Cystic fibrosis carrier screening at first diagnosis of pregnancy in general practice.
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Carrier State↗
Biomedical subjects
A Wallace
Publications and source records attributed to A Wallace.
Beyond silver staining.
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Electrophoresis, Polyacrylamide Gel↗
Ultramicrodetection of proteins in polyacrylamide gels.
Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.
Electrophoresis, Polyacrylamide Gel↗
Characterization of Chilean, Bolivian, and Argentinian Trypanosoma cruzi populations by restriction endonuclease and isoenzyme analysis.
Ninety-one Chilean, 15 Bolivian, and 9 Argentinian Trypanosoma cruzi stocks, isolated from various hosts and vectors, were characterized by schizodeme analysis with EcoRI and MspI endonucleases. The three major similar pattern groups that emerged from this sample correlated with results of isoenzyme analysis. This result confirms previous work and supports the hypothesis of the clonal structure of natural populations of T. cruzi, fully defined at the level of isoenzyme analysis, quantitative kinetoplast DNA restriction fragment length polymorphism, and kinetoplast DNA hybridization analysis. In Chile, sylvatic and domestic cycles of T. cruzi transmission appear to be mainly independent: genetically different families of natural clones are specific to these cycles. Nevertheless, the possibility of overlap remains unclear. Results described here indicate that natural clones inhabiting Chilean regions appear genetically related to the natural clones identified in neighboring countries. In Chile the more frequently sampled parasite types are natural clone 39 and a genetically closely related clone NP13. In this work an evaluation of T. cruzi natural clone mixtures in T. cruzi stocks from Chile was performed for the first time by schizodeme analysis before and after serial transfer in mouse maintenance. The results indicate that six of nine stocks are composed of two or more natural clones. This observation raises the relevant question of whether specific T. cruzi natural clones generate different clinical features of Chagas' disease.
Animals↗
Haemagglutinins and fimbriae of soft rot Erwinias.
Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c. carotovora and E.c. atroseptica.
Animals↗
Generation and characterization of a monoclonal antibody specific for the major thiol-activated cysteine proteinase of Porphyromonas gingivalis W83.
An IgM monoclonal antibody specified for the thiol-activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno-electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol-activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol-activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.
Antibodies, Bacterial↗
Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells. A model for scatter factor modulation.
Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional cytokine produced by embryonic fibroblasts and other mesenchymal cells that affects the growth and/or the movement of certain epithelia. Here we report that expression of scatter factor activity by MRC 5 cells, a strain of normal human embryonic lung fibroblasts, is greatly reduced as a result of co-culture of these cells with SVK14, an SV40-transformed human keratinocyte cell line. Using a cDNA probe to the beta chain of human HGF, we have found that the fall in SF activity in MRC 5/SVK14 co-cultures is accompanied by the loss of SF/HGF transcripts. As the inhibition of SF activity coincides with the disappearance of SF/HGF transcript, we conclude that inhibition of the SF activity expressed by MRC 5 cells by co-culture with SVK14 involves transcriptional regulation.
Animals↗
Studying DNA modifications and DNA-protein interactions in vivo. A window onto the native genome.
The study of native genomes has been greatly facilitated by the use of direct genomic sequencing and footprinting strategies. This review provides an overview of the techniques involved and presents some highlights of the biological results obtained with these methods.
Animals↗
Racial differences in drug response: isoproterenol effects before and after propranolol.
The aim of this study was to determine in young, healthy men the relative contribution of pharmacodynamic factors inherent between two groups known to respond differently to hypertensive therapy. Black (n = 10) and white (n = 10) men received an isoproterenol sensitivity test before and after propranolol (0.1 mg/kg, then 50 micrograms/min). There were greater increases (twofold) in systolic BP following the 1.0- and 1.5-microgram isoproterenol dose (P less than 0.05) in the black group. During propranolol there were no differences in free (1)-propranolol concentrations between the groups; however, propranolol decreased resting heart rate in the white group more than in the black group (P less than 0.05). Cardiac index decreased less in the black group compared to the white group (P less than 0.05). Following the second isoproterenol challenge, there again were greater increases in systolic BP in the black group at both the 10- and the 20-micrograms isoproterenol dose (P less than 0.05). Our study has highlighted the importance of cross-racial studies in evaluating drug effects.
Adult↗
Profiles: White House nurse. Interview by Marlene Jezierski.
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Government↗
Use of fragments of hirudin to investigate thrombin-hirudin interaction.
Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.
Antithrombin III↗
Cloning and expression of a Porphyromonas (Bacteroides) gingivalis protease gene in Escherichia coli.
A genomic library of Bacteroides gingivalis W83 chromosomal DNA was constructed in the Escherichia coli lambda (lambda) vector EMBL 4. Three recombinant lambda phages expressing a cloned protease were identified in the library. All three lambda phages contained cloned overlapping DNA fragments from the same region of the chromosome and encoded the same cloned protease. The cloned protease was expressed poorly using its own promoter in E. coli.
Bacteroides↗
Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice.
Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.
Animals↗
Trypanosoma cruzi: a possible control of transfusion-induced Chagas' disease by phenolic antioxidants.
The following phenolic antioxidant food additives were evaluated against Trypanosoma cruzi epimastigotes: BHT, BHA, gallic acid and its methyl, propyl, octyl, and lauryl esters, 2,4-di-tert-butyl-6-(4-methoxybenzyl)-phenol, 4,4'-isopropilidenediphenol, and protocatechuic acid and its ethyl ester. The inhibition of the respiration; the changes in motility, shape, and lysis of the parasites; and the human blood hemolysis caused by these chemicals were studied. Human blood samples experimentally contaminated with 2000 or 150,000 trypomastigotes per milliliter were freed of parasites after treatment for 24 hr at 4 degrees C with 5 or 10 mM BHT (2,6-di-tert-butyl-4-hydroxytoluene), respectively. Consequently, BHT and other phenolic compounds deserve further study to determine their role in preventing the transmission of Chagas' disease by blood transfusion.
Animals↗
Racial differences in baseline cyclic adenosine monophosphate concentrations per million T-lymphocytes and protein concentrations.
beta-Adrenergic blockers are less efficacious as monotherapy for the treatment of hypertension in blacks as compared with whites. Because beta-adrenergic stimulation and blockade differ between racial groups, biochemical differences in the beta-adrenergic pathway may exist. It is the intent of this report to show underlying similarities and differences, at least in part, in the beta-adrenergic pathway (e.g., baseline cAMP and protein concentrations) using the T-lymphocyte as the model system. A total of 20 (n = 10 black, n = 10 white) normotensive male volunteers were recruited, begun on a low-sodium diet to normalize serum catecholamines, and blood was collected for lymphocyte beta-receptor isotherm binding experiments and cAMP determination. There were no differences in Bmax, sites per cell, or kd. Basal cAMP concentrations were significantly higher in the black group (16.0 +/- 9.8 pmol/10(6) cells) compared with the white group (7.0 +/- 1.8 pmol/10(6) cells) (p less than 0.05). Protein levels from the lymphocyte suspension were also higher in the black group (1,081.0 +/- 367.7 micrograms/ml) compared with the white group (766.8 +/- 220.4 micrograms/ml) (p less than 0.05). Normalization of cAMP for protein yielded 83.2 +/- 55.4 fmol/micrograms protein in the black group and 56.6 +/- 29.8 fmol/micrograms protein in the white group (p = 0.11). Altered protein levels may be a confounding variable in studies of this type. Further work is necessary to identify the nature and significance of this protein elevation, its relationship to the adenylate cyclase system in lymphocytes, and the source of the cAMP elevation noted herein.
Adrenergic beta-Antagonists↗
Contribution of the N-terminal region of hirudin to its interaction with thrombin.
Hirudin is a 65-residue polypeptide that specifically inhibits thrombin by forming a tight, noncovalent complex with the enzyme. The role of the two amino-terminal valine residues and the N-terminal alpha-amino group of hirudin in the formation of the complex has been investigated by site-directed mutagenesis and chemical modification. Replacement of the two N-terminal valyl residues of recombinant hirudin by polar amino acids resulted in an increase in the inhibition constant (KI). In contrast, replacement of these residues by hydrophobic amino acids had little effect on the value for KI. These results demonstrated that the hydrophobic nature of the N-terminal residues of hirudin was important for its interaction with thrombin. Addition of a single amino acid to the N-terminus of hirudin resulted in a marked increase in the value of KI. A similar effect was observed when the positive charge of the alpha-amino group was removed by acetylation. In contrast, amidination of this group, which preserves the positive charge, resulted in a less pronounced increase in the value of KI. Thus, it appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.
Amino Acid Sequence↗
Effect of heparin on the glia-derived-nexin-thrombin interaction.
In order to determine the specificity of the interaction between thrombin and glia-derived nexin (GdN), the inactivation of proteolytically modified human thrombin species by GdN has been studied. The second-order rate constants for the inactivation of alpha-, beta T-, gamma T- and epsilon-thrombin by GdN were 1.41, 0.63, 0.33 and 1.91 microM-1.s-1 respectively. The kinetic properties of gdN were also investigated in the presence of different types of heparin, fractionated according to antithrombin III-binding affinity. Association rate constants of both gdN and antithrombin III with alpha-thrombin were obtained using unfractionated, low- and high-affinity heparin types. The different heparin types gave optimal rates of inhibition at similar heparin concentrations for both inhibitors. At optimal heparin concentrations, the rate of inactivation of alpha-thrombin by GdN was 0.5-1.2 nM-1.s-1, which suggests that, under these conditions, the interaction is diffusion-controlled.
Amyloid beta-Protein Precursor↗
Management of proximal humeral fractures.
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Humans↗