Effect of ionizing radiation on the functional state of the vascular-thrombocytic component of the hemostasis system

The influence of ionizing radiation (gamma-rays 60Co) on aggregation activity of the vascular wall and functional (aggregation) platelet activity was studied in the course of the development of acute radiation sickness. The decrease in the aggregation properties of the vascular wall and high functional activity of platelets were inversely proportional, correlating with the periods of acute radiation sickness development and depending on the radiation dose. It is suggested that the changes detected may play a role in the pathogenesis of the development of the postirradiation thrombohemorrhagic syndrome.     

The role of protons in a medium in gamma-radiation-induced damage of nucleic acids

The study of the combined effect of gamma-radiation and acid medium (pH 7.0-2.0) on DNA and RNA showed that the radiation-induced injury to nucleic acids increased with increasing concentration of H+-ions in the medium up to pH values below which protons exerted a protective action. Irradiation of native DNA in acid medium, as compared to neutral one, increased not only the number of injured bases but also the average size of the induced local defect in the secondary structure. It was shown that the proton sensitization was determined both by the number of protonated bases and by the degree of ordering the polynucleotide chain.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

Volatile fatty acids as indicators of process imbalance in anaerobic digestors

In continuously stirred tank reactor experiments, with manure as substrate at thermophilic temperatures, the use of volatile fatty acids (VFA) as process indicators was investigated. Changes in VFA level were shown to be a good parameter for indicating process instability. The VFA were evaluated according to their relative changes caused by changes in hydraulic loading, organic loading or temperature. Butyrate and isobutyrate together were found to be particularly good indicators. Butyrate and isobutyrate concentrations increased significantly 1 or 2 days after the imposed perturbation, which makes these acids suitable for process monitoring and important for process control of the anaerobic biological system. In addition it was shown in a batch experiment that VFA at concentrations up to 50 mM did not reduce the overall methane production rate. This showed that VFA accumulation in anaerobic reactors was the result of process imbalance, not the cause of inhibition, thus justifying the use of VFA as process indicators.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Lipid composition of thylakoid membranes of cadmium treated wheat seedlings.

Cadmium (200 ppm) applied through the rooting medium to 30-day-old wheat plants decreased chlorophyll content, net CO2 exchanges and PSII activity by 34, 54 and 43% respectively. Thylakoid total lipids, total glycolipids, total phospholipids and total neutral lipids decreased by 22, 23, 12 and 25%, respectively, under cadmium treatment. Thylakoid membrane glycolipids had three major constituents, viz. monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulphoquinovosyl diacylglycerol. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol contents decreased by 32 and 27%, respectively, under cadmium. Cadmium application also decreased the concentration of phosphatidyl glycerol and phosphatidyl choline to the extent of about 57 and 31%, respectively. On the other hand, phosphatidic acid and free fatty acids content showed an increase. These compositional changes in thylakoid membranes might be responsible for reduced PSII activity and rate of photosynthesis as observed under cadmium treatment.