Specific inactivation of lysosomal glycosidases in living fibroblasts by the corresponding glycosylmethyl-p-nitrophenyltriazenes.

Glicentin (a highly purified 100-amino acid peptide with glucagon-like immunoreactivity from porcine gut) was subjected to limited digestion with trypsin and carboxypeptidase B, and the resulting peptides were studied by gel filtration and region-specific glucagon radioimmunoassays. Similar digests of glucagon and purified fragments of glucagon were studied in parallel. Glicentin gave rise to peptides that corresponded closely to the 1-17 and 19-29 fragments of glucagon. Also, 125I-labelled glicentin and 125I-labelled glucagon gave rise to identical fragments after trypsin treatment. On the basis of this and other evidence [Jacobsen, Demandt, Moody & Sundby (1977) Biochim. Biophys. Acta 493, 452-459] it is concluded that glicentin contains the entire glucagon sequence at residues number 64-92 and thus fulfills one of the requirements for being a 'proglucagon'.     

Liposome-cell interactions. A rapid assay for cells in suspension culture

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interaction. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.     

Carcinogen aflatoxin B1 is located preferentially in internucleosomal deoxyribonucleic acid following exposure in vivo in rainbow trout

The purpose of this work was to investigate the distribution in chromatin of deoxyribonucleic acid (DNA) adducts of aflatoxin B1, following exposure in vivo. Rainbow trout were injected intraperitoneally with radiolabeled aflatoxin B1, a potent procarcinogen known to readily induced hepatocellular carcinomas in these fish. After maximum incorporation, liver nuclei were prepared and digested with micrococcal nuclease. Mono-, di-, and trinucleosomal fractions were purified from several stages of nuclease digestion, and the lengths and specific activities of their DNA were determined. The results indicate that aflatoxin B1 is approximately 5 times as likely on a per nucleotide basis to localize on internucleosomal (linker) DNA as on nucleosomal core DNA in this system.     

The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.     

Production of rhodomycins inStreptomyces griseoruber 4620

The strainStreptomyces griseoruber 4620 produces, besides the anthracycline antibiotics beromycins, some other anthracyclines of the rhodomycin type. Twelve isolates exhibiting a higher antibiotic activity (up to 2.5×), as compared to the parent strain, were obtained after a spontaneous selection. The following species were isolated from the hydrolysate of mycelial extract: β-rhodomycinone, β-isorhodomycinone, α₂-rhodomycinone and 10-deoxy-β-rhodomycinone, which has not yet been described.     

Anhydrobiotic Coiling of Nematodes in Soil

Nematodes of three genera (Acrobeloides sp., Aphelenchus avenae, and Scutellonema brachyurum) were induced to coil and enter anhydrobiosis in drying soil of two types: sandy loam and loamy sand. Coiling was studied in relationship to soil moisture characteristics. Coiling and the physiological state of anhydrobiosis occurred before the water in sandy soils reached a water potential of -15 bars. Coiling was maximum at 3-6 bars, depending on the soil type and nematode species. It appeared that induction of coiling and anhydrohiosis were determined by the physical forces exerted by the water film surrounding the nematode, which, for these three species, was 6-9 monomolecular layers of water, rather than the % moisture and relative humidity of the soil per se.     

Effects of cycloheximide on the uterine refractory state induced by 'nidatory' oestrogen in rats.

After priming with oestradiol, ovariectomized rats were given 6 days of progesterone treatment in which two doses of 50 ng oestradiol were given on Days 3 and 6. This basic treatment allows the oestradiol-induced (1st injection) disappearance of uterine sensitivity to decidual stimuli to occur. Cycloheximide could not mimic oestrogen action in the production of the uterine refractory state. However, a high dose (500 micrograms per animal) of this inhibitor given with the first injection of oestradiol allowed the uterus to remain in a neutral state and to respond to decidual induction after the second dose of oestradiol. By delaying the injection of cycloheximide after the first oestrogen treatment, protein synthesis requisite to the occurrence of uterine refractoriness would not take place within 12 h after the 'nidatory' oestrogen injection.