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At least 811 records · Page 45Linked to original sources
PubMed1989

Structural basis for the high activation energy of spectrin self-association.

The association of spectrin hetero-dimer (alpha beta) to the tetramer (alpha 2 beta 2, which predominates in the cell) is marked by an exceptionally high activation energy, so that the reaction does not proceed measurably in the cold. We have tested the hypothesis that this is due to intra-dimer association between the alpha- and beta-chain ends, which must be broken before tetramers can form. Two mutant univalent spectrins with association defects at the alpha and beta ends, respectively, and incapable therefore of intra-dimer bonding, were found to associate rapidly with one another at 4 degrees C. The bimolecular rate constant is greater than for the association of normal dimers by 6 orders of magnitude.

Elliptocytosis, Hereditary↗
PubMed1999

An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition.

By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes.

Calorimetry, Differential Scanning↗
PubMed2006

Reduced expression of the melanocortin-1 receptor in human liver during brain death.

OBJECTIVE: There is evidence that brain death has detrimental effects on peripheral organs. Clinical and experimental studies on organ donors showed marked inflammation in tissue samples of livers and kidneys collected during brain death. The inflammatory reaction is characterized by release of cytokines and inflammatory cell infiltration. Because melanocortins and their receptors are significant modulators of inflammation, we hypothesized that downregulation of melanocortin receptors during brain death could contribute to enhance inflammation. METHODS: Using real-time polymerase chain reaction (PCR) analysis, we determined expression of melanocortin receptors in liver biopsies obtained from brain-dead organ donors before cold ischemia and in normal liver tissue during resection of benign focal lesions of the liver. Tissue biopsies were also analyzed for expression of intercellular adhesion molecule-1 (ICAM-1), which has a central function in inflammatory cell migration. RESULTS: Expression of melanocortin-1 receptor (MC1R) mRNA was markedly reduced in liver samples obtained from brain-dead organ donors compared to hepatic tissue collected during resection of benign focal lesions of the liver. Conversely, expression of the adhesion molecule ICAM-1 was significantly increased in livers of brain-dead organ donors. CONCLUSIONS: Disruption of the endogenous anti-inflammatory circuit based on MC1R could contribute to tissue damage during brain death.

Adult↗
PubMed2002

Expression of neurotensin receptors in human corneal keratocytes.

PURPOSE: The purpose of the study was to investigate whether cultured human keratocytes express the neurotensin receptors (NTR1, NTR2, and NTR3), to determine the presence of neurotensin (NT) in keratocytes, and to assess the influence of NT on these cells. METHODS: Human keratocytes were cultured in medium treated with various concentrations (10(-7)-10(-9) M) of JMV449 (a weakly degradable NT agonist). Cell proliferation and viability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis was studied by nucleus labeling with a fluorescent dye and cold light fluorometry. NT, NTR1, NTR2, and NTR3 mRNA were detected in human keratocytes by means of reverse transcriptase-polymerase chain reaction (RT-PCR). NTR1 protein was detected by Western blot analysis. Functionality of NTR1 was assessed by intracellular calcium ([Ca2+]i) measurement with a dynamic imaging microscopy system. RESULTS: RT-PCR and Western blot analysis showed the expression of the NTR1 (mRNA and protein) and NTR3 mRNA in human corneal keratocytes. NT and NTR2 mRNA were undetectable. JMV449 induced a rapid and transient [Ca2+]i increase in human corneal keratocytes that was blocked by the specific antagonist SR48692. JMV449 significantly increased cell proliferation and viability after 72, 96, and 120 hours of culture, with a maximum effect at 10(-7) M (P < 0.005). Finally, JMV449 decreased keratocyte apoptosis, whatever the concentration used (P < 0.005). CONCLUSIONS: These results indicate that cultured human keratocytes express NTR1 and NTR3 and that NT may exert physiological effects on cornea such as regulation of keratocyte proliferation and apoptosis.

Apoptosis↗
PubMedDate not supplied

[Listeria monocytogenes detection in different food products and environmental samples from a large chain of supermarkets in the city of Bahía Blanca (Argentina)].

This work on Listeria monocytogenes detection in different foods was carried out between January 2002 and July 2003. Ninety cold-served cooked meats, sliced and packaged by different methods and 132 pieces of soft cheeses were studied. These products were analyzed using the presence/absence in 25 g criterion. L. monocytogenes was not found either in foods sliced over the counter or in controlled cheeses, but it was found in 10% of sliced cold-served foods and 5% of cut and cold-served meats vacuum packaged. These results led us to investigate the presence of these pathogen bacteria in different environmental samples. A hundred and fifteen points were swabbed including processing lines, raw materials, tools, and refrigerators. L. monocytogenes was found in 13.2% of the analyzed samples: 5% in packaging sector, 6.7% in meat processing lines and 1.5% in personalized sales. These results showed the presence of sites where the microorganism may reside and create reservoirs, so that routinary measures of hygiene and disinfection were increased.

Animals↗
PubMed1990

[Localization of Entamoeba histolytica antigens using IgA].

The presence of IgA anti-Entamoeba histolytica antibodies has been demonstrates in intestinal secretions and serum of human amebiasis. We investigated which are the cellular components of trophozoites that react with IgA anti-ameba antibodies from immune serum, colostrum and human milk. The cellular localization of such antigens was accomplished by an indirect immunoperoxidase technique using anti-human IgA (alpha chain specific) labeled with peroxidase, both for light and transmission electron microscopy. Intracellular antigens were localized after permeating the parasites with cold acetone (-10 degrees C) for 3 min. and cryosections of 1 micron thick. The antigens that react with IgA antibodies from immune serum, colostrum and human milk were located in the plasma membrane and the internal portion of some cytoplasmic vesicles. So far, it is unknown what is the biological function of IgA in human amebiasis but in other systems it protects against certain parasites.

Animals↗
PubMed1980

An epidemic chain model.

An epidemic chain model is developed by assuming a beta distribution for the probability of being infected by contact with a given infective from the same household. This model includes, as a particular case, the epidemic chain model corresponding to the stochastic Kermack-McKendrick model and, as a limiting case, the Reed-Frost chain binomial model. The advantages of the more general model are illustrated with an application to household data for the common cold. Finally, it is shown how the coefficient of variation of the duration of the infectious period may be estimated without any direct observations on this duration.

Common Cold↗
PubMed1994

Properties and structure of spermidine acetyltransferase in Escherichia coli.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

Acetyl Coenzyme A↗
PubMed1994

Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex.

Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.

Adrenal Cortex↗
PubMed1999

Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition.

CspB is a small acidic protein of Bacillus subtilis, the induction of which is increased dramatically in response to cold shock. Although the exact functional role of CspB is unknown, it has been demonstrated that this protein binds single-stranded deoxynucleic acids (ssDNA). We addressed the question of the effect of base composition on the CspB binding to ssDNA by analyzing the thermodynamics of CspB interactions with model oligodeoxynucleotides. Combinations of four different techniques, fluorescence spectroscopy, gel shift mobility assays, isothermal titration calorimetry, and analytical ultracentrifugation, allowed us to show that: 1) CspB can preferentially bind poly-pyrimidine but not poly-purine ssDNA templates; 2) binding to T-based ssDNA template occurs with high affinity (K(d(25 degrees C)) approximately 42 nM) and is salt-independent, whereas binding of CspB to C-based ssDNA template is strongly salt-dependent (no binding is observed at 1 M NaCl), indicating large electrostatic component involved in the interactions; 3) upon binding each CspB covers a stretch of 6-7 thymine bases on T-based ssDNA; and 4) the binding of CspB to T-based ssDNA template is enthalpically driven, indicating the possible involvement of interactions between aromatic side chains on the protein with the thymine bases. The significance of these results with respect to the functional role of CspB in the bacterial cold shock response is discussed.

Bacterial Proteins↗
PubMed1975

[High titer cold agglutinins with anti-pr specificity after rubella infection (author's transl)].

High titer cold agglutinins (CA) after rubella infection are reported. When the rubella exanthema disappeared the clinical aspect of a cold agglutinin disease was observed. Three weeks after the appearance of the cutaneous eruption the CA titer reached a maximum of 1/8000, to then continuously fall off to normal values within 20 weeks. Double diffusion tests showed that the isolated CA were IgM proteins that possess only chi-type light chains. In spite of normal protein- and immunoelectrophoresis patterns obtained with whole serum samples, the isolated CA showed restricted electrophoretic mobility and a deformation of the precipitate typical for monoclonal immunoglobulins. In contrast to the common anti-I specificity of IgM CA, the IgM CA described showed anti-Pr specificity. Possible interrelations between CA specificities and types of germs inducing reactive cold agglutination are discussed.

Adult↗
PubMed1984

Evaluation of nine different fixatives. 1. Preservation of immunoglobulin isotypes, J chain, and secretory component in human tissues.

Eight cross-linking fixatives were tested for their ability to preserve IgG, IgA, IgM and IgD isotypes, J chain, and secretory component (SC) in paraffin-embedded specimens of human tonsils and colonic mucosa. The results were compared with the antigenic preservation afforded by cold 96% ethanol (with or without inclusion of a prefixation 48-hr washing period). A semiquantitative immunofluorescence scoring system was applied. In relation to the cytoplasmic scores of 3.0 assigned to IgG and IgA immunocytes after ethanol fixation, the following median scores were obtained with the other fixatives: routine formalin, 1.3 (IgG and IgA); glutaraldehyde (1%)-formalin, 0.3 (IgG and IgA); Baker's formol calcium, 2.3 (IgG) and 1.5 (IgA); formol sublimate, 1.0 (IgG) and 1.2 (IgA); acetic acid (2%)-formol saline, 2.0 (IgG and IgA); Bouin's fluid and Susa fixative, 0 (IgG) and 1.3 (IgA); and carbodiimide, 2.3 (IgG) and 2.0 (IgA). Most aldehyde-based fixatives afforded poorer result for IgA immunocytes when an anti-alpha-chain reagent of restricted specificity was applied. The result was usually slightly better for IgM cells than for IgG cells, but IgM did not resist fixation in Bouin's fluid and Susa fixative to the same extent as IgA. IgD immunocytes were poorly revealed except after fixation with Baker's formol calcium, carbodiimide, and acetic acid-formol saline, which afforded median fluorescence scores of 1.0-1.8 compared with 3.0 after ethanol fixation. J chain in IgA immunocytes was most intensely stained in sections of ethanol-fixed tissue denatured in acid urea (median score, 3) but was also well revealed after fixation with formalin, acetic acid-formol saline, Bouin's fluid or Susa fixative (median scores, 1.8-2.0). The results with most of the cross-linking fixatives were much less favorable for IgA, IgM, J chain and SC in colonic crypt epithelium than in immunocytes. Only carbodiimide afforded fluorescence scores for IgA and SC approaching those obtained after ethanol fixation (2.0 and 1.5 vs. 3.0 and 2.8). The effect of proteolytic unmasking depended on the fixative - not only for different antigens and locations in the tissue, but also for the same antigen in apparently similar cells.

Colon↗
PubMed1999

Plasmapheresis and paraproteinemia: cryoprotein-induced diseases, monoclonal gammopathy, Waldenström's macroglobulinemia, hyperviscosity syndrome, multiple myeloma, light chain disease, and amyloidosis.

Therapeutic plasmapheresis has been in widespread use as either a primary or adjunctive therapy in the United States since the 1960s. There are several types of plasmapheresis procedures used to treat various diseases. Plasma exchange with a centrifugal plasma separator using replacement fluid such as human albumin solution is the most widely used method in the United States. Other forms of plasmapheresis include membrane plasma separation, membrane fractionation, cryofiltration apheresis, immunoadsorption, and chemical affinity column pheresis. Therapeutic plasmapheresis has been used for the treatment of paraproteinemia to remove harmful paraproteins. Paraproteinemia is a disease classification in which abnormal or large amounts of plasma proteins such as cryoproteins or immunoglobulins are produced. In most cases, plasmapheresis is used in combination with corticosteroids and immunosuppressive drugs to prevent production of abnormal proteins or to treat the underlying disease. Cryoprotein-induced diseases, which include cryoglobulinemia, cryofibrinogenemia, and cold IgM antibody agglutinin with cryoglobulin properties, are a subclass of paraproteinemia. Other categories of paraproteinemia include monoclonal gammopathy, Waldenström's macroglobulinemia, hyperviscosity syndrome, multiple myeloma, light chain disease, and amyloidosis. Some of these diseases may be interrelated, and they may be associated with one another. In this review paper, we discuss the role of plasmapheresis in the specific classes of paraproteinemia in the United States, including our own experience.

Amyloidosis↗
PubMed2004

BRAF mutations are not an alternative explanation for the molecular etiology of ras-mutation negative cold thyroid nodules.

Activation of the RAS/RAF/MEK/ERK/MAP kinase pathway is a known mediator of signaling that results in cellular proliferation. Moreover, this activation can lead to a growth advantage of tumor cells. Therefore, mitogenic mutations in the RAS family of oncogenes are detectable in a significant percentage in most tumors. Moreover, mutations in the BRAF gene have recently been suggested as an alternate predominant cause of colorectal and papillary thyroid cancers without ras mutations. Similar to neoplasms of other organs mutations of all three ras genes can be found in thyroid tumors. In our set of 40 cold thyroid adenoma and adenomatous nodules ras mutations were detected in only a single case. We therefore tested these hypofunctional tumors for BRAF mutations. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 11 and 15 of the BRAF gene were polymerase chain reaction (PCR) amplified and denaturing gradient gel electrophoresis was used to screen for mutations. No point mutation could be found in the two exons of the BRAF gene. Our study suggests that BRAF mutations are rather rare in solitary cold adenomas and adenomatous nodules and do not explain the molecular etiology of ras mutation-negative cold thyroid nodules.

Exons↗
PubMed1995

Differential regulation of epidermal growth factor and transforming growth factor-alpha messenger ribonucleic acid in the rat anterior pituitary and hypothalamus induced by stresses.

Evidence has shown that epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are present in the anterior pituitary as well as the hypothalamus, and that EGF can influence the function of pituitary cells, particularly corticotropes in vivo and in vitro. However, little is known about their exact functional roles and how they are regulated in these two areas. The present study was designed to determine if EGF and TGF alpha messenger RNA (mRNA) are expressed in the rat anterior pituitary and hypothalamus and how stress conditions such as cold, ether, or restraint affect their local expression. A sensitive mRNA detection method, the ribonuclease protection assay, detected both EGF and TGF alpha mRNA in the rat anterior pituitary and hypothalamus. Reverse transcription-polymerase chain reaction (RT-PCR) further showed the presence of EGF and TGF alpha mRNA in these two areas and several other rat tissues (submandibular gland, liver, kidney, lung cerebral cortex, and testis). No TGF alpha mRNA was found in the kidney, however. EGF mRNA was up-regulated in the anterior pituitary after 30 min acute cold stress (CS) and restrainer-restraint stress (RS) but not 30 min after ether stress (2 min, ES), novelty stress (NS), or tape-restraint stress (TS). Further analysis showed that EGF mRNA expression decreased after 1 h CS (1C) and then increased after 3 h CS (3C). In contrast, TGF alpha mRNA in the anterior pituitary and hypothalamus and hypothalamic EGF mRNA did not show significant changes in response to either acute stresses (CS, ES, RS, TS, NS) or longer CS (1C, 3C). Our results suggest that 1) EGF, is up-regulated after some stresses; 2) increased pituitary EGF mRNA in response to stresses varies with the type of stress; and 3) pituitary TGF alpha and hypothalamic EGF and TGF alpha may be not involved in the stress response.

Adrenocorticotropic Hormone↗
PubMed2006

A multiplex RT-PCR method for screening of reassortant live influenza vaccine virus strains.

A system based on reverse transcription polymerase chain reaction (RT-PCR) of the RNA genome was established to identify genetic composition of influenza viruses generated by reassortment between an attenuated donor virus and virulent wild type virus. The primers were designed, by multiple sequence alignment of variable regions, specific for cold-adapted donor virus HTCA-A 101, as compared to other influenza A viruses. The specificity of each primer set was confirmed and the primers were combined to perform RT-PCR in multiplex manner. The multiplex PCR was adopted to distinguish the 6:2 reassortant viruses containing six internal genome segments of attenuated donor virus and two surface antigens of virulent strain from the wild type viruses. The method allowed us to optimize the reassorting process on a routine basis and to confirm the selection of reassortant clones efficiently. The method is suitable for analyzing the contribution of specific gene segments for growth and attenuating characteristics and for generation of live attenuated vaccine by annual reassortment.

Adaptation, Physiological↗
PubMed1980

Cold-sensitive mutations in beta and beta' subunit gene affecting the interaction of RNA polymerase with promoters.

RNA polymerases with a cold-sensitive activity were purified from seven mutants of Escherichia coli. Subunit reconstitution experiments have shown that RNA polymerases from three mutants (Rpob262, RpoB264, and RpoB265) owed their cold sensitivity to alterations in the beta subunit. Three mutants (RpoC3, RpoC263, and RpoC267) were shown to be defective in the beta' subunit and one (RpoBC266) in both beta and beta' subunits. Two mutations (rpoC3 and rpoC263) reduced the level of RNA polymerase reconstitution. RNA polymerases from RpoC3 and RpoBC266 mutants are defective in RNA chain elongation at 6 degree C, while all the other mutants are defective in RNA polymerase-promoter interaction. most mutant RNA polymerases differ from the wild-type enzyme in transcription selectivity. The results obtained in this study indicate that both beta and beta' subunits are involved in RNA chain elongation and promoter binding and selection.

Bacteriocin Plasmids↗