The androgen-dependent rat prostate protein,probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1

Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”. This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J. M.).     

Monoclonal antibody production in hollow fiber bioreactors using serum-free medium

Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems.     

Reproductive anatomy, manipulation of ovarian activity and non-surgical embryo recovery in suni (Neotragus moschatus zuluensis)

Marked disparity in the uterine horn dimensions and relative degrees of caruncle development in suni suggested that exclusive or predominant dextral implantation occurs in association with bilateral ovulatory activity. Daily urinary measurements of pregnanediol-3 alpha-glucuronide revealed an oestrous cycle of approximately 21 days in length. Ovarian activity was controlled for synchronization of oestrus by using progestagen-impregnated intravaginal sponges and multiple ovulations were induced by using exogenous gonadotrophin therapy. An effective transcervical uterine catheterization technique was developed for the non-surgical collection of embryos. The efficiency of embryo recovery performed 5 days after sponge removal was 50.0%.     

Rates of synonymous substitution in plant nuclear genes

Summary The rate of synonymous nucleotide substitution in nuclear genes of higher plants has been estimated. The rate varies among genes by a factor of up to two, in a manner that is not immediately explicable in terms of base composition or codon usage bias. The average rate, in both monocots and dicots, is about four times higher than that in chloroplast genes. This leads to an estimated absolute silent substitution rate of 6 × 10^–9 substitutions per site per year that falls within the range of average rates (2–8 × 10^–9) seen in different mammalian nuclear genomes.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.     

An unusual thiol-driven fumarate reductase in Methanobacterium with the production of the heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3-phosphate

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Plant-soil interactions in primary succession at Hawaii Volcanoes National Park

Summary Plant nutrient status and physiological processes were examined in relation to soil nutrient characteristics under individuals of five species colonizing a young cinder deposit in Hawaii Volcanoes National Park. Two exotic species, Buddeleja asiatica and Myrica faya, had high photosynthetic rates and high nitrogen concentrations and relatively easily decomposed leaves; soils under them had high concentrations of nitrogen, cations, and organic matter and high rates of net nitrogen mineralization. At the other extreme, the natives Metrosideros polymorpha and Vaccinium reticulatum had low plant concentrations and photosynthetic rates, and low concentrations and turnover rates of N in the soil. Thus, a strong correlation exists between soil processes and plant processes, suggesting a positive feedback cycle.