Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Studies on the Protection Test of Pertussis Vaccine. Immunization Period and Protectivity

In order to confirm the data reported in the previous papers, variously prepared pertussis vaccines were employed in the present investigation. Pertussis organisms grown either on a solid or in a liquid semisynthetic medium were treated by: (1) heating at 56 C for 30 min, (2) storage in 0.1% formalin at 37 C for 5 days, (3) storage in 0.1% formalin at 25 C for 5 days, and (4) simple addition of sodium ethyl-mercuri thiosalicylate (merthiolate) as a preservative. Freeze-dried vaccines were made from these preparations four and a half months later. Mice were immunized intraperitoneally and challenged intracerebrally with a virulent strain of Bordetella pertussis 10 or 17 days later. The data were analyzed statistically assuming the probit corresponding to the percentage of survivors at any dose be a linear function of the logarithms of the dose. The 50% effective doses (ED₅₀) and slopes of each vaccine were found to be uniform. More accurate estimates of ED₅₀ were obtained by employing a pooled slope in each experiment. From these ED₅₀ values, the relative potency was estimated by comparing the value of a vaccine to that of a dried merthiolate-vaccine. For vaccines derived from solid cultures, with an immunization period of 17 days, the relative potency of the vaccine heated at 56 C was 0.63 (95% fiducial limits=0.52 to 0.76); the value for the formalinized vaccine at 37 C was 0.40 (0.30 to 0.53) and one at 25 C was 0.51 (0.34 to 0.77). Vaccines derived from liquid cultures showed a relative potency of 20 to 50% less than that of corresponding vaccine derived from a solid culture. The potency obtained for the 17 day immunization period was usually higher than that for the 10 day period. Using the overall-pooled slope, an experimental design which will be appropriate for statistical analysis is discussed.     

Glycosaminoglycans and proteoglycans synthesized by rat limb buds during prechondrogenic and chondrogenic stages

The sulfated glycosaminoglycans synthesized in the forelimb plates of rats on days 12, 13, 14, and 15 of gestation were characterized by their susceptibility to various glycosaminoglycan lyases. On days 12 and 13, heparan sulfate accounted for approximately 65% of the newly synthesized sulfated glycosaminoglycans. Small amounts of dermatan sulfate and chondroitin sulfates were also observed. On day 14, the relative amount of chondroitin 4-sulfate began to increase, there being a compensatory decrease in the amount of heparan sulfate. 35S-Sulfate-labeled material was extracted from day-13 forelimb plates with 4 M guanidine/HCl without proteolysis. Using ultracentrifugation on a sucrose density gradient, the extract was separated into two peaks: a light peak (L) mainly composed of heparan sulfate, and a faster-sedimenting peak (M) mainly composed of chondroitin sulfate. The cartilage-type proteoglycan (H) was first detectable on day 14 of gestation, indicating that chondrogenesis in rat forelimb plates starts on day 14 of gestation. In addition to these previously identified glycosaminoglycans or proteoglycans, we isolated an unknown component in the glycosaminoglycan preparations obtained from limb plates during these developmental stages. This component was not found in glycosaminoglycan preparations obtained either from the brain or tail of rat fetuses at the same stages.     

Isolation and characterization of a novel reassortant between avian Ty-1 and simian RRV rotaviruses.

A reassortant, TyRh, was isolated after coinfection of MA104 cells with avian Ty-1 and simian RRV rotaviruses. Hybridization and serological studies showed that the reassortant's 4th gene, which encodes Vp4, was derived from the simian RRV rotavirus parent, whereas the remaining 10 genes were derived from the avian Ty-1 rotavirus parent.     

Hydrolysis of beta-D-glucopyranosyl fluoride to alpha-D-glucose catalyzed by Aspergillus niger alpha-D-glucosidase

Aspergillus niger alpha-D-glucosidase, crystallized and free of detectable activity for beta-D-glucosides, catalyzes the slow hydrolysis of beta-D-glucopyranosyl fluoride to form alpha-D-glucose. Maximal initial rates, V, for the hydrolysis of beta-D-glucosyl fluoride, p-nitrophenyl alpha-D-glucopyranoside, and alpha-D-glucopyranosyl fluoride are 0.27, 0.75, and 78.5 mumol.min-1.mg-1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from beta-D-glucosyl fluoride and not from a contaminating trace of alpha-D-glucosyl fluoride, and is catalyzed by the alpha-D-glucosidase and not by an accompanying trace of beta-D-glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase. Present findings provide new evidence that alpha-glucosidases are not restricted to alpha-D-glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.     

Light controls stamen elongation via cryptochromes,phytochromes and COP1 through HY5 and HYH

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 – oppositely to ARF8 – directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far‐red light receptors PHYA/PHYB. In conclusion, different light qualities – sequentially perceived by specific photoreceptors – and the downstream COP1–HY5/HYH module finely tune auxin‐induced stamen elongation and thus male fertility.     

Site-Specific RNA Cleavage Using Terpyridine·Cu(II)-Linked 2′-O-Methyloligonucleotides

Abstract

2′-Deoxy- and 2′-O-methyl-5′-O-terpyridyl derivatives of adenosine and cytidine were synthesized and used to construct 5′-end-modified oligonucleotides. These antisense agents complexed with Cu(II) exclusively cleaved a complementary RNA oligomer at the site opposite the terpyridine-nucleoside residue. We also found that the terpyridine·Cu(II) moiety stabilizes 2′-O-methyl RNA duplex. These suggest that after RNA hybridization, the terpyridine moiety is close to the RNA strand, presumably in an end capping manner.