01

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wavelengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases. Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the /sup 14/C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed. Although indoles are able to split the dimers in RNA, they fail to photoreactivate uv-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactive viral RNA when irradiated at 313 nm, possibly because of side reactions.

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

Directory of Open Access Journals (Sweden)

Martin Zühlsdorf

2015-07-01

Full Text Available Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137; both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

Management of Biogas spent slurry for hastening the composting of agro residues

Directory of Open Access Journals (Sweden)

G. S. Geeta

2015-04-01

Full Text Available The demand for energy and the fertilizers are ever increasing. Organic farming has many advantages looking to the environment pollution, unproductive soil, less yields etc. By installation of a biogas plant serves both the purposes of meeting the fuel as well as obtaining manures. The organic manures need to be added in bulk to meet the nutrient demands of the crop as it is not in concentrated form like chemical fertilizers. Hence, biogas spent slurry is the best alternate for hastening the compost preparation of abundantly available crop residues as well as obtaining enriched compost as conventional method takes long time. Moreover, slurry is composed of major nutrients besides enzymes and a rich microflora. Based on the preliminary results, the present study was conducted at farmer’s field to know whether slurry could be used for degradation of agro residues. One ton of crop residues that included banana waste, sunflower and maize waste, leaf litter of horticultural crops were inoculated individually with 60 L of spent slurry along with consortia of degrading fungi and P-solubilising bacteria. After a retention period of 60 days, nutrients were analysed. The cultures along with slurry indicated 1.5 - 1.96% N with reduction in C:N ratio between 1.6 - 1.82. The micronutrients also increased. Thus, it was concluded that efficient use of spent slurry can be made besides utilising the crop residues and the product for organic cultivation.

Carbamylated monomeric allergoids as a therapeutic option for sublingual immunotherapy of dust mite- and grass pollen-induced allergic rhinoconjunctivitis: a systematic review of published trials with a meta-analysis of treatment using Lais® tablets.

Science.gov (United States)

Mösges, R; Ritter, B; Kayoko, G; Allekotte, S

2010-10-01

Lais® allergoid tablets contain allergens that are modified by carbamylation. Due to their modified chemical structure, they are suitable for sublingual immunotherapy (SLIT) (13, 16, 17, 24). Based on their small molecule size of 12 to 40 kDa, they can be easily absorbed via the oral mucosa (1). In this review, we studied the efficacy of SLIT with carbamylated monomeric allergoid tablets in the treatment of grass pollen- and dust mite-induced allergic rhinoconjunctivitis on the basis of symptom and medication score improvements. Following a selective internet and databank search, six trials-some placebo-controlled-regarding the treatment of grass pollen- (n = 266) and dust mite-induced (n = 241) allergic rhinoconjunctivitis were used to draw conclusions regarding the clinical efficacy of allergoid tablets. The primary endpoints in these trials were decreases in the need for allergy medications and/or reductions in the occurrence of rhinoconjunctivitis symptoms. Data was recorded from patient diaries regarding their symptoms and medications used and conclusions were then drawn about the effectiveness and tolerabieity of Lais® tablets. The average improvement in symptom score in three trials of grass pollen allergy treatment was 34% in comparison to the placebo group. The treatment of dust mite-induced rhinoconjunctivitis produced an average symptom score improvement of 22% compared to the placebo or control groups. The intake of symptomatic rescue medication during allergoid tablet therapy declined. Treatment of grass pollen allergies and dust mite-induced rhinoconjunctivitis showed an average medication score improvement of 49% and 24%, respectively. Few side effects were documented in the trials and predominantly local effects were observed. Severe systemic side effects did not occur. On the basis of the trial results summarized in this review, we suggest that SLIT using Lais® sublingual tablets is an effective and well-tolerated form of treatment.

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells

International Nuclear Information System (INIS)

Li, Xiting; Shu, Rong; Liu, Dali; Jiang, Shaoyun

2010-01-01

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 μg/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.

A 170kDa multi-domain cystatin of Fasciola gigantica is active in the male reproductive system.

Science.gov (United States)

Geadkaew, Amornrat; Kosa, Nanthawat; Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

2014-09-01

Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often 120kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization. Copyright © 2014 Elsevier B.V. All rights reserved.

The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing

Science.gov (United States)

Fang, Qing; Indzhykulian, Artur A; Mustapha, Mirna; Riordan, Gavin P; Dolan, David F; Friedman, Thomas B; Belyantseva, Inna A; Frolenkov, Gregory I; Camper, Sally A; Bird, Jonathan E

2015-01-01

The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture. DOI: http://dx.doi.org/10.7554/eLife.08627.001 PMID:26302205

10 CFR 431.82 - Definitions concerning commercial packaged boilers.

Science.gov (United States)

2010-01-01

... gases. Manufacturer of a commercial packaged boiler means any person who manufactures, produces... 10 Energy 3 2010-01-01 2010-01-01 false Definitions concerning commercial packaged boilers. 431.82... COMMERCIAL AND INDUSTRIAL EQUIPMENT Commercial Packaged Boilers § 431.82 Definitions concerning commercial...

Residual stresses

International Nuclear Information System (INIS)

Macherauch, E.

1978-01-01

Residual stresses are stresses which exist in a material without the influence of external powers and moments. They come into existence when the volume of a material constantly changes its form as a consequence of mechanical, thermal, and/or chemical processes and is hindered by neighbouring volumes. Bodies with residual stress are in mechanical balance. These residual stresses can be manifested by means of all mechanical interventions disturbing this balance. Acoustical, optical, radiological, and magnetical methods involving material changes caused by residual stress can also serve for determining residual stress. Residual stresses have an ambivalent character. In technical practice, they are feared and liked at the same time. They cause trouble because they can be the cause for unexpected behaviour of construction elements. They are feared since they can cause failure, in the worst case with catastrophical consequences. They are appreciated, on the other hand, because, in many cases, they can contribute to improvements of the material behaviour under certain circumstances. But they are especially liked for their giving convenient and (this is most important) mostly uncontrollable explanations. For only in very few cases we have enough knowledge and possibilities for the objective evaluation of residual stresses. (orig.) [de

Effects of food processing on pesticide residues in fruits and vegetables: a meta-analysis approach.

Science.gov (United States)

Keikotlhaile, B M; Spanoghe, P; Steurbaut, W

2010-01-01

Pesticides are widely used in food production to increase food security despite the fact that they can have negative health effects on consumers. Pesticide residues have been found in various fruits and vegetables; both raw and processed. One of the most common routes of pesticide exposure in consumers is via food consumption. Most foods are consumed after passing through various culinary and processing treatments. A few literature reviews have indicated the general trend of reduction or concentration of pesticide residues by certain methods of food processing for a particular active ingredient. However, no review has focused on combining the obtained results from different studies on different active ingredients with differences in experimental designs, analysts and analysis equipment. In this paper, we present a meta-analysis of response ratios as a possible method of combining and quantifying effects of food processing on pesticide residue levels. Reduction of residue levels was indicated by blanching, boiling, canning, frying, juicing, peeling and washing of fruits and vegetables with an average response ratio ranging from 0.10 to 0.82. Baking, boiling, canning and juicing indicated both reduction and increases for the 95% and 99.5% confidence intervals. Copyright 2009 Elsevier Ltd. All rights reserved.

22 CFR 226.82 - Program income.

Science.gov (United States)

2010-04-01

... Relations AGENCY FOR INTERNATIONAL DEVELOPMENT ADMINISTRATION OF ASSISTANCE AWARDS TO U.S. NON-GOVERNMENTAL ORGANIZATIONS Additional Provisions For Awards to Commercial Organizations § 226.82 Program income. The... commercial organization. ...

Preliminary results of the round-robin testing of F82H

Energy Technology Data Exchange (ETDEWEB)

Shiba, K.; Yamanouchi, N.; Tohyama, A.

1996-10-01

Preliminary results of metallurgical, physical and mechanical properties of low activation ferritic steel F82H (IEA heat) were obtained in the round-robin test in Japan. The properties of IEA heat F82H were almost the same as the original F82H.

42 CFR 82.0 - Background information on this part.

Science.gov (United States)

2010-10-01

... 82.0 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY... EMPLOYEES OCCUPATIONAL ILLNESS COMPENSATION PROGRAM ACT OF 2000 Introduction § 82.0 Background information on this part. The Energy Employees Occupational Illness Compensation Program Act (EEOICPA), 42 U.S.C...

Percutaneous closure of postoperative ventricular septal residual left-to-right shunt with the China made device

International Nuclear Information System (INIS)

Qin Yongwen; Zhao Xianxian; Wu Hong; Ding Jijun; Cao Jiang; Zheng Xin

2007-01-01

Objective: To evaluate the feasibility and efficacy of percutaneous transcatheter closure of residual ventricular septal defect (VSD)after surgical closure using China made-nitinol VSD device. Methods: Transcatheter closure was attempted in 11 patients (5 males, 6 females)with a residual VSD following surgical closure. The mean residual VSD narrowest diameter was(5.82 ± 2.09) mm (range from 3 to 9 mm)by echocardiography. A 6 F-9 F delivery sheath was advanced across the residual VSD over a guidewire from femoral vein to deploy the occluder under guidance of left ventriculography and transthoracic echocardiography. Results: The left ventriculography showed membranous part aneurism-like residual VSD in 8 patients and funnel type in 3 cases. There were multiple outlet in 5 cases and one outlets in 6 cases, with mean residual VSD narrowest diameter of (6.09 ± 1.58) mm (range from 3 to 9 mm)measured by left ventriculography. The diameter of occluder was (9.18 ± 2.79) mm (range from 8 to 12 mm). Complete closure of the defect was obtained in 10 cases, and another small residual shunt still remained in one case who had four outlets been treated by 2 occluders. No aortic valvular regurgitation occured in all patients except 1 patient presented complete atrioventricular block within 3 days after the procedure and recovered 2 weeks later with intravenous steroids therapy; and no other complications occured. The fluoroscopy time was(16.91 ± 4.23) min (range from 8 to 30 min). During follow-up from 1 m - 4 y, only 1 case showed residual shunt, and the other had no episodes of endocarditis, thromboembolism, hemolysis, infectious endocarditis, displacement of the occluder and aortic valvular regurgitation. Conclusion: Transcatheter closure of postoperative ventricular septal residual left-to-right shunt with China made-nitinol occluder is safe and effective. (authors)

Evaluation of residue-residue contact predictions in CASP9

KAUST Repository

Monastyrskyy, Bohdan

2011-01-01

This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures being the precision in recognizing contacts and the difference between the distribution of distances in the subset of predicted contact pairs versus all pairs of residues in the structure. The emphasis is placed on the prediction of long-range contacts (i.e., contacts between residues separated by at least 24 residues along sequence) in target proteins that cannot be easily modeled by homology. Although there is considerable activity in the field, the current analysis reports no discernable progress since CASP8.

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Energy Technology Data Exchange (ETDEWEB)

Ren, Yunli; Wang, Jianji; Liu, Zhen; Ren, Yunlai; Li, Guozhi [School of Chemical Engineering and Pharmaceutics, Henan University of Science and Technology, Luoyang 471039, Henan (China)

2009-12-15

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose. (author)

Lipoamino acid-based micelles as promising delivery vehicles for monomeric amphotericin B.

Science.gov (United States)

Serafim, Cláudia; Ferreira, Inês; Rijo, Patrícia; Pinheiro, Lídia; Faustino, Célia; Calado, António; Garcia-Rio, Luis

2016-01-30

Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug amphotericin B (AmB). The micellar solubilisation of AmB by a gemini lipoamino acid (LAA) derived from cysteine and its equimolar mixtures with the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC), as well as the aggregation sate of the drug in the micellar systems, was studied under biomimetic conditions (phosphate buffered-saline, pH 7.4) using UV-vis spectroscopy. Pure surfactant systems and equimolar mixtures were characterized by tensiometry and important parameters were determined, such as critical micelle concentration (CMC), surface tension at the CMC (γCMC), maximum surface excess concentration (Γmax), and minimum area occupied per molecule at the water/air interface (Amin). Rheological behaviour from viscosity measurements at different shear rates was also addressed. Solubilisation capacity was quantified in terms of molar solubilisation ratio (χ), micelle-water partition coefficient (KM) and Gibbs energy of solubilisation (ΔGs°). Formulations of AmB in micellar media were compared in terms of drug loading, encapsulation efficiency, aggregation state of AmB and in vitro antifungal activity against Candida albicans. The LAA-containing micellar systems solubilise AmB in its monomeric and less toxic form and exhibit in vitro antifungal activity comparable to that of the commercial formulation Fungizone. Copyright © 2015 Elsevier B.V. All rights reserved.

Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

Science.gov (United States)

Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

2017-12-01

Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent

A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

OpenAIRE

Yannariello-Brown, J; Madri, J A

1990-01-01

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the se...

40 CFR 82.154 - Prohibitions.

Science.gov (United States)

2010-07-01

... substance for use in motor vehicle air conditioners in accordance with 40 CFR part 82, subpart G; (4) The... wholly or in part of a class I or class II substance for use in motor vehicle air conditioners pursuant... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PROTECTION OF...

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

Science.gov (United States)

Permyakov, Serge E; Pershikova, Irina V; Khokhlova, Tatyana I; Uversky, Vladimir N; Permyakov, Eugene A

2004-05-18

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.

The 82-plex plasma protein signature that predicts increasing inflammation

DEFF Research Database (Denmark)

Tepel, Martin; Beck, Hans C; Tan, Qihua

2015-01-01

The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney....... The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P signature (P ....001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein...

High-spin states in 82Sr

International Nuclear Information System (INIS)

Baktash, C.; Halper, M.L.; Garcia Bermudez, G.J.

1989-01-01

As recent theoretical calculations that predicted the onset of superdeformation in the A ≅ 80 region, the 52 Cr( 34 S,2p2n) reaction at 130 MeV beam energy was employed to populate the high-spin states in 82 Sr. The detection system consisted of the ORNL Compton-Suppression Spectrometer System (18 Ge detectors), the Spin Spectrometer, and the 4 φ CsI Dwarf Ball of Washington University. Off-line analysis of the proton-gated data resulted in nearly 170 million Ge-Ge pairs, which were mostly due to the 2p2n channel. A decay scheme extending to spin I=27h has been established. No strong evidence for the presence of superdeformed states in 82 Sr was found in a preliminary analysis of the data. (Author) [es

7 CFR 3.82 - Procedures for salary offset: types of collection.

Science.gov (United States)

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Procedures for salary offset: types of collection. 3.82 Section 3.82 Agriculture Office of the Secretary of Agriculture DEBT MANAGEMENT Federal Salary Offset § 3.82 Procedures for salary offset: types of collection. A debt will be collected in a lump-sum...

STS-82 Pilot Scott Horowitz arrives for TCDT

Science.gov (United States)

1997-01-01

STS-82 Pilot Scott J. 'Doc' Horowitz arrives at KSCs Shuttle Landing Facility in a T-38 jet from Houston, TX. Horowitz and the other six crew members are at KSC to participate in the Terminal Countdown Demonstration Test (TCDT), a dress rehearsal for launch. The crew aboard the Space Shuttle Discovery on STS-82 will conduct the second Hubble Space Telescope servicing mission. The 10-day flight is targeted for a Feb. 11 liftoff.

Design Function and Structure of a Monomeric CLC Transporter

Energy Technology Data Exchange (ETDEWEB)

L Robertson; L Kolmakova-Partensky; C Miller

2011-12-31

Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual - the generation of the kilowatt pulses with which electric fish stun their prey - to the quotidian - the acidification of endosomes, vacuoles and lysosomes. The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl{sup -} channel and later confirmed by crystal structures of bacterial Cl{sup -}/H{sup +} antiporters, is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl{sup -} and H{sup +} antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity, we cannot declare as definitive a 'parallel-pathways picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl{sup -}/H{sup +} exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not

Primary structure of a 14 kDa basic structural protein (Lm-76) from the cuticle of the migratory locust, Locusta migratoria

DEFF Research Database (Denmark)

Andersen, Jens S.; Andersen, S O; Højrup, P

1993-01-01

The complete amino acid sequence of a 14 kDa structural protein (LM-76) isolated from pharate cuticle of the locust, Locusta migratoria, was determined by Edman degradation of the intact protein and enzymatically derived peptides. Plasma desorption and electrospray mass spectrometry was used as a...

Ultrafast polarized fluorescence measurements on monomeric and self-associated melittin

NARCIS (Netherlands)

Pandit, A.; Larsen, O.F.A.; Stokkum, van I.H.M.; Grondelle, van R.; Kraayenhof, R.; Amerongen, van H.

2003-01-01

The anisotropic and magic-angle fluorescence decay of the single tryptophan (Trp) residue of melittin, a bee venom peptide, was investigated by time-resolved fluorescence anisotropy using a streak camera setup. The peptide was dissolved either in distilled water or in Hepes/NaOH buffer containing

21 CFR 82.5 - General specifications for straight colors.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 1 2010-04-01 2010-04-01 false General specifications for straight colors. 82.5... LISTING OF CERTIFIED PROVISIONALLY LISTED COLORS AND SPECIFICATIONS General Provisions § 82.5 General specifications for straight colors. No batch of a straight color listed in subpart B, C, or D shall be certified...

Identifying Tm-C82 isomers with density functional theory calculations

International Nuclear Information System (INIS)

Zheng Limin; He Hongqing; Yang Minghui; Zeng Qun; Yang Mingli

2010-01-01

Density functional theory calculations have been performed to study the geometrical and electronic properties of endohedral metallofullerene Tm-C 82 isomers. Three energetically favorable isomers (with C s , C 2 and C 2v symmetry, respectively) are identified which are consistent with the nuclear magnetic resonance (NMR) observations. The simulated ultraviolet photoelectron spectra (UPS) based on the three structures agree well with the measurements. Particularly, the parent cage of the experimentally observed Tm-C 82 isomer with C s symmetry is newly assigned, which matches the experiments better than early assignments. In addition, strong interaction between an endohedral Tm atom and the C 82 cage is discussed and is thought to be responsible for the dramatic change in the relative stability of C 82 isomers when Tm is encapsulated.

Synthesis of 6-[18F]fluoro-PBR28, a novel radiotracer for imaging the TSPO 18 kDa with PET

International Nuclear Information System (INIS)

Damont, A.; Boisgard, R.; Kuhnast, B.; Lemee, F.; Raggiri, G.; Tavitian, B.; Dolle, F.; Boisgard, R.; Tavitian, B.; Scarf, A.M.; Scarf, A.M.; Kassiou, M.; Da Pozzo, E.; Martini, C.; Selleri, S.; Kassiou, M.; Tavitian, B.; Kassiou, M.

2011-01-01

6-Fluoro-PBR28 (N-(6-fluoro-4-phenoxypyridin-3-yl)-N-(2-methoxybenzyl)acetamide), a fluorinated analogue of the recently developed TSPO 18 kDa ligand PBR28, was synthesized and labelled with fluorine- 18. 6-Fluoro-PBR28 and its 6-chloro/6-bromo counterparts were synthesized in six chemical steps and obtained in 16%, 10% and 19% overall yields, respectively. Labelling with fluorine-18 was performed in one single step (chlorine/bromine-for-fluorine heteroaromatic substitution) using a Zymate-XP robotic system affording HPLC-purified, ready-to-inject, 6-[ 18 F]fluoro-PBR28 (≥95% radiochemically pure). Non decay-corrected overall yields were 9-10% and specific radioactivities ranged from 74 to 148 GBq/μmol. In vitro binding experiments, dynamic μPET studies performed in a rat model of acute neuro-inflammation (unilaterally, AMPA-induced, striatum-lesioned rats) and ex vivo autoradiography on the same model demonstrated the potential of 6-[ 18 F]fluoro-PBR28 to image the TSPO 18 kDa using PET. (authors)

Identification and localization of a soluble antigen, Ag2, of 136 kDa from Plasmodium falciparum in vitro cultures

DEFF Research Database (Denmark)

Jakobsen, P H; Grellier, P; Theander, T G

1991-01-01

as a duplet with molecular masses of 136 and 120 kDa when tested by immunoblotting. Immunoprecipitation experiments on Triton X-100 extracted antigens from synchronized cultures showed that the antigen was synthesized in the schizont stage. Ag2 was located near the surface of schizonts in the parasitophorous...

Crystal structure of the flavoenzyme PA4991 from Pseudomonas aeruginosa

Energy Technology Data Exchange (ETDEWEB)

Jacewicz, Agata; Schnell, Robert; Lindqvist, Ylva; Schneider, Gunter, E-mail: gunter.schneider@ki.se [Karolinska Institutet, S-171 77 Stockholm (Sweden)

2016-01-22

PA4991 is a FAD-dependent oxidoreductase from the pathogen P. aeruginosa that is essential for virulence and survival in the infected host. The structure of this enzyme, determined to 2.4 Å resolution, reveals that PA4991 belongs to the GR{sub 2} family of flavoenzymes. The locus PA4991 in Pseudomonas aeruginosa encodes an open reading frame that has been identified as essential for the virulence and/or survival of this pathogenic organism in the infected host. Here, it is shown that this gene encodes a monomeric FAD-binding protein of molecular mass 42.2 kDa. The structure of PA4991 was determined by a combination of molecular replacement using a search model generated with Rosetta and phase improvement by a low-occupancy heavy-metal derivative. PA4991 belongs to the GR{sub 2} family of FAD-dependent oxidoreductases, comprising an FAD-binding domain typical of the glutathione reductase family and a second domain dominated by an eight-stranded mixed β-sheet. Most of the protein–FAD interactions are via the FAD-binding domain, but the isoalloxazine ring is located at the domain interface and interacts with residues from both domains. A comparison with the structurally related glycine oxidase and glycerol-3-phosphate dehydrogenase shows that in spite of very low amino-acid sequence identity (<18%) several active-site residues involved in substrate binding in these enzymes are conserved in PA4991. However, enzymatic assays show that PA4991 does not display amino-acid oxidase or glycerol-3-phosphate dehydrogenase activities, suggesting that it requires different substrates for activity.

Crystal structure of the flavoenzyme PA4991 from Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Jacewicz, Agata; Schnell, Robert; Lindqvist, Ylva; Schneider, Gunter

2016-01-01

PA4991 is a FAD-dependent oxidoreductase from the pathogen P. aeruginosa that is essential for virulence and survival in the infected host. The structure of this enzyme, determined to 2.4 Å resolution, reveals that PA4991 belongs to the GR 2 family of flavoenzymes. The locus PA4991 in Pseudomonas aeruginosa encodes an open reading frame that has been identified as essential for the virulence and/or survival of this pathogenic organism in the infected host. Here, it is shown that this gene encodes a monomeric FAD-binding protein of molecular mass 42.2 kDa. The structure of PA4991 was determined by a combination of molecular replacement using a search model generated with Rosetta and phase improvement by a low-occupancy heavy-metal derivative. PA4991 belongs to the GR 2 family of FAD-dependent oxidoreductases, comprising an FAD-binding domain typical of the glutathione reductase family and a second domain dominated by an eight-stranded mixed β-sheet. Most of the protein–FAD interactions are via the FAD-binding domain, but the isoalloxazine ring is located at the domain interface and interacts with residues from both domains. A comparison with the structurally related glycine oxidase and glycerol-3-phosphate dehydrogenase shows that in spite of very low amino-acid sequence identity (<18%) several active-site residues involved in substrate binding in these enzymes are conserved in PA4991. However, enzymatic assays show that PA4991 does not display amino-acid oxidase or glycerol-3-phosphate dehydrogenase activities, suggesting that it requires different substrates for activity

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene.

Science.gov (United States)

Kato, Yasuo; Nomura, Taiji; Ogita, Shinjiro; Takano, Maki; Hoshino, Kazuhiro

2013-12-01

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81% identity and exhibited less than 60% identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.

Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions

International Nuclear Information System (INIS)

Georgatos, S.D.; Weber, K.; Geisler, N.; Blobel, G.

1987-01-01

Using solution binding assays, the authors found that a 45-kDa fragment of 125 I-labelled desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, they demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin

Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum

International Nuclear Information System (INIS)

Leberer, E.; Charuk, J.H.M.; MacLennan, D.H.; Green, N.M.

1989-01-01

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1,308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH 2 -terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca 2+ . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45 C 2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca 2+ with the membrane. The authors propose that this lumenal Ca 2+ binding glycoprotein of the sarcoplasmic reticulum be designated sarcalumenin

A novel 35 kDa frog liver acid metallophosphatase.

Science.gov (United States)

Szalewicz, A; Radomska, B; Strzelczyk, B; Kubicz, A

1999-04-12

The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases.

Purification and cloning of the two domain glyoxalase I from wheat bran

DEFF Research Database (Denmark)

Johansen, K.S.; Svendsen, I.; Rasmussen, S.K.

2000-01-01

induced by desiccation of the resurrection grass Sporobulus stapfianus, suggesting a role for glyoxalase in de- or rehydration of plant tissue. The 37 kDa wheat enzyme belongs to a group of monomeric glyoxalases and is composed of two similar halves each representing the full-length human glyoxalase I...... U/mg protein (1U = 1 mu mol S-lactoyl glutathione formed/min). Degenerate primers were designed and used for PCR-RACE-based cloning of the corresponding composite cDNA sequence (AJ243528). The wheat bran glyoxalase I amino acid sequence is very similar to the translated sequence of a RNA transcript...

40 CFR 82.162 - Certification by owners of recovery and recycling equipment.

Science.gov (United States)

2010-07-01

... and recycling equipment. 82.162 Section 82.162 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PROTECTION OF STRATOSPHERIC OZONE Recycling and Emissions Reduction § 82.162 Certification by owners of recovery and recycling equipment. (a) No later than August 12...

Results on charm hadroproduction from CERN experiment WA82

Energy Technology Data Exchange (ETDEWEB)

Antinori, F.; Barberis, D.; Beusch, W.; Davenport, M.; Dufey, J.P.; French, B.R.; Harrison, K.; Jacholkowski, A.; Kirk, A.; Lamanna, E.; Lassalle, J.C.; Muller, F.; Redaelli, N.; Roda, C.; Weymann, M. (CERN, Geneva (Switzerland)); Forino, A.; Gessaroli, R.; Mazzanti, P.; Quareni, A.; Viaggi, F. (Dipartimento di Fisica and INFN, Bologna (Italy)); Anselmi, R.; Casanova, V.; Dameri, M.; Hurst, R.; Novelli, P.; Osculati, B.; Rossi, L.; Tomasini, G. (Dipartimento di Fisica and INFN, Genova (Italy)); Buys, A.; Grard, F.; Legros, P. (Universite de Mons-Hainaut and IISN, Mons (Belgium)); Adamovich, M.; Alexandrov, Y.; Kharlamov, S.; Nechaeva, P.; Zavertyaev, M. (Lebedev Physical Institute, Moscow (Russian Federation))

1992-02-01

Experiment WA82 has collected data from 1987 to 1989 with the [Omega][prime] spectrometer at the CERN SPS. The aim of WA82 was a high statistics study of charm hadroproduction, using a silicon microstrip vertex detector and an impact parameter trigger. Latest results on the nuclear dependence of charm production and on the [ital x][sub [ital F

A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

DEFF Research Database (Denmark)

Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

2007-01-01

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

Identification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH as a binding protein for a 68-kDa Bacillus thuringiensis parasporal protein cytotoxic against leukaemic cells

Directory of Open Access Journals (Sweden)

Nadarajah Vishna

2010-11-01

Full Text Available Abstract Background Bacillus thuringiensis (Bt, an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa, human breast cancer (MCF-7 and colon cancer (HT-29 suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells. Methods Bt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell. Results Anion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18 for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH. Double immunofluorescence staining showed

Identification of a 48 kDa tubulin or tubulin-like C6/36 mosquito cells protein that binds dengue virus 2 using mass spectrometry

International Nuclear Information System (INIS)

Chee, H.-Y.; AbuBakar, Sazaly

2004-01-01

Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to β-tubulin and α-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells

A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

Science.gov (United States)

Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

Results on charm hadroproduction from CERN experiment WA82

International Nuclear Information System (INIS)

Antinori, F.; Barberis, D.; Beusch, W.; Davenport, M.; Dufey, J.P.; French, B.R.; Harrison, K.; Jacholkowski, A.; Kirk, A.; Lamanna, E.; Lassalle, J.C.; Muller, F.; Redaelli, N.; Roda, C.; Weymann, M.; Forino, A.; Gessaroli, R.; Mazzanti, P.; Quareni, A.; Viaggi, F.; Anselmi, R.; Casanova, V.; Dameri, M.; Hurst, R.; Novelli, P.; Osculati, B.; Rossi, L.; Tomasini, G.; Buys, A.; Grard, F.; Legros, P.; Adamovich, M.; Alexandrov, Y.; Kharlamov, S.; Nechaeva, P.; Zavertyaev, M.

1992-01-01

Experiment WA82 has collected data from 1987 to 1989 with the Ω' spectrometer at the CERN SPS. The aim of WA82 was a high statistics study of charm hadroproduction, using a silicon microstrip vertex detector and an impact parameter trigger. Latest results on the nuclear dependence of charm production and on the x F distributions of D + and D - mesons are presented and discussed

Action of Monomeric/Gemini Surfactants on Free Cells and Biofilm of Asaia lannensis

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Anna Koziróg

2017-11-01

Full Text Available We investigated the biological activity of surfactants based on quaternary ammonium compounds: gemini surfactant hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide (C6, synthesized by the reaction of N,N-dimethyl-N-dodecylamine with 1,6-dibromohexane, and its monomeric analogue dodecyltrimethylammonium bromide (DTAB. The experiments were performed with bacteria Asaia lannensis, a common spoilage in the beverage industry. The minimal inhibitory concentration (MIC values were determined using the tube standard two-fold dilution method. The growth and adhesive properties of bacterial cells were studied in different culture media, and the cell viability was evaluated using plate count method. Both of the surfactants were effective against the bacterial strain, but the MIC of gemini compound was significantly lower. Both C6 and DTAB exhibited anti-adhesive abilities. Treatment with surfactants at or below MIC value decreased the number of bacterial cells that were able to form biofilm, however, the gemini surfactant was more effective. The used surfactants were also found to be able to eradicate mature biofilms. After 4 h of treatment with C6 surfactant at concentration 10 MIC, the number of bacterial cells was reduced by 91.8%. The results of this study suggest that the antibacterial activity of the gemini compound could make it an effective microbiocide against the spoilage bacteria Asaia sp. in both planktonic and biofilm stages.

Action of Monomeric/Gemini Surfactants on Free Cells and Biofilm of Asaia lannensis.

Science.gov (United States)

Koziróg, Anna; Kręgiel, Dorota; Brycki, Bogumił

2017-11-22

We investigated the biological activity of surfactants based on quaternary ammonium compounds: gemini surfactant hexamethylene-1,6-bis-( N,N -dimethyl- N -dodecylammonium bromide) (C6), synthesized by the reaction of N,N -dimethyl- N- dodecylamine with 1,6-dibromohexane, and its monomeric analogue dodecyltrimethylammonium bromide (DTAB). The experiments were performed with bacteria Asaia lannensis , a common spoilage in the beverage industry. The minimal inhibitory concentration (MIC) values were determined using the tube standard two-fold dilution method. The growth and adhesive properties of bacterial cells were studied in different culture media, and the cell viability was evaluated using plate count method. Both of the surfactants were effective against the bacterial strain, but the MIC of gemini compound was significantly lower. Both C6 and DTAB exhibited anti-adhesive abilities. Treatment with surfactants at or below MIC value decreased the number of bacterial cells that were able to form biofilm, however, the gemini surfactant was more effective. The used surfactants were also found to be able to eradicate mature biofilms. After 4 h of treatment with C6 surfactant at concentration 10 MIC, the number of bacterial cells was reduced by 91.8%. The results of this study suggest that the antibacterial activity of the gemini compound could make it an effective microbiocide against the spoilage bacteria Asaia sp. in both planktonic and biofilm stages.

30 CFR 203.82 - What is MMS's authority to collect this information?

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 2 2010-07-01 2010-07-01 false What is MMS's authority to collect this information? 203.82 Section 203.82 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF THE INTERIOR... Reports § 203.82 What is MMS's authority to collect this information? The Office of Management and Budget...

A survey of residual analysis and a new test of residual trend.

Science.gov (United States)

McDowell, J J; Calvin, Olivia L; Klapes, Bryan

2016-05-01

A survey of residual analysis in behavior-analytic research reveals that existing methods are problematic in one way or another. A new test for residual trends is proposed that avoids the problematic features of the existing methods. It entails fitting cubic polynomials to sets of residuals and comparing their effect sizes to those that would be expected if the sets of residuals were random. To this end, sampling distributions of effect sizes for fits of a cubic polynomial to random data were obtained by generating sets of random standardized residuals of various sizes, n. A cubic polynomial was then fitted to each set of residuals and its effect size was calculated. This yielded a sampling distribution of effect sizes for each n. To test for a residual trend in experimental data, the median effect size of cubic-polynomial fits to sets of experimental residuals can be compared to the median of the corresponding sampling distribution of effect sizes for random residuals using a sign test. An example from the literature, which entailed comparing mathematical and computational models of continuous choice, is used to illustrate the utility of the test. © 2016 Society for the Experimental Analysis of Behavior.

Crystallization and preliminary crystallographic analysis of Gre2p, an NADP+-dependent alcohol dehydrogenase from Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Breicha, Klaus; Müller, Marion; Hummel, Werner; Niefind, Karsten

2010-01-01

The alcohol dehydrogenase Gre2p from S. cerevisiae catalyses the stereospecific reduction of a variety of different keto compounds and can therefore be applied as a valuable biocatalyst. The crystallization of the complex of Gre2p with NADP + and its preliminary X-ray analysis are described. Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP + . Crystals of a Gre2p complex with NADP + were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P2 1 . The current diffraction resolution is 3.2 Å. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

Science.gov (United States)

Tanaka, Tetsuya S; Ikenishi, Kohji

2002-02-01

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

Directory of Open Access Journals (Sweden)

Chenggu Cai

Full Text Available Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2 and Tas1r3 (taste receptor type 1 member 3. Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

Science.gov (United States)

Cai, Chenggu; Jiang, Hua; Li, Lei; Liu, Tianming; Song, Xuejie; Liu, Bo

2016-01-01

Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1989-01-01

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage

40 CFR 82.4 - Prohibitions for class I controlled substances.

Science.gov (United States)

2010-07-01

... violation of this subpart. Any person selling unused class I controlled substances produced or imported... substances. 82.4 Section 82.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Prohibitions for class I controlled substances. (a)(1) Prior to January 1, 1996, for all Groups of class I...

Structural Variation of Alpha-synuclein with Temperature by a Coarse-grained Approach with Knowledge-based Interactions (Postprint)

Science.gov (United States)

2015-07-01

Alzheimer disease. ASN1,3 has been extensively studied as an intrinsically disordered (unstructured) protein with a monomeric conformation compa- rable to a...changes in these physical quantities occur by avoiding the low and high temperature extremes. Typical snapshots from the simulations as a function of...mobile as the residue-residue interactions become less impor- tant and the protein assumes a self- avoiding walk (SAW) or random coil conformation (vida

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

Science.gov (United States)

Neela, Venkata Sanjeev Kumar; Devalraju, Kamakshi Prudhula; Pydi, Satya Sudheer; Sunder, Sharada Ramaseri; Adiraju, Kameswara Rao; Singh, Surya Satyanarayana; Anandaraj, M P J S; Valluri, Vijaya Lakshmi

2016-09-01

Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients. © The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.

Science.gov (United States)

Macias, Alba T; Williamson, Douglas S; Allen, Nicola; Borgognoni, Jenifer; Clay, Alexandra; Daniels, Zoe; Dokurno, Pawel; Drysdale, Martin J; Francis, Geraint L; Graham, Christopher J; Howes, Rob; Matassova, Natalia; Murray, James B; Parsons, Rachel; Shaw, Terry; Surgenor, Allan E; Terry, Lindsey; Wang, Yikang; Wood, Mike; Massey, Andrew J

2011-06-23

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides

Directory of Open Access Journals (Sweden)

Regina C. Adão

2012-06-01

Full Text Available Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2 of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans, as well as its mutated monomers (CH1-S and CH2-S and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1 bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S in which the cystein (Fig.1 a residues of each chain were changed by serin residues (Fig. 1 b. The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC

40 CFR 80.82 - Butane blending.

Science.gov (United States)

2010-07-01

... FUELS AND FUEL ADDITIVES Reformulated Gasoline § 80.82 Butane blending. A refiner for any refinery that produces gasoline by blending butane with conventional gasoline or reformulated gasoline or RBOB may meet... paragraph (b)(1) of this section, the refiner may: (i) Blend the butane with conventional gasoline, or...

40 CFR 82.65 - Temporary exemptions.

Science.gov (United States)

2010-07-01

...) PROTECTION OF STRATOSPHERIC OZONE Ban on Nonessential Products Containing Class I Substances and Ban on Nonessential Products Containing or Manufactured With Class II Substances § 82.65 Temporary exemptions. (a) Any... January 1, 1996. (e) Any person selling or distributing, or offering to sell or distribute, any product...

Early cytokine modulation after the rapid induction phase of sublingual immunotherapy with mite monomeric allergoids.

Science.gov (United States)

Di Gioacchino, M; Perrone, A; Petrarca, C; Di Claudio, F; Mistrello, G; Falagiani, P; Dadorante, V; Verna, N; Braga, M; Ballone, E; Cavallucci, E

2008-01-01

The influence of different treatment schedules of sublingual immunotherapy (SLIT) in activating IL-10-producing T-cells, crucial in inducing allergen-specific tolerance, is not completely understood. The present work was designed to evaluate allergen driven interleukin release by mononuclear cells in the early phase of SLIT, after application of different induction schemes. Twenty mite-allergic patients were enrolled, 10 (group A) treated with a traditional 98 day induction scheme and 10 (group B) with a 16 day scheme with monomeric allergoid vaccine. At the end of the induction phase, the cumulative doses taken by group A and group B patients were equivalent to 50.5 and 50.3 microg of mite group 1 allergens, respectively. The release of Th1-, Th2- and Treg-related interleukins was assessed in culture supernatants of 5 microg/ml Der-p1-stimulated mononuclear cells, isolated before and after the induction phases. No relevant treatment-related side effects were observed. Interleukin release was similar in the two groups at the enrolment. Non-stimulated and Der p 1 stimulated release of studied cytokines was similar in the two groups at enrolment. Der p 1 stimulation significantly increased IL-10 release (pallergoids are utilized.

Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

International Nuclear Information System (INIS)

Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

1988-01-01

The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32 P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32 P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice

The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

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Jasdave S Chahal

Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

Malignant progressive tumor cell clone exhibits significant up-regulation of cofilin-2 and 27-kDa modified form of cofilin-1 compared to regressive clone.

Science.gov (United States)

Kuramitsu, Yasuhiro; Wang, Yufeng; Okada, Futoshi; Baron, Byron; Tokuda, Kazuhiro; Kitagawa, Takao; Akada, Junko; Nakamura, Kazuyuki

2013-09-01

QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p<0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p<0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4.

Science.gov (United States)

Virginia, Agnes; Rachmawati, Heni; Riani, Catur; Retnoningrum, Debbie S

2016-01-01

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 μg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.

50 CFR 82.6 - Submission of proposals.

Science.gov (United States)

2010-10-01

... (MARINE MAMMAL PROTECTION ACT OF 1972) Application for Grants § 82.6 Submission of proposals. (a) Preapplication forms may be submitted by any potential grantee in order to (1) establish communication between...

RESRAD, Residual Radioactive Material Guideline Implementation

International Nuclear Information System (INIS)

1998-01-01

This code system is designed to calculate site-specific residual radioactive material guidelines, and radiation dose and excess cancer risk to an on-site resident (maximally exposed individual). A guideline is a radionuclide concentration or level of radioactivity that is acceptable if a site is to be used without radiological restrictions. Guidelines are expressed as concentrations of residual radionuclides in soil. Soil is unconsolidated earth material, including rubble and debris that may be present. The guidelines are based on the following principles: (1) the total effective dose equivalent should not exceed 100 mrem/yr for all plausible land uses and 30 mrem/yr for current and likely future land uses and (2) doses should be kept as low as reasonably achievable (ALARA). Nine environmental pathways are considered: direct exposure, inhalation of dust and radon, and ingestion of plant foods, meat, milk, aquatic foods, soil, and water. CCC-0552/04: A - Description of program or function: RESRAD-BUILD Version 2.36 is a pathway analysis model designed to evaluate the potential radiological dose incurred by an individual who works or lives in a building contaminated with radioactive material. The radioactive material in the building structure can be released into the indoor air by mechanisms such as diffusion (radon gas), mechanical removal (decontamination activities), or erosion (removable surface contamination). In the June 1998 update, RESRAD was updated to Version 5.82 and RESRAD-BUILD was updated to version 2.36. The following notes highlight new features: RESRAD5.82 (4/30/98): - Allow plot data to be exported to tab-delimited text file - Corrected Installation problem to Windows 3.1 - Corrected plotting problem for soil guidelines RESRAD-BUILD2.36 (4/9/98): - Corrected problem with simultaneously changing number of wall regions and their parameters - Added OK button to uncertainty window - Made sure first uncertainty variable in added on first try See the

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

Science.gov (United States)

Liu, Xiaoqing; Xiu, Zhilong; Hao, Ce

2009-05-01

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

Dynamic changes in the secondary structure of ECE-1 and XCE account for their different substrate specificities

Directory of Open Access Journals (Sweden)

Ul-Haq Zaheer

2012-11-01

Full Text Available Abstract Background X-converting enzyme (XCE involved in nervous control of respiration, is a member of the M13 family of zinc peptidases, for which no natural substrate has been identified yet. In contrast, it’s well characterized homologue endothelin-converting enzyme-1 (ECE-1 showed broad substrate specificity and acts as endopeptidase as well as dipeptidase. To explore the structural differences between XCE and ECE-1, homology model of XCE was built using the complex structure of ECE-1 with phosphoramidon (pdb-id: 3DWB as template. Phosphoramidon was docked into the binding site of XCE whereas phosphate oxygen of the inhibitor was used as water molecule to design the apo forms of both enzymes. Molecular dynamics simulation of both enzymes was performed to analyze the dynamic nature of their active site residues in the absence and presence of the inhibitor. Results Homology model of XCE explained the role of non-conserved residues of its S2’ subsite. Molecular dynamics (MD simulations identified the flexible transitions of F149/I150, N566/N571, W714/W719, and R145/R723 residues of ECE-1/XCE for the strong binding of the inhibitor. Secondary structure calculations using DSSP method reveals the folding of R145/R723 residue of ECE-1/XCE into β-sheet structure while unfolding of the S2’ subsite residues in aECE-1 and sustained compact folding of that of aXCE. The results evaluated are in good agreement with available experimental data, thus providing detailed molecular models which can explain the structural and specificities differences between both zinc peptidases. Conclusions Secondary structure changes of both enzymes during the simulation time revealed the importance of β-sheet structure of R145/R723 for its binding with the terminal carboxylate group of the inhibitor. Unfolding of the α-helix comprising the S2’ subsite residues in aECE-1 correlate well with its endopeptidase activity while their compact folding in aXCE may

Evaluation of residue-residue contact predictions in CASP9

KAUST Repository

Monastyrskyy, Bohdan; Fidelis, Krzysztof; Tramontano, Anna; Kryshtafovych, Andriy

2011-01-01

This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures

Anglo-Australian Telescope 1981/82

International Nuclear Information System (INIS)

1982-01-01

Operations of the Board for the period July 81 to June 82 are summarised. Major areas under study are galaxies, quasi-stellar objects, stars, supernova remnants and interstellar material. Research on dwarf novae, an infrared view of our galactic centre, the nucleus of the Seyfert galaxy NGC 1068, galaxies at great distances, the highest redshift quasar, the origin of quasar absorption lines and the unseen mass in our galaxy is briefly reported

Physical immobilization of 60 kDa chaperonin linked lipase from pseudomonas aeruginosa BN-1

International Nuclear Information System (INIS)

Syed, M.N.; Mehmood, S.; Bashir, A.; Ashraf, F.

2012-01-01

Abstract: The 60 kDa chaperone linked lipase from Pseudomonas aeruginosa was subjected to physical adsorption on silica 60 and acrylic beads. It was found that higher enzyme loading was achieved on silica gel than acrylic bead. The half life of immobilized enzyme was greater compared to the free enzyme. The adsorption of the enzyme onto a solid phase also resulted in increased thermo and solvent stability. It was observed that soluble enzyme showed maximum stability at 70 degree C while immobilized enzyme showed stability up to 80 degree C for 45 minutes. The stability of immobilized enzyme increased up to 48 hours from 24 hours against different organic solvent at 1.0 M concentration. It was noted that enzyme immobilized on acrylic beads have greater reusability compared to silica immobilized enzyme. (author)

28 CFR 544.82 - General program characteristics.

Science.gov (United States)

2010-07-01

... Section 544.82 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT..., state departments of education, or other recognized accrediting educational organizations; (2... through a sponsoring accredited educational institution; (3) General Educational Development tests...

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

DEFF Research Database (Denmark)

Dziegiel, M; Borre, Mette; Petersen, E

1992-01-01

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

[Zymography--method for quantitation of activity on gelatinase A (pro-MMP-2, 72 kDa) and gelatinase B (pro-MMP-9, 92 kDa) in serum of patients with breast cancer].

Science.gov (United States)

Sliwowska, Izabela; Kopczyński, Zygmunt

2007-01-01

Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on polyacrylamide gels impregnated with a protein substrate (gelatin, casein), which is degraded by the proteases. It is already widely used for research on extracellular matrix degrading enzymes, in particular the matrix metalloproteinases (MMPs). The aim of this study was to evaluate the zymography used to estimate gelatinase A (pro-MMP-2, 72 kDa) and gelatinase B (pro-MMP-9, 92 kDa) in serum of women with breast cancer. The study was conducted on the serum of 90 female with breast cancer aged 32-85 years (average 57.2 year) and in a group of 30 women with benign breast diseases aged 34-87 years (average 55.4 year). The results showed significantly higher activity ofgelatinase A and gelatinase B in the group of women with breast cancer that in the control group. The activity of gelatinase A was over 0.48 AU/ml in 50 women (55.6%) and gelatinase B in 46 women in this group (51.1%). Further analysis showed a strong correlation between activity of gelatinase A and B and advance stage of breast cancer, lymph node status, and tumour burden. An elevated activity of gelatinases in patients with breast cancer confirms the role of these enzymes in the development of such tumours. There was no significant difference in the gelatinases activity between the serum samples from patients with breast cancer and those from women with benign breast diseases. The diagnostic sensitivity of this procedure used to estimate gelatinase A and gelatinase B activity carried out 56% and 51%, and the diagnostic specificity 75% and 77%, respectively. Zymography is a simple, sensitive, and functional assay for analysing proteolytic activity of gelatinase A and gelatinase B. The diagnostic accuracy of gelatinases evaluation is limited by the lack of sensitivity and specificity in early stage of the disease and in differentiation of breast cancer from benign diseases. Measuring their activity in serum

Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

Science.gov (United States)

Waldmann, Herman

2014-01-01

One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

Science.gov (United States)

Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

2015-05-01

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

Conteúdo de peptídeos e avaliação morfofisiológica dos espermatozóides do epidídimo e ejaculado de bovinos Peptides content and morphophisiological evaluation of epididymis and ejaculated sperm in bovine

Directory of Open Access Journals (Sweden)

Antonio Emidio D. Feliciano Silva

2003-12-01

Full Text Available Objetivou-se com este estudo a identificação de alguns fatores protéicos envolvidos na qualidade funcional dos espermatozóides epididimais (SPZEP e ejaculados (SPZEJ de bovinos. Foram avaliadas as características morfofisiológicas e analisado o conteúdo peptídico destas estruturas de 11 animais mestiços Nelore, de 24 a 30 meses de idade. As avaliações morfofisiológicas foram motilidade progressiva (MOT, %, vigor, patologias espermáticas, integridade acrossômica e da cromatina. Foi observado que, os SPZEJ, na média, apresentaram MOT maior do que os SPZEP, 72,3 e 46,4%, respectivamente. Considerando as patologias espermáticas, taxas de defeitos maiores (DEFMAI, menores (DEFMEN e totais (DEFTOT, houve diferença significativa entre as taxas dos DEFMEN e DEFTOT dos SPZEP e SPZEJ, sendo, em média, 91,1 e 8,5% e 95,4 e 11,8%, respectivamente. As taxas dos DEFMEN e DEFTOT dos SPZEP foram maiores em função da presença de espermatozóides com gotas citoplasmáticas distais. A análise das protéinas dos SPZEP e SPZEJ foi realizada por espectrometria de massa, método MALDI-TOF (matrix -assisted laser desorption/ionization - time of flight, e revelou presença de peptídeos de massa molecular variando de 1,1 a 26,3 kDa nos SPZEJ e de 1,1 a 11,6 kDa nos SPZEP. Foram identificados peptídeos de 10,6 e 13,4 kDa somente nos SPZEJ e de 6,8 kDa somente nos SPZEP. Foi observada relação do peptídeo de massa molecular de 7,4 kDa dos SPZEP e de 4,7 kDa dos SPZEJ, com a MOT Ê 80%, destas estruturas. Os resultados sugerem o envolvimento destes peptídeos nos processos funcionais das células espermáticas do epidídimo e ejaculado. O estudo utilizou o método MALDI/TOF para espectrômetro de massa, para identificar peptídeos em espermatozóides do epidídimo de bovinos, pela primeira vez no País.The objective of the study was to identify some protein factors involved in bovine epididymis (SPZEP and ejaculated (SPZEJ sperm functional quality

Effects of initial microstructure and helium production on radiation hardening in F82H Steels

Energy Technology Data Exchange (ETDEWEB)

Okubo, N.; Wakai, E.; Takada, F.; Jitsukawa, S. [Japan Atomic Energy Agency, Naga-gun, Ibaraki-ken (Japan); Katoh, Y. [Oak Ridge Noational Laboratory, TN (United States)

2007-07-01

Full text of publication follows: Fission neutron irradiation to steels doped with isotope boron-10 is frequently conducted to study effects of the helium production on mechanical properties. The intrinsic mechanical properties of F82H steels could have been changed due to the boron doping. Recently, we reported that co-doping with boron and nitrogen to F82H (F82H+B+N) improved the mechanical properties of F82H doped only with boron. The mechanical properties of F82H+B+N are successfully comparable with the non-doped F82H before irradiation. In order to evaluate the effects of initial microstructure and helium production on radiation hardening, F82H and F82H+B+N were irradiate d Specimens used in this study were standard F82H martensitic steels, F82H steels doped with 60 mass ppm {sup 10}B and 200 ppm N (F82H+10B+N) and F82H steels doped with 60 mass ppm {sup 11}B and 200 ppm N (F82H+11B+N). Initial microstructures were changed by tempering conditions, and the tempering temperatures were at 700, 750 and 780 deg. C. Irradiation was performed at nominally 250 deg. C to 2 dpa in JMTR. Tensile properties were measured for the specimens before and after irradiation. Change of yield stress due to the irradiation in the F82H+11B+N steels depended strongly on the initial microstructure and hardness before irradiation. The radiation hardening due to helium production in the F82H+10B+N steels was less than 60 MPa in these experiments. Size of dimple in the fracture surface of specimen with helium production was larger than that with non-helium production. (authors)

Very proton-rich nuclei with N asymptotically equals 82

International Nuclear Information System (INIS)

Nolte, E.

1984-01-01

The proton-rich nuclei with N asymptotically equals 82 show beautifully properties, which are perfectly described by the nuclear shell model. Some of these properties are the occurrence of seniority isomerism in the proton-rich N=82 isotones and the perfect description of the corresponding life times by the seniority scheme as well as the observation of favoured Gamow-Teller β transitions in this nuclear region and the dependence of the corresponding ft values on the number of the envolved nucleons. (author)

Modulation of basophils' degranulation and allergy-related enzymes by monomeric and dimeric naphthoquinones.

Directory of Open Access Journals (Sweden)

Brígida R Pinho

Full Text Available Allergic disorders are characterized by an abnormal immune response towards non-infectious substances, being associated with life quality reduction and potential life-threatening reactions. The increasing prevalence of allergic disorders demands for new and effective anti-allergic treatments. Here we test the anti-allergic potential of monomeric (juglone, menadione, naphthazarin, plumbagin and dimeric (diospyrin and diosquinone naphthoquinones. Inhibition of RBL-2H3 rat basophils' degranulation by naphthoquinones was assessed using two complementary stimuli: IgE/antigen and calcium ionophore A23187. Additionally, we tested for the inhibition of leukotrienes production in IgE/antigen-stimulated cells, and studied hyaluronidase and lipoxidase inhibition by naphthoquinones in cell-free assays. Naphthazarin (0.1 µM decreased degranulation induced by IgE/antigen but not A23187, suggesting a mechanism upstream of the calcium increase, unlike diospyrin (10 µM that reduced degranulation in A23187-stimulated cells. Naphthoquinones were weak hyaluronidase inhibitors, but all inhibited soybean lipoxidase with the most lipophilic diospyrin, diosquinone and menadione being the most potent, thus suggesting a mechanism of competition with natural lipophilic substrates. Menadione was the only naphthoquinone reducing leukotriene C4 production, with a maximal effect at 5 µM. This work expands the current knowledge on the biological properties of naphthoquinones, highlighting naphthazarin, diospyrin and menadione as potential lead compounds for structural modification in the process of improving and developing novel anti-allergic drugs.

Effects of post weld heat treatment and weld overlay on the residual stress and mechanical properties in dissimilar metal weld

International Nuclear Information System (INIS)

Campos, Wagner R.C.; Ribeiro, Vladimir S.; Vilela, Alisson H.F.; Almeida, Camila R.O.; Rabello, Emerson G.

2017-01-01

The object of this work is a dissimilar metal weld (DMW) pipe joint between carbon steel (A-106 Gr B) and stainless steel (A-312 TP316L) pipes and filler metals of Nickel alloy (82/182), which find wide application in the field of chemical, oil, petroleum industries, fossil fuel and nuclear power plant. A lot of the failures that have occurred in dissimilar metal welded are affected greatly by residual stresses. Residual stress is often a cause of premature failure of critical components under normal operation of welded components. Several methods have been tested and developed for removing the tensile residual stresses. The aim of the methods is to reduce the tensile stress state or to create compressive stresses at a predefined area, such as the inner surface of a welded pipe joint. Post weld heat treatment (PWHT) and weld overlay (WOL) are two of the residual stress mitigation methods which reduce the tensile residual stress, create compressive stresses and arrest crack initiation and crack growth. The technique used to substantially minimized or eliminated this failure development in the root weld is the post weld heat treatments (stress relief heat treatment) or the weld overlay. In this work was studied the effectiveness in reducing internal residual stress in dissimilar metal welded pipe joints subjected to post weld heat treatment and weld overlay, measurement by hole-drilling strain-gage method of stress relaxation. Also held was mechanical characterization of the welded pipe joint itself. (author)

Effects of post weld heat treatment and weld overlay on the residual stress and mechanical properties in dissimilar metal weld

Energy Technology Data Exchange (ETDEWEB)

Campos, Wagner R.C.; Ribeiro, Vladimir S.; Vilela, Alisson H.F.; Almeida, Camila R.O.; Rabello, Emerson G., E-mail: wrcc@cdtn.br, E-mail: camilarezende.cr@gmail.com, E-mail: egr@cdtn.br, E-mail: vladimirsoler@hotmail.com, E-mail: ahfv02@outlook.com [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

2017-07-01

The object of this work is a dissimilar metal weld (DMW) pipe joint between carbon steel (A-106 Gr B) and stainless steel (A-312 TP316L) pipes and filler metals of Nickel alloy (82/182), which find wide application in the field of chemical, oil, petroleum industries, fossil fuel and nuclear power plant. A lot of the failures that have occurred in dissimilar metal welded are affected greatly by residual stresses. Residual stress is often a cause of premature failure of critical components under normal operation of welded components. Several methods have been tested and developed for removing the tensile residual stresses. The aim of the methods is to reduce the tensile stress state or to create compressive stresses at a predefined area, such as the inner surface of a welded pipe joint. Post weld heat treatment (PWHT) and weld overlay (WOL) are two of the residual stress mitigation methods which reduce the tensile residual stress, create compressive stresses and arrest crack initiation and crack growth. The technique used to substantially minimized or eliminated this failure development in the root weld is the post weld heat treatments (stress relief heat treatment) or the weld overlay. In this work was studied the effectiveness in reducing internal residual stress in dissimilar metal welded pipe joints subjected to post weld heat treatment and weld overlay, measurement by hole-drilling strain-gage method of stress relaxation. Also held was mechanical characterization of the welded pipe joint itself. (author)

[{sup 18}F]DPA-714, [{sup 18}F]PBR111 and [{sup 18}F]FEDAA1106-Selective radioligands for imaging TSPO 18 kDa with PET: Automated radiosynthesis on a TRACERLAb FX-FN synthesizer and quality controls

Energy Technology Data Exchange (ETDEWEB)

Kuhnast, Bertrand, E-mail: bertrand.kuhnast@cea.fr [CEA, I2BM, Service Hospitalier Frederic Joliot, 4 Place du General Leclerc, F-91401, Orsay Cedex (France); Damont, Annelaure; Hinnen, Francoise; Catarina, Tony; Demphel, Stephane; Le Helleix, Stephane; Coulon, Christine; Goutal, Sebastien; Gervais, Philippe; Dolle, Frederic [CEA, I2BM, Service Hospitalier Frederic Joliot, 4 Place du General Leclerc, F-91401, Orsay Cedex (France)

2012-03-15

Imaging of TSPO 18 kDa with PET is more and more considered as a relevant biomarker of inflammation in numerous diseases. Development of new radiotracers for TSPO 18 kDa has seen acceleration in the last years and the challenge today is to make available large amounts of such a radiotracer in compliance with GMP standards for application in humans. We present in this technical note automated productions of [{sup 18}F]DPA-714, [{sup 18}F]PBR111 and [{sup 18}F]FEDAA1106, three promising radiotracers for TSPO 18 kDa imaging, using a TRACERlab FX-FN synthesizer. This note also includes the quality control data of the validation batches for the manufacturing qualification of clinical production of [{sup 18}F]DPA-714. - Highlights: Black-Right-Pointing-Pointer Protein TSPO 18 kDa is recognized as a biomarker of inflammation involved in many diseases. Black-Right-Pointing-Pointer Radiotracers targeting TSPO prepared in compliance with GMPs are mandatory as new imaging tools. Black-Right-Pointing-Pointer An automated radiosynthesis of promising radiotracers and full QC have been implemented.

Radiofrequency ablation of renal tumours: diagnostic accuracy of contrast-enhanced ultrasound for early detection of residual tumour

International Nuclear Information System (INIS)

Hoeffel, Christine; Pousset, Maud; Elie, Caroline; Timsit, Marc-Olivier; Mejean, Arnaud; Merran, Samuel; Tranquart, Francois; Khairoune, Ahmed; Helenon, Olivier; Correas, Jean-Michel; Joly, Dominique; Richard, Stephane

2010-01-01

To evaluate the diagnostic accuracy of contrast-enhanced ultrasound (CEUS) in the early detection of residual tumour after radiofrequency ablation (RFA) of renal tumours. Patients referred to our institution for RFA of renal tumours prospectively underwent CEUS and computed tomography (CT) or magnetic resonance imaging (MRI) before, within 1 day and 6 weeks after treatment. Identification of residual tumour was assessed by three blinded radiologists. Reference standard was CT/MRI performed at least 1 year after RFA. A total of 66 renal tumours in 43 patients (median age 62 years; range 44-71.5) were studied. Inter-reader agreement (κ value) was 0.84 for CEUS. Prevalence of residual disease was 19%. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), respectively, were as follows: 64% [confidence interval (CI) 39-84], 98% [CI 91-100], 82% [CI 52-95] and 92% [CI 83-97] on 24-h CEUS; 79% [CI 52-92], 100% [CI 94-100], 100% [CI 74-100] and 95% [CI 87-100] on 6-week CEUS; 79% [CI 52-92], 95% [CI 86-98], 79% [CI 52-92] and 95% [CI 86-98] on 24-h CT/MRI; and 100% [CI 72-100], 98% [CI 90-100], 91% [CI 62-98] and 100% [CI 93-100] on 6-week CT/MRI. CEUS has high specificity for the early diagnosis of residual tumour after renal RFA. (orig.)

Pesticide residues in canned foods, fruits, and vegetables: the application of Supercritical Fluid Extraction and chromatographic techniques in the analysis.

Science.gov (United States)

El-Saeid, Mohamed H

2003-12-11

Multiple pesticide residues have been observed in some samples of canned foods, frozen vegetables, and fruit jam, which put the health of the consumers at risk of adverse effects. It is quite apparent that such a state of affairs calls for the need of more accurate, cost-effective, and rapid analytical techniques capable of detecting the minimum concentrations of the multiple pesticide residues. The aims of this paper were first, to determine the effectiveness of the use of Supercritical Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC) techniques in the analysis of the levels of pesticide residues in canned foods, vegetables, and fruits; and second, to contribute to the promotion of consumer safety by excluding pesticide residue contamination from markets. Fifteen different types of imported canned and frozen fruits and vegetables samples obtained from the Houston local food markets were investigated. The major types of pesticides tested were pyrethroids, herbicides, fungicides, and carbamates. By using these techniques, the overall data showed 60.82% of the food samples had no detection of any pesticide residues under this investigation. On the other hand, 39.15% different food samples were contaminated by four different pyrethroid residues +/- RSD% ranging from 0.03 +/- 0.005 to 0.05 +/- 0.03 ppm, of which most of the pyrethroid residues were detected in frozen vegetables and strawberry jam. Herbicide residues in test samples ranged from 0.03 +/- 0.005 to 0.8 +/- 0.01 ppm. Five different fungicides, ranging from 0.05 +/- 0.02 to 0.8 +/- 0.1 ppm, were found in five different frozen vegetable samples. Carbamate residues were not detected in 60% of investigated food samples. It was concluded that SFE and SFC techniques were accurate, reliable, less time consuming, and cost effective in the analysis of imported canned foods, fruits, and vegetables and are recommended for the monitoring of pesticide contaminations.

Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

Directory of Open Access Journals (Sweden)

Robert Kalescky

2016-04-01

Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

Evaluation of some residual bioactivities of microencapsulated Phaseolus lunatus protein fraction with carboxymethylated flamboyant (Delonix regia gum/sodium alginate

Directory of Open Access Journals (Sweden)

Mukthar Sandovai-Peraza

2014-12-01

Full Text Available Recent studies have shown the beneficial effect of peptides, an unexploited source could be Phaseolus lunatus being an important raw material for those functional products in order to improve their utilization. In addition to improve the beneficial effect of bioactive peptides the microencapsulation could be a way to protect the peptides against the environment to which they are exposed. P. lunatus protein fraction (<10 kDa of weight was encapsulated using a blend of carboxymethylated flamboyant gum (CFG and sodium alginate (SA at different concentrations of CaCl2 and hardening times. After in vitro digestion of microcapsules the residual activity, in the intestinal system, both inhibition of agiotensin-converting enzyme (I-ACE and antioxidant activity obtained were in a range of 0.019-0.136 mg/mL and 570.64-813.54 mM of TEAC respectively. The microencapsulation employed CFG/SA blends could be used controlled delivery of peptide fractions with potential use as a nutraceutical or therapeutic agents.

Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

International Nuclear Information System (INIS)

Haas, R.; Marshall, T.L.; Rosenberry, T.L.

1988-01-01

The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [ 125 ]I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine

Integrating biological treatment of crop residue into a hydroponic sweetpotato culture

Science.gov (United States)

Trotman, A. A.; David, P. P.; Bonsi, C. K.; Hill, W. A.; Mortley, D. G.; Loretan, P. A.

1997-01-01

Residual biomass from hydroponic culture of sweetpotato [Ipomoea batatas (L.) Lam.] was degraded using natural bacterial soil isolates. Sweetpotato was grown for 120 days in hydroponic culture with a nutrient solution comprised of a ratio of 80% modified half Hoagland solution to 20% filtered effluent from an aerobic starch hydrolysis bioreactor. The phytotoxicity of the effluent was assayed with `Waldmann's Green' lettuce (Lactuca sativa L.) and the ratio selected after a 60-day bioassay using sweetpotato plants propagated vegetatively from cuttings. Controlled environment chamber experiments were conducted to investigate the impact of filtrate from biological treatment of crop residue on growth and storage root production with plants grown in a modified half Hoagland solution. Incorporation of bioreactor effluent, reduced storage root yield of `Georgia Jet' sweetpotato but the decrease was not statistically significant when compared with yield for plants cultured in a modified half Hoagland solution without filtrate. However, yield of `TU-82-155' sweetpotato was significantly reduced when grown in a modified half Hoagland solution into which filtered effluent had been incorporated. Total biomass was significantly reduced for both sweetpotato cultivars when grown in bioreactor effluent. The leaf area and dry matter accumulation were significantly (P < 0.05) reduced for both cultivars when grown in solution culture containing 20% filtered effluent.

HindIII RFLP on chromosome 8 detected with a calbindin 27 kDa cDNA probe, HBSC21

Energy Technology Data Exchange (ETDEWEB)

Parmentier, M; Vassart, G

1988-10-11

A 1.8 kb for EcoRI fragment of the human calbindin cDNA clone HBSC21 was subcloned into M13mp18 and used as a probe. HindIII identifies a 2 allele polymorphism with a band at 4.7 kb (A1) and a band at 4.3 kb (A2). A constant band is located at 5.3 kb. The calbindin 27 kDa gene was assigned to chromosome 8 using chinese hamster-human and mouse-human cell hybrids. Co-dominant segregation was demonstrated in 3 families (total of 20 individuals).

Effect of crop sequence and crop residues on soil C, soil N and yield of maize

International Nuclear Information System (INIS)

Shafi, M.; Bakht, J.; Attaullah; Khan, M.A.

2010-01-01

Improved management of nitrogen (N) in low N soils is critical for increased soil productivity and crop sustainability. The objective of the present study was to evaluate the effects of residues incorporation, residues retention on soil surface as mulch, fertilizer N and legumes in crop rotation on soil fertility and yield of maize (Zea may L.). Fertilizer N was applied to maize at the rate of 160 kg ha/sup -1/, and to wheat at the rate of 120 kg ha/sup -1/ or no fertilizer N application. Crop rotation with the sequence of maize after wheat (Triticum aestivum L.), maize after lentil (Lens culinaris Medic) or wheat after mash bean (Vigna mungo L.) arranged in a split plot design was followed. Post-harvest incorporation of crop residues and residues retention on soil surface as mulch had significantly (p=0.05) affected grain and stover yield during 2004 and 2005. Two years average data revealed that grain yield was increased by 3.31 and 6.72% due to mulch and residues incorporation. Similarly, stover yield was also enhanced by 5.39 and 10.27% due to the same treatment respectively. Mulch and residues incorporation also improved stover N uptake by 2.23 and 6.58%, respectively. Total soil N and organic matter was non significantly (p=0.05) increased by 5.63 and 2.38% due to mulch and 4.13, 7.75% because of crop residues incorporation in the soil. Maize grain and stover yield responded significantly (p=0.05) to the previous legume (lentil) crop when compared with the previous cereal crop (wheat). The treatment of lentil - maize(+N), on the average, increased grain yield of maize by 15.35%, stover yield by 16.84%, total soil N by 10.31% and organic matter by 10.17%. Similarly, fertilizer N applied to the previous wheat showed carry over effect on grain yield (6.82%) and stover yield (11.37%) of the following maize crop. The present study suggested that retention of residues on soil surface as mulch, incorporation of residues in soil and legume (lentil - maize) rotation

Gas-phase synthesis and structure of monomeric ZnOH: a model species for metalloenzymes and catalytic surfaces.

Science.gov (United States)

Zack, Lindsay N; Sun, Ming; Bucchino, Matthew P; Clouthier, Dennis J; Ziurys, Lucy M

2012-02-16

Monomeric ZnOH has been studied for the first time using millimeter and microwave gas-phase spectroscopy. ZnOH is important in surface processes and at the active site of the enzyme carbonic anhydrase. In the millimeter-wave direct-absorption experiments, ZnOH was synthesized by reacting zinc vapor, produced in a Broida-type oven, with water. In the Fourier-transform microwave measurements, ZnOH was produced in a supersonic jet expansion of CH(3)OH and zinc vapor, created by laser ablation. Multiple rotational transitions of six ZnOH isotopologues in their X(2)A' ground states were measured over the frequency range of 22-482 GHz, and splittings due to fine and hyperfine structure were resolved. An asymmetric top pattern was observed in the spectra, showing that ZnOH is bent, indicative of covalent bonding. From these data, spectroscopic constants and an accurate structure were determined. The Zn-O bond length was found to be similar to that in carbonic anhydrase and other model enzyme systems.

Residual stress measurements in the dissimilar metal weld in pressurizer safety nozzle of nuclear power plant

International Nuclear Information System (INIS)

Campos, Wagner R.C.; Rabello, Emerson G.; Mansur, Tanius R.; Scaldaferri, Denis H.B.; Paula, Raphael G.; Souto, Joao P.R.S.; Carvalho Junior, Ideir T.

2013-01-01

Weld residual stresses have a large influence on the behavior of cracking that could possibly occur under normal operation of components. In case of an unfavorable environment, both stainless steel and nickel-based weld materials can be susceptible to stress-corrosion cracking (SCC). Stress corrosion cracks were found in dissimilar metal welds of some pressurized water reactor (PWR) nuclear plants. In the nuclear reactor primary circuit the presence of tensile residual stress and corrosive environment leads to so-called Primary Water Stress Corrosion Cracking (PWSCC). The PWSCC is a major safety concern in the nuclear power industry worldwide. PWSCC usually occurs on the inner surface of weld regions which come into contact with pressurized high temperature water coolant. However, it is very difficult to measure the residual stress on the inner surfaces of pipes or nozzles because of inaccessibility. A mock-up of weld parts of a pressurizer safety nozzle was fabricated. The mock-up was composed of three parts: an ASTM A508 C13 nozzle, an ASTM A276 F316L stainless steel safe-end, an AISI 316L stainless steel pipe and different filler metals of nickel alloy 82/182 and AISI 316L. This work presents the results of measurements of residual strain from the outer surface of the mock-up welded in base metals and filler metals by hole-drilling strain-gage method of stress relaxation. (author)

EROP-Moscow: specialized data bank for endogenous regulatory oligopeptides.

Science.gov (United States)

Zamyatnin, A A

1991-07-01

At present we are familiar with several hundred endogenous oligopeptides involved in the functioning of the nervous, immune and endocrine systems. The information on their structures and functions is contained in a specially created data bank. Data on 865 regulatory oligopeptides with a total of 17,723 amino acid residues have been collected. Most molecules have about 10 amino acid residues, although the range chosen was from 2 to 50. The results obtained permitted us to define the difference between oligo- and polypeptides.

Dehydration kinetics of boehmite in the temperature range 723-873 K

Energy Technology Data Exchange (ETDEWEB)

Xu, Bingan, E-mail: bingan.xu@csiro.au [A.J. Parker Cooperative Research Centre for Integrated Hydrometallurgy Solutions, CSIRO Light Metals Flagship (Division of Process Science and Engineering), PO Box 7229, Karawara, WA 6152 (Australia); Smith, Peter [A.J. Parker Cooperative Research Centre for Integrated Hydrometallurgy Solutions, CSIRO Light Metals Flagship (Division of Process Science and Engineering), PO Box 7229, Karawara, WA 6152 (Australia)

2012-03-10

Highlights: Black-Right-Pointing-Pointer The dehydration of boehmite is a complex topotactic process. Black-Right-Pointing-Pointer The process is chemical control with a diffusion complication as the reaction progresses. Black-Right-Pointing-Pointer Al cation diffusion is the rate-limiting step. Black-Right-Pointing-Pointer The reaction rates predicted by the model-free method agree well with the measured values. - Abstract: The dehydration kinetics of boehmite prepared from the hydrothermal transformation of gibbsite has been comprehensively studied to clarify the mechanism of the dehydration process using a thermogravimetric technique under both isothermal and non-isothermal conditions. Selected residues were examined by XRD, SEM, TEM and selected area electron diffraction (SAED). Results indicate that the dehydration process is a topotactic reaction which brings about {gamma}-Al{sub 2}O{sub 3} with porous microstructure and poor crystallinity. The kinetic data were analysed using the model-free (isoconversional) methods. The results indicate that the activation energy of the dehydration process depends on the reaction extent, signifying the dehydration process is chemical controlled with a diffusion complication as the reaction extent increases. Prediction of the dehydration extent was accomplished using the kinetic parameter of the non-isothermal process and the isoconversional method. The predicted rate is in a reasonably good agreement with the measured values. Finally the mechanism of the dehydration process is discussed based on the auxiliary results from the physical examination of the reacted residues and the kinetic behaviour.

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

Science.gov (United States)

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

Cross-sections of spallation residues produced in 1A GeV 208Pb on proton reactions

International Nuclear Information System (INIS)

Wlazlo, W.; Uniwersytet Jagiellonski, Cracow; Enqvist, T.; Armbruster, P.

2000-02-01

Spallation residues produced in 1 GeV per nucleon 208 Pb on proton reactions have been studied using the fragment separator facility at GSI. Isotopic production cross-sections of elements from 61 Pm to 82 Pb have been measured down to 0.1 mb with a high accuracy. The recoil kinetic energies of the produced fragments were also determined. The obtained cross-sections agree with most of the few existing gamma-spectroscopy data. Data are compared with different intranuclear-cascade and evaporation-fission models. Drastic deviations were found for a standard code used in technical applications. (orig.)

ASSOCIATION BETWEEN THE PRESENCE OF A 38 kDa FACTOR IN THE SEMINAL PLASMA AND INHIBITION OF SPERM MOTILITY IN JUNDIÁ FISH Rhamdia quelen

Directory of Open Access Journals (Sweden)

Vinicius Farias Campos

2010-06-01

Full Text Available Protein factors have been identified in the seminal plasmaof fish and mammal species and, in some situations, associatedto sperm quality indicators. However, for jundiá fish (Rhamdiaquelen, such factors and those potential associations remainunknown. In the present study, we aimed to identify some proteinfactors present in the seminal plasma of jundiá fish and to evaluatetheir association to sperm motility. SDS-PAGE was used to identify14 bands, with molecular weight ranging from 217.1 to 7.1 kDa.Sperm motility was evaluated for 21 males. Four protein bands(81.5; 60.4; 33.6; and 25.5 kDa were present in all seminal plasmasamples. One protein band with molecular weight of 38.3 kDa wasassociated to reduced sperm motility of jundiá (P<0.01, since itwas detected in 91.4% of the samples having motility lower than80%. These results suggest that this seminal protein band associatedto lower sperm motility may be considered a potential biochemicalmarker for sperm quality.

Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis.

Science.gov (United States)

Quissell, D O; Deisher, L M

1992-04-01

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.

Pleiotropic benefit of monomeric and oligomeric flavanols on vascular health--a randomized controlled clinical pilot study.

Science.gov (United States)

Weseler, Antje R; Ruijters, Erik J B; Drittij-Reijnders, Marie-José; Reesink, Koen D; Haenen, Guido R M M; Bast, Aalt

2011-01-01

Cardiovascular diseases are expanding to a major social-economic burden in the Western World and undermine man's deep desire for healthy ageing. Epidemiological studies suggest that flavanol-rich foods (e.g. grapes, wine, chocolate) sustain cardiovascular health. For an evidenced-based application, however, sound clinical data on their efficacy are strongly demanded. In a double-blind, randomized, placebo-controlled intervention study we supplemented 28 male smokers with 200 mg per day of monomeric and oligomeric flavanols (MOF) from grape seeds. At baseline, after 4 and 8 weeks we measured macro- and microvascular function and a cluster of systemic biomarkers for major pathological processes occurring in the vasculature: disturbances in lipid metabolism and cellular redox balance, and activation of inflammatory cells and platelets. In the MOF group serum total cholesterol and LDL decreased significantly (P ≤ 0.05) by 5% (n = 11) and 7% (n = 9), respectively in volunteers with elevated baseline levels. Additionally, after 8 weeks the ratio of glutathione to glutathione disulphide in erythrocytes rose from baseline by 22% (n = 15, Pbenefit of an 8 weeks supplementation with 200 mg/d MOF in humans. ClinicalTrials.gov NCT00742287.

Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

International Nuclear Information System (INIS)

Li, Dongyang; Ying, Yibin; Wu, Jian; Niessner, Reinhard; Knopp, Dietmar

2013-01-01

We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL −1 ). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immuno detection. (author)

Residual gas analysis

International Nuclear Information System (INIS)

Berecz, I.

1982-01-01

Determination of the residual gas composition in vacuum systems by a special mass spectrometric method was presented. The quadrupole mass spectrometer (QMS) and its application in thin film technology was discussed. Results, partial pressure versus time curves as well as the line spectra of the residual gases in case of the vaporization of a Ti-Pd-Au alloy were demonstrated together with the possible construction schemes of QMS residual gas analysers. (Sz.J.)

Assessing the Availability of Wood Residues and Residue Markets in Virginia

OpenAIRE

Alderman, Delton R. Jr.

1998-01-01

A statewide mail survey of primary and secondary wood product manufacturers was undertaken to quantify the production and consumption of wood residues in Virginia. Two hundred and sixty-six wood product manufacturers responded to the study and they provided information on the production, consumption, markets, income or disposal costs, and disposal methods of wood residues. Hardwood and pine sawmills produce approximately 66 percent of Virginia's wood residues. Virginia's wood product man...

Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ2 conformation by intra-residue NOEs

International Nuclear Information System (INIS)

Miyanoiri, Yohei; Takeda, Mitsuhiro; Jee, JunGoo; Ono, Akira M.; Okuma, Kosuke; Terauchi, Tsutomu; Kainosho, Masatsune

2011-01-01

Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U– 13 C, 15 N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the 13 C– 13 C and 13 C– 1 H spin coupling networks (Kainosho et al. in Nature 440:52–57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3- 2 H 2 ; δ1,ε3,η2- 13 C 3 ; ε1- 15 N]-indole ring ([ 12 C γ, 12 C ε2 ] SAIL-Trp), which provides a more robust way to correlate the 1 H β , 1 H α , and 1 H N to the 1 H δ1 and 1 H ε3 through the intra-residue NOEs. The assignment of the 1 H δ1 / 13 C δ1 and 1 H ε3 / 13 C ε3 signals can thus be transferred to the 1 H ε1 / 15 N ε1 and 1 H η2 / 13 C η2 signals, as with the previous type of SAIL-Trp, which has an extra 13 C at the C γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was 1 H β2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ 2 angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [ 12 C γ , 12 C ε2 ] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.

Intracoronary use of rubidium-82

International Nuclear Information System (INIS)

Harper, P.V.; Ryan, J.W.; Al-Sadir, J.; Chua, K.G.; Resnekov, L.; Nierinckx, R.; Loberg, M.

1985-01-01

Rubidium-82 (75 sec) is well suited for myocardial localization studies performed by intracoronary injection in connection with coronary angiography. Rubidium ion is efficiently extracted from the blood so that blood background and radiation to other organs such as the kidneys are greatly reduced, as is the amount of activity required to make satisfactory images. Pilot studies in 15 patients have resulted in no complications. Feasibility has been demonstrated for pacing studies and for drug intervention with nitroglycerine. Uptake defects correlate well with vascular lesions

50 CFR 82.13 - Ownership of property.

Science.gov (United States)

2010-10-01

...) FINANCIAL ASSISTANCE-WILDLIFE SPORT FISH RESTORATION PROGRAM ADMINISTRATIVE PROCEDURES FOR GRANTS-IN-AID... assignment of title and any compensations consistent with the terms of Federal Management Circular 74-7 or other appropriate referenced Federal Management Circulars cited in § 82.3 of this part. ...

Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

DEFF Research Database (Denmark)

Creixell, Pau; Schoof, Erwin M.; Tan, Chris Soon Heng

2012-01-01

in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different......; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues...... mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our...

Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area.

Science.gov (United States)

Elanga Ndille, Emmanuel; Doucoure, Souleymane; Poinsignon, Anne; Mouchet, François; Cornelie, Sylvie; D'Ortenzio, Eric; DeHecq, Jean Sébastien; Remoue, Franck

2016-12-01

Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes. Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation. Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species.

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis.

Science.gov (United States)

Riaz, Muhammad; Rashid, Muhammad Hamid; Sawyer, Lindsay; Akhtar, Saeed; Javed, Muhammad Rizwan; Nadeem, Habibullah; Wear, Martin

2012-01-01

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS-PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k cat = 343 and 727 s -1 , K m = 0.25 and 0.16 mg mL -1 , k cat / K m (specificity constant) = 1374 and 4510 mg mL -1 s -1 , respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.

Sterically crowded monomeric neutral bis(benzamidinato) compounds of aluminium, [PhC(NSiMe(3))(2)](2)AlX (X=Cl, H); X-ray crystal structure of [PhC(NSiMe(3))(2)]2AlH

NARCIS (Netherlands)

Duchateau, R; Meetsma, A; Teuben, JH

1996-01-01

AlCl3 reacts with [PhC(NSiMe(3))(2)]Li(OEt(2)) to afford the bis(N,N'-bis(trimethylsilyl)benzamidinato)aluminium chloro compound which, on treatment with KBEt(3)H, yields the structurally characterized monomeric hydride derivative, [PhC(NSiMe(3))(2)]2AlH, whose reactivity towards unsaturated

Quantitative myocardial blood flow with Rubidium-82 PET

DEFF Research Database (Denmark)

Hagemann, Christoffer E; Ghotbi, Adam A; Kjær, Andreas

2015-01-01

Positron emission tomography (PET) allows assessment of myocardial blood flow in absolute terms (ml/min/g). Quantification of myocardial blood flow (MBF) and myocardial flow reserve (MFR) extend the scope of conventional semi-quantitative myocardial perfusion imaging (MPI): e.g. in 1) identificat......Positron emission tomography (PET) allows assessment of myocardial blood flow in absolute terms (ml/min/g). Quantification of myocardial blood flow (MBF) and myocardial flow reserve (MFR) extend the scope of conventional semi-quantitative myocardial perfusion imaging (MPI): e.g. in 1...... global MFR and major adverse cardiovascular events (MACE), and together with new diagnostic possibilities from measuring the longitudinal myocardial perfusion gradient, cardiac (82)Rb PET faces a promising clinical future. This article reviews current evidence on quantitative (82)Rb PET's ability...

Weld residual stress according to the ways of heat input in the simulation of weld process using finite element analysis

International Nuclear Information System (INIS)

Yang, Jun Seog; Lee, Kyoung Soo; Park, Chi Yong

2008-01-01

This paper is to discuss distribution of welding residual stresses of a ferritic low alloy steel nozzle with dissimilar metal weld using Alloy 82/182. Two Dimensional (2D) thermo-mechanical finite element analyses are carried out to simulate multi-pass welding process on the basis of the detailed and fabrication data. On performing the welding analysis generally, the characteristics on the heat input and heat transfer of weld are affected on the weld residual stress analyses. Thermal analyses in the welding heat cycle process is very important process in weld residual stress analyses. Therefore, heat is rapidly input to the weld pass material, using internal volumetric heat generation, at a rate which raises the peak weld metal temperature to 2200 .deg. C and the base metal adjacent to the weld to about 1400 .deg. C. These are approximately the temperature that the weld metal and surrounding base materials reach during welding. Also, According to the various ways of applying the weld heat source, the predicted residual stress results are compared with measured axial, hoop and radial through-wall profiles in the heat affected zone of test component. Also, those results are compared with those of full 3-dimensional simulation

28 CFR 65.82 - Procedure for requesting a Presidential determination of an immigration emergency.

Science.gov (United States)

2010-07-01

... determination of an immigration emergency. 65.82 Section 65.82 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) EMERGENCY FEDERAL LAW ENFORCEMENT ASSISTANCE Immigration Emergency Fund § 65.82 Procedure for requesting a Presidential determination of an immigration emergency. (a) The President may make a...

36 CFR 223.82 - Contents of advertisement.

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Contents of advertisement... SALE AND DISPOSAL OF NATIONAL FOREST SYSTEM TIMBER Timber Sale Contracts Advertisement and Bids § 223.82 Contents of advertisement. (a) A timber sale advertisement shall include the following information...

Detailed monitoring of two biogas plants and mechanical solid-liquid separation of fermentation residues.

Science.gov (United States)

Bauer, Alexander; Mayr, Herwig; Hopfner-Sixt, Katharina; Amon, Thomas

2009-06-01

The Austrian "green electricity act" (Okostromgesetz) has led to an increase in biogas power plant size and consequently to an increased use of biomass. A biogas power plant with a generating capacity of 500 kW(el) consumes up to 38,000 kg of biomass per day. 260 ha of cropland is required to produce this mass. The high water content of biomass necessitates a high transport volume for energy crops and fermentation residues. The transport and application of fermentation residues to farmland is the last step in this logistic chain. The use of fermentation residues as fertilizer closes the nutrient cycle and is a central element in the efficient use of biomass for power production. Treatment of fermentation residues by separation into liquid and solid phases may be a solution to the transport problem. This paper presents detailed results from the monitoring of two biogas plants and from the analysis of the separation of fermentation residues. Furthermore, two different separator technologies for the separation of fermentation residues of biogas plants were analyzed. The examined biogas plants correspond to the current technological state of the art and have designs developed specifically for the utilization of energy crops. The hydraulic retention time ranged between 45.0 and 83.7 days. The specific methane yields were 0.40-0.43 m(3)N CH(4) per kg VS. The volume loads ranged between 3.69 and 4.00 kg VS/m(3). The degree of degradation was between 77.3% and 82.14%. The screw extractor separator was better suited for biogas slurry separation than the rotary screen separator. The screw extractor separator exhibited a high throughput and good separation efficiency. The efficiency of slurry separation depended on the dry matter content of the fermentation residue. The higher the dry matter content, the higher the proportion of solid phase after separation. In this project, we found that the fermentation residues could be divided into 79.2% fluid phase with a dry matter

Story of rubidium-82 and advantages for myocardial perfusion PET imaging

Directory of Open Access Journals (Sweden)

Jean-Francois eChatal

2015-09-01

Full Text Available Rubidium-82 has a long story, starting in 1954. After preclinical studies in dogs showing that myocardial uptake of this radionuclide was directly proportional to myocardial blood flow, clinical studies were performed in the 80s leading to an approval in the USA in 1989. From that time thousands of patients have been tested and their results have been reported in 3 meta-analyses. Pooled patient-based sensitivity and specificity were respectively 0.91 and 0.90. By comparison with 99mTc-SPECT, 82Rb-PET had a much better diagnostic accuracy, especially in obese patients with BMI (Body Mass Index ≥30 kg/m2 (85% versus 67% with SPECT and in women with large breasts. A great advantage of 82Rb-PET is its capacity to accurately quantify myocardial blood flow. Quite importantly it has been recently shown that coronary flow reserve is associated with adverse cardiovascular events independently of luminal angiographic severity. Moreover coronary flow reserve is a functional parameter particularly useful in the estimate of microvascular dysfunction such as in diabetes mellitus. Due to the very short half-life of rubidium-82, the effective dose calculated for a rest/stress test is roughly equivalent to the annual natural exposure and even less when stress-only is performed with a low activity compatible with a good image quality with the last generation 3D PET scanners.There is still some debate on the relative advantages of 82Rb-PET with regard to 99mTc-SPECT. For the last ten years, great technological advances substantially improved performances of SPECT with its accuracy getting closer to this of 82Rb/PET. Currently the main advantages of PET are its capacity to accurately quantify myocardial blood flow and to deliver a low radiation exposure.

Evaluation of residue-residue contact prediction in CASP10

KAUST Repository

Monastyrskyy, Bohdan

2013-08-31

We present the results of the assessment of the intramolecular residue-residue contact predictions from 26 prediction groups participating in the 10th round of the CASP experiment. The most recently developed direct coupling analysis methods did not take part in the experiment likely because they require a very deep sequence alignment not available for any of the 114 CASP10 targets. The performance of contact prediction methods was evaluated with the measures used in previous CASPs (i.e., prediction accuracy and the difference between the distribution of the predicted contacts and that of all pairs of residues in the target protein), as well as new measures, such as the Matthews correlation coefficient, the area under the precision-recall curve and the ranks of the first correctly and incorrectly predicted contact. We also evaluated the ability to detect interdomain contacts and tested whether the difficulty of predicting contacts depends upon the protein length and the depth of the family sequence alignment. The analyses were carried out on the target domains for which structural homologs did not exist or were difficult to identify. The evaluation was performed for all types of contacts (short, medium, and long-range), with emphasis placed on long-range contacts, i.e. those involving residues separated by at least 24 residues along the sequence. The assessment suggests that the best CASP10 contact prediction methods perform at approximately the same level, and comparably to those participating in CASP9.

Infrared studies of Seyfert galaxies and of the irregular galaxy M82

International Nuclear Information System (INIS)

Rodriguez Espinosa, J.M.

1985-01-01

Middle and far infrared studies of the irregular galaxy M82 and of Seyfert galaxies are presented. M 82 was observed spectrophotometrically from 8 to 13 microns at 6 different positions selected across its 10μm emitting region. The observations show that the mid-IR emitting region is fairly homogeneous and that similar physical processes are responsible for the emission observed throughout the central region of M82. A model is proposed to explain the 8 to 13μm spectrum of M82. A model accumulates 10 5 orion units in the central region of M82. The proposed model explains satisfactorily most of the observed properties of M82 from x-ray to radio wavelengths. It is also suggested that a similar model may be applied to other active nuclei, like the emission line galaxy NGC 1614 and the classical Seyfert 1 galaxy NGC 7469. For Seyfert galaxies, the dat analyzed are drawn from the recently released IRAS catalog. It is found that Seyfert galaxies are strong far infrared sources but, unlike the near and mid-IR emission from these sources, the far-IR emission does not appear to be produced by the active nucleus. Rather it is shown that the observed far-IR properties of Seyfert galaxies are consistent with their far-IR emission being produced in intense episodes of star formation taking place in or near the central regions of these galaxies

Interface strength measurement of tungsten coatings on F82H substrates

International Nuclear Information System (INIS)

Kim, Hyoungil; El-Awady, Jaafar; Gupta, Vijay; Ghoniem, Nasr; Sharafat, Shahram

2009-01-01

In the current work, hot isostatic pressing is adopted to deposit tungsten coatings on F82H substrates. The interface strength of the W/F82H samples is measured using the Laser Spallation technique and the microstructure is analyzed to determine the strength of the coating. Finally, the failure mechanisms of the hot isostatic pressing versus vacuum plasma spraying tungsten coatings and their different failure strengths are compared. It is concluded that the hot isostatic pressing process ensures a good adhesion for the W/F82H interface while the vacuum plasma spraying process results in relatively lower failure strength for the W-coating itself due to the high porosity in the coating.

Proceedings of the 7. biennial residue-to-revenue residual wood conference 2007

International Nuclear Information System (INIS)

Raulin, J.

2007-01-01

This conference provided information on the highest and best use of residual wood, which is quickly becoming a valuable commodity. Issues concerning forest residues, sawmill wastes, agricultural residues and urban organic materials were discussed along with trends in Canadian surplus mill waste production. The evolving nature and technologies of the biomass business were highlighted with particular focus on how to generate energy and save money through the use of residual wood. Residual wood energy projects and developments in Canada, North America and Europe were outlined along with biomass development in relation to forest fires and insect disturbances. Cogeneration technologies using wood wastes for thermal heat, steam and electricity were also presented, along with transportation fuel technologies for the production of ethanol. It was noted that with the rising cost of energy, the forest industry is seeking energy solutions based on the use of residual wood. The range of economically practical residual wood solutions continues to grow as energy prices increase. The conference was attended by more than 200 delegates from the forest industry, suppliers and government representatives, to discuss policies and procedures currently in place. Industry investment is being stimulated by the potential for biofuels and biochemicals, as well as the co-operation between the forest and energy sectors. The conference featured 23 presentations, of which 12 have been catalogued separately for inclusion in this database. refs., tabs., figs

Saturation behavior of irradiation hardening in F82H irradiated in the HFIR

Energy Technology Data Exchange (ETDEWEB)

Hirose, T. [Blanket Engineering Group, Japan Atomic Energy Agency, Naka, Ibaraki (Japan); Shiba, K.; Tanigawa, H.; Ando, M. [Japan Atomic Energy Agency, Tokai-mura, Naga-gun, Ibaraki-ken (Japan); Klueh, R.L. [Oak Ridge National Laboratory, TN (United States); Stoller, R. [ORNL - Oak Ridge National Laboratory, Materials Science and Technology Div., Oak Ridge, AK TN (United States)

2007-07-01

Full text of publication follows: Post irradiation tensile tests on reduced activation ferritic/martensitic steel, F82H have been conducted over the past two decades using Japan Materials Testing Reactor (JMTR) of JAEA, and Fast Flux Testing Facility (FFTF) of PNNL and High Flux Isotope Reactor (HFIR) of ORNL, USA, under Japan/US collaboration programs. According to these results, F82H does not demonstrate irradiation hardening above 673 K up to 60 dpa. The current study has been concentrated on hardening behavior at temperature around 573 K. A series of low temperature irradiation experiment has been conducted at the HFIR under the international collaborative research between JAEA/US-DOE. In this collaboration, the irradiation condition is precisely controlled by the well matured capsule designing and instrumentation. This paper summarizes recent results of the irradiation experiments focused on F82H and its modified steels compared with the irradiation properties database on F82H. Post irradiation tensile tests have been conducted on the F82H and its modified steels irradiated at 573 K and the dose level was up to 25 dpa. According to these results, irradiation hardening of F82H is saturated by 9 dpa and the as-irradiated 0.2 % proof stress is less than 1 GPa at ambient temperature. The deterioration of total elongation was also saturated by 9 dpa irradiation. The ductility of some modified steels which showed larger total elongation than that of F82H before irradiation become the same level as that of standard F82H steel after irradiation, even though its magnitude of irradiation hardening is smaller than that of F82H. This suggests that the more ductile steel demonstrates the more ductility loss at this temperature, regardless to the hardening level. The difference in ductility loss behavior between various tensile specimens will be discussed as the ductility could depend on the specimen dimension. (authors)

50 CFR 82.2 - Purpose of regulations.

Science.gov (United States)

2010-10-01

... animals within species and populations of marine mammals at the optimum carrying capacity of their habitat. ... (MARINE MAMMAL PROTECTION ACT OF 1972) Introduction § 82.2 Purpose of regulations. The Marine Mammal..., subject to such terms and conditions as he deems necessary, and after review by the Marine Mammal...

Early risk stratification using Rubidium-82 positron emission tomography in STEMI patients

DEFF Research Database (Denmark)

Ghotbi, Adam Ali; Hasbak, Philip; Nepper-Christensen, Lars

2018-01-01

BACKGROUND: Assessment of infarct size after myocardial infarction is predictive of subsequent morphological changes and clinical outcome. This study aimed to assess subacute post-intervention Rubidium-82 ((82)Rb)-PET imaging in predicting left ventricle ejection fraction, regional wall motion, a...

Low-dose myocardial blood flow imaging using 82Rb-PET (RUBILOW 2.0)

DEFF Research Database (Denmark)

Hoff, Camilla Molich; Tolbod, Lars Poulsen; Harms, Hans

2017-01-01

Aim: Relative and absolute measures of myocardial blood flow (MBF) can be derived from a 82Rb PET/CT scan using list mode data to extract static, gated and dynamic PET series. High doses of 82Rb are used to maximize image quality in especially static images. High doses require large eluate volumes......, may degrade scanner dead time performance at first pass and decreases generator lifetime. Lower doses will expand the availability of 82Rb PET/CT and lower doses to personnel and patients. The aim of the current study was to examine MBF with a low dose (LD) 82Rb protocol compared to the institution......’s standard (STD) protocol. Methods: Twenty-one patients referred to 82Rb PET were included in the study. Patients were examined during rest and stress with repeated LD (740 MBq) and STD dose (1110 MBq) Rb-PET during the same imaging session. Regional and global rest and stress MBF values, total perfusion...

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Anke K Trilling

Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

Report on Security 82

International Nuclear Information System (INIS)

Anon.

1982-01-01

The 1982 trade fair Security 82 presented a variety of equipment and systems for protection and security in general. Apart from physical protection devices to prevent theft, a great number of fire protection devices, transmitting apparatus, locator systems, metal detectors, protective devices against signal interception, and complete alarm systems were exhibited and explained. By means of specifications and brief descriptions, sometimes with figures, predominantly passive and active infrared sensors, light and microwave sensors, ultrasound and microwave Doppler effect devices for indoor and outdoor physical protection, as well as other alarm systems and fire alarm systems or entry control systems are presented in this report. Design and functioning mode of the equipment presented are illustrated by explaining the fields of employment. (orig.) [de

Double beta decay of 82Se

International Nuclear Information System (INIS)

Elliott, S.R.; Hahn, A.A.; Moe, M.K.; Nelson, M.A.; Vient, M.A.

1992-01-01

The two-neutrino double beta decay of 82 Se has been measured during a 20 244 h run resulting in a half-life of 1.08 -0.06 +0.26 x10 20 years (68% C.L.). No candidate events for the zero-neutrino double beta decay during 21 924 h results in a half-life limit of 2.7x10 22 years at the 68% confidence level

40 CFR 82.19 - Apportionment of baseline consumption allowances for class II controlled substances.

Science.gov (United States)

2010-07-01

... allowances for class II controlled substances. 82.19 Section 82.19 Protection of Environment ENVIRONMENTAL... Consumption Controls § 82.19 Apportionment of baseline consumption allowances for class II controlled... Ineos Fluor Americas HCFC-22 2,546,305 Kivlan & Company HCFC-22 2,081,018 MDA Manufacturing HCFC-22 2...

A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

Science.gov (United States)

Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

2016-03-01

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.