Studies in vitro of the nature and synthesis of the cell wall ofChlamydia trachomatis

The possibility thatChlamydia trachomatis contains peptidoglycan was examined by three methods. Preincubation of chlamydia with enzymes known to cleave peptidoglycan had no adverse effect on the subsequent development. Immunofluorescence studies with antistreptococcal peptidoglycan antisera failed to show any cross reactions with chlamydial antigens. The antichlamydial activity of anti-cell-wall antimicrobials was examined; lactams proved the most active, and cycloserine and bacitracin also showed antichlamydial activity. Alaphosphin, phosphomycin, and vancomycin showed no antichlamydial activity at the concentrations tested.     

A radioimmunoassay for 6-keto- prostaglandin F1α

A radioimmunoassay for 6-keto-prostaglandin F_1α has been developed. The assay is accurate and sensitive but since the antiserum cross-reacts 5–10% with prostaglandins (PGs) of the E and F series, solvent extraction and thin layer chromatography are required fo absolute specifity. The assay has been validated by comparison with a radiochemical assay and by the use of an inhibitor of 6-keto PGF_1α formation, 15-hydroperoxy arachidonic acid. 6-Keto PGF_1α was found to have a low cross reaction with antisera directed against PGE₂, PGF_2α and thromboxane B₂.     

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.     

Proteolytic processing of chromogranin A in cultured chromaffin cells

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.     

Processing of precursor interleukin 1 beta and inflammatory disease

The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.