Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.     

Montane plant communities of Fynbos Biome

The vegetation of the mountains of the Fynbos Biome is classified and described, mostly on the basis of vegetation structure and higher taxa. Various gradients can be recognized. A gradient in soil conditions runs from coarse textured, nutrient-poor soils in non-grassy fynbos (Mountain Fynbos) through grassy fynbos and grassy non-fynbos (Eastern Fynbos and Grasslands & Grassy Shrubland) to finer textured and less nutrient-poor soils in the non-fynbos Karroid & Renoster Shrubland. Another gradient of significance can be likened to the tropical gradient running from the dry conditions of hot semi-deserts to savannas or grasslands to woodlands. At the dry extreme an open shrubland occurs (Karroid & Renoster Shrubland, Asteraceous Fynbos), at intermediate positions a herbland occurs (Restioid Fynbos) and at the wet extreme another type of shrubland occurs (Ericaceous Fynbos). This kind of moisture gradient occurs on individual mountains with the xeric end being at the base of the north slopes, and the mesic end being on the upper south aspects. Soil characteristics are closely linked to this gradient; the better developed soils being found on the south aspects. Another moisture gradient is apparent in the vegetation. This gradient runs from the mesic southern coastal mountains to the north west where summer droughts are extremely severe although total rainfall may be similar.Nomenclature follows that used in the Government Herbarium, Stellenbosch.I thank William Bond, Richard Cowling, John Rushworth, Eddy van der Maarel and Marinus Werger for critical comments and discussion.     

Barotolerant variant of Streptococcus faecalis with reduced sensitivity to glucose catabolite repression

Physiological characterization of the APR-11 variant of Streptococcus faecalis ATCC 9790 revealed that the variant has reduced sensitivity to glucose catabolite repression. This reduced sensitivity was indicated by the synthesis of enzymes for catabolism of lactose or arginine in cultures growing at 0.1, 40, or 70 MPa in media with levels of glucose highly repressive for the parent strain. Reduced catabolite repression appeared to be due to reduced activity of the glucose-specific, phosphotransferase system in APR-11 cells. Conversion of pyruvate to lactate or to acetate and ethanol did not appear to be altered in the variant. The APR-11 variant produced a greater final yield of biomass than the parent at all pressures tested, and its barotolerance was especially marked in media with low levels of glucose and high levels of lactose in which derepression of the lactose catabolic system was necessary for full growth. Overall, the greater barotolerance of the APR-11 strain appeared to be due to its enhanced capacity for catabolism related to its reduced sensitivity to catabolite repression by glucose.     

Geminate recombination of CO in rabbit, opossum, and adult hemoglobins

The geminate recombination of CO with Hb following dissociation by a 10-ns laser pulse has been studied as a function of pH (9.2 and 7.0 without inositol hexaphosphate and 6.0 with inositol hexaphosphate) and temperature (5-35 degrees C). The hemoglobins studied included adult, Rothschild, rabbit, opossum, and carp. Despite significant differences in their structural and functional properties, the first four of these hemoglobins show similar trends in the yields, rates, and activation energies of the geminate recombination. The nature of the "cage recombination" in hemoglobin is discussed in the light of such findings. Neither a slow diffusion model nor a model based upon a specific non-heme binding site accounts for the observations.     

Relationship between progesterone receptor binding and progestin biological activity

The binding affinities of a series of steroidal compounds for the hamster uterine progesterone receptor were determined using two sets of incubation conditions. These competitive binding conditions were designed to deduce the relative rates of ligand dissociation from the progesterone receptor. The progestin activity of these compounds was also determined in a bioassay employing the measurement of diamine oxidase in the traumatized hamster uterus. Steroids could be classified into two categories based on either an increase or decrease in relative binding affinity (RBA) with increasing time of competitive incubation. The mean (+/- SEM) progestin biopotency for the compounds having an increase in RBA was 120 +/- 18 (progesterone = 100), while the biopotency for compounds having a decrease in RBA was only 44 +/- 17. This difference was significant (P less than 0.01). Linear regression analyses revealed significant correlations between the RBAs and progestin biopotencies. Compounds showing a decrease in RBA with increasing time of incubation did not have antiprogestin activity. Kinetic studies of this type should be useful for selecting compounds with potent agonistic activity, but cannot unequivocally predict antihormonal activity.     

Ivermectin: an update

Ivermecan was introduced as an antiparasitic agent in 1981. It is now registered for animal-health use in 35 countries and is being evaluated for possible use in man. This review summarises its antiparasitic efficacy and apparent mode of action. Additional information is given in previous review articles.     

Isolation of the gene encoding yeast DNA polymerase I

A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.     

Enhanced enzymatic hydrolysis of lignocellulosic biomass pretreated by low-pressure steam autohydrolysis

Summary The use of low-pressure steam autohydrolysis in the pretreatment of corn stover and hybrid poplar has been assessed. In terms of yield of prehydrolyzed solids, minimal by-product formation and extent of subsequent enzymatic saccharification, the results of low-pressure steam pretreatment were found to be as good as or better than those reported for more severe pretreatment processes. Almost complete saccharification of the cellulose in the prehydrolyzed biomass solids was obtained within 24h with a commercial cellulase preparation — Celluclast. The presence of grinding elements (glass beads) during the enzymatic hydrolysis was found to increase the extent of saccharification by 40% to 50% over controls without any grinding elements.     

Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis.

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.