Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.     

Structure and function of hemoglobin in antarctic fishes and evolutionary implications

Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)     

Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.

DNA from several mammals, including humans, was found to contain one or more restriction enzyme digested DNA fragments which hybridized to the mouse VpreB gene under stringencies demonstrating at least 70% nucleotide sequence homologies, indicating that the VpreB locus may be widespread and highly conserved among mammals. A human VpreB genomic clone was isolated and sequenced. Two exons and the intervening intron are spaced almost identically as in the mouse VpreB1 gene, and show 76% sequence homology to the mouse gene. As in the mouse VpreB1 gene, the 5' end of the human VpreB gene contains characteristic features of Ig domains, while the 3' end is Ig non-related. This 3' Ig non-related structure of the VpreB gene(s) may, therefore, have existed before the speciation of humans and mice over 65 million years ago. Sequences encoding the entire putative second framework region and a stretch in the third framework region are identical in human and mouse VpreB. the human VpreB gene appears to be selectively expressed in human pre-B cell lines as an 0.85 kb poly(A)+ RNA. Its expression promises to be a useful marker for the detection of normal and malignant human pre-B lymphocytes.     

Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.     

Lethal activity of miconazole in Neisseria gonorrhoeae grown in pure and mixed cultures

Abstract Low concentrations (e.g. 2 × 10⁻⁶ M) of an imidazole derivative anti-fungal agent, miconazole, were lethal for the Gram-negative, facultative aerobic pathogen Neisseria gonorrhoeae grown either alone or in mixed culture with the yeast Candida albicans . Electron microscopic observation of Neisseria cells exposed to miconazole showed the presence of blebs in the outer wall and areas of separation between the wall and the cytoplasmic membrane. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell lysates did not reveal differences in major outer membrane proteins between the treated and the untreated cells of any one strain. Imidazole derivatives are frequently used in the treatment of candidiasis. Our in vitro results show that low concentrations of one of them, miconazole, can be bactericidal for N. gonorrhoeae , a bacterium that can colonise sites of the human body where Candida is often found.     

Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae

S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.     

Morphometric characteristics of hepatocellular dysplasia

Morphometric study of liver biopsies from six entities (normal tissue, post-hepatitis cirrhosis, post-alcoholic cirrhosis, cancer-related cirrhosis, hepatocellular adenoma and hepatocellular adenocarcinoma) confirmed that this technique can be a valuable adjunct to histopathologic study in the examination of such specimens. As expected, measurements in cirrhotic nodules showed two populations of cells. The so-called "large dysplastic cells" had nuclear and cellular areas close to those of normal hepatocytes and should thus be considered to be hyperplastic elements, not precancerous elements. The smaller dysplastic cells had morphometric values close to those of the corresponding hepatocellular carcinomas, indicating that these cells are the truly precancerous ones. Therefore, while the study confirmed that hepatic cirrhosis is a precancerous lesion, it also showed that the term hepatocellular dysplasia must be restricted to the smaller type of cells found in such nodules.     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.