Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new,extreme thermophilic,anaerobic bacterium

Two strains, JW 200 and JW 201, of an extreme thermophilic, non-spore-forming anaerobic bacterium were isolated from alkaline and slightly acidic hot springs located in Yellowstone National Park. Both strains were peritrichously flagellated rods. Cell size varied from 0.5–0.8 by 4–100 m; coccoid-shaped cells of about 1 m in diameter frequently occurred. Division was often unequal. Spheroplast-like forms were visible at the late logarithmic growth phase. The Gram reaction was variable. The DNA base composition of the two strains was between 37 and 39 mol% guanine plus cytosine as determined by buoyant density measurements and approximately 32% by the thermal denaturation method. The main fermentation products from hexoses were ethanol and CO₂. Growth occurred between 37 and 78°C and from pH 4.4 to 9.8. The name Thermoanaerobacter ethanolicus gen. nov., spec. nov. was proposed for the two, new isolates. Strain JW 200 was designated as the type strain.A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Los Angeles, CA, 1979 (J. Wiegel and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol., 1979, 163, p. 105) and at the 27th IUPAC Congress Helsinki, 1979 (L. G. Ljungdahl and J. Wiegel, Abstracts p. 546)     

Genetic Evaluation and Utilization (GEU) program

Summary The Genetic Evaluation and Utilization (GEU) program of the International Rice Research Institute (IRRI) is an interdisciplinary program for the improvement of rice crops. Scientists trained in diverse disciplines such as plant breeding, plant pathology, entomology, agronomy, cereal chemistry, plant physiology, and soil chemistry work together and contribute their specialized skills to this joint endeavor. The program has five interrelated components: (1) germ plasm collection and conservation, (2) research in disciplinary areas, (3) development of improved germ plasm, (4) distribution, evaluation and exchange of germ plasm internationally, (5) training of young scientists.Over forty thousand rice varieties from different countries are being maintained in the IRRI germ plasm bank. These varieties have been screened for grain quality, resistance to various diseases and insects, and tolerance to various environmental stresses such as drought, high and low temperatures and problem soils. Donor parents for resistances to each of the problem areas have been identified. These parents were utilized for developing improved germ plasm. Varieties with resistance to as many as five diseases and five insect species have been developed. These multiple resistant varieties are grown on millions of hectares of rice land. Seeds of improved breeding materials are exchanged internationally and 194 scientists from different countries have been trained in rice improvement work.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Studies on the mode of action of calciferol: Biological activity of 1α,24R,25-trihydroxyvitamin D3 in the chick

The biological activity of 1α,24R,25-trihydroxyvitamin D₃ [1α,24R,25(OH)₃D₃] was elevated in comparison to the hormonally active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], in the rachitic chick in terms of its ability to (a) stimulate intestinal calcium absorption, (b) mobilize bone calcium, (c) induce intestinal calcium binding protein, (d) modulate the level of enzyme activity of the renal 25-OH-D₃-1-hydroxylase system, and (e) interact with the intestinal cystosol-chromatin receptor system for the 1α,25(OH)₂D₃ receptor system. In each of these assays, the relative ratio of activity of 1α,24R,25(OH)₃D₃ to 1α,25(OH)₂D₃was (a) 25–50, (b) ca. 20, (c) 10, (d) 50, and (e) 36%, respectively.