Direct detection of recombinant gene expression by two genetically engineered yeasts in soil on the transcriptional and translational levels.

The expression of a recombinant gene by yeasts seeded into soil samples was directly measured by analyzing transcripts and gene product occurrences in soil extracts. Two yeast species, Saccharomyces cerevisiae WHL292 and Hansenula polymorpha LR9-Apr4, both engineered by a synthetic gene sequence encoding the mammalian peptide aprotinin, produced and secreted this peptide in batch cultures at concentrations of 90 and 64 ng ml-1, respectively. In S. cerevisiae, the aprotinin gene was located on plasmid p707 and expressed constitutively. H. polymorpha carried the gene chromosomally integrated, and its expression was inducible by methanol. To detect aprotinin transcripts, cells were directly lysed in the soil samples and the crude lysates were hybridized to oligo(dT)-coated magnetized polystyrene beads (Dynabeads). After separation and purification in a magnetic field, aprotinin mRNA was detected by reverse transcriptase PCR with aprotinin gene-specific primers. Transcripts from 10 cells g of soil-1 were sufficient for detection. When 10(7) cells of S. cerevisiae were inoculated into soil, aprotinin mRNA was detectable during the first 4 days. Addition of methanol and a combined nutrient solution was necessary to induce aprotinin gene expression of H. polymorpha in soil. Aprotinin could be detected directly in soil extracts by an indirect enzyme-linked immunosorbent assay with monoclonal aprotinin-specific antibodies. The detection threshold was 45 pg g of soil-1. In presterilized soil inoculated with S. cerevisiae (10(6) CFU g-1), aprotinin accumulated during the first 10 days to 12 ng g of soil-1 and then remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

The stability of A1,B29-adipoylinsulin to chemical and enzymatic degradation.

The degradation of A1,B29-adipolyinsulin by partial hydrolysis, by disulphide-bond exchange, by chymotrypsin and by a cytosolic enzyme fraction of rat liver was measured and compared with that of A1,B29-diacetylinsulin and insulin. The cross-linked insulin is, in general, more resistant than insulin itself or the diacetylated derivative. However, in the case of enzymatic degradation, A1,B29-diacetylinsulin resembles more the cross-linked derivative than insulin. This provides evidence that the properties of A1,B29-adipoylinsulin could not in all circumstances be attributed to its 'proinsulin-like' structure.

Radioimmunological determination of cyproterone acetate.

A sensitive and specific radioimmunoassay for cyproterone acetate in serum has been developed. Antiserum was raised in rabbits by immunization with a conjugate of cyproterone acetate-11 alpha-hemisuccinate and bovine serum albumin. Radioactive tracers with high specific activities were synthesized by coupling the hemisuccinate with [3H]-tyrosine methyl ester of [125I]-iodohistamine. Competition studies with substances structurally related to cyproterone acetate have revealed that the structures of ring A and B are mainly responsible for binding to the antibody.

Continuous oral low-dosage cyproterone acetate for fertility regulation in the male? A trend analysis in 15 volunteers.

PIP: The effects of cyproterone acetate (CPA), administered orally in dos es of either 10 or 20 mg/day for 26 weeks, were studied in 15 healthy male volunteers. During treatment, there was a decrease in sperm density and in normal-shaped sperm and an increase in pathological and immature forms, an increase in dead spermatozoa, reduced motility of spermatozoa, an in vitro decrease in the speed of sperm transport, and decreased ability of spermatozoa to penetrate ovulatory cervical mucus. Blood plasma levels of testosterone declined to about 40% of basal values, though there were no effects on sex behavior. Plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were unaltered, while seminal plasma alkaline phosphatase values were considerably increased. Seminal plasma levels of fructose and sialic acid levels were unchanged, while the cortisol-binding capacity of transcortin was markedly increased. The results indicate that CPA has potential as a fertility control agent in males, though further study on a mass phase 3 scale is required before final conclusions can be made.^ieng

A controlled study with Dihydergot on patients with orthostatic dysregulation.

(1) 36 outpatients with a manifest orthostatic syndrome were the subject of a double-blind study to investigate the influence of Dihydergot and a placebo over a 4-week period. (2) In the majority of subjects, the dosage was 1 tablet (2.5 mg) 3 times a day. (3) In the test group with Dihydergot (17 subjects), a normalization of circulatory regulation occurred in all cases. (4) In contrast, the placebo group (19 patients) showed no changes. (5) The subjective complaints of which only a part can be attributed to the orthostatic syndrome, largely disappeared through the treatment with Dihydergot, while those in the placebo group were not altered. This may be explained by the central stabilizing effect of Dihydergot which complements the hemodynamic properties.

A method for radioimmunological assay of glisoxepide (author's transl). / Eine Methode zur radioimmunologischen Bestimmung von Glisoxepid.

A radioimmunoassay for the antidiabetic drug 3-(4-[beta-(5-methyl-isoxazol-3-carboxyamido)-ethyl]-benzenesulfonyl)-ureido-piperidine-4-carboxic acid (glisoxepide, I, Pro-Diaban) is described. Antisera were obtained by immunizing rabbits with a piperidine carboxylic acid analogue of glisoxepide coupled to bovine serum albumine. The carboxylic acid analogue coupled to [3H]-1-tyrosine methyl ester was used as the tracer. Antisera with high sensitivity were obtained. With some glisoxepide analogues the specificity of the antisera was investigated. For good binding the isoxazole and the benzene ring as well as the appropriate distance between both rings are essential. The assay can be carried out without extraction of the compound from the serum. Dextran coated charcoal is used for separation of free and bound glisoxepide.