Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.     

NMR studies of cytochrome P-450scc. Effects of steroid binding on water proton access to the active site of the ferric enzyme

Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450 (-450scc) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe3+ center, and the long electron spin relaxation time of the heme iron, tau s approximately 0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is approximately 2.5 A, a distance consistent with a water molecule binding directly to the heme iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to approximately 4 A, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the complex with (22R)-22-hydroxycholesterol, a distance of approximately 2.7 A is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450scc with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, or (20R)-20-phenyl-5-pregnene-3 beta,20-diol, also yielded distances of approximately 2.5 A and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenylglyoxal modification of the Photosystem II reaction center

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance (ESR), have been used in conjunction with fluorescence-induction and dye-reduction assays to monitor electron transport in Photosystem II (PS II) subchloroplast particles incubated with the covalent modifier, phenylglyoxal. Phenylglyoxal-modified digitonin (D-10) particles from spinach are characterized by a high initial fluorescence yield (Fi) and an abolition of the variable component of fluorescence (Fv); an inhibition of PS-II-mediated reduction of dichlorophenol indophenol (DPIP) by sym-diphenylcarbazide; an abolition of flash-induced absorption transients (t1/2 greater than 2 microseconds) at 820 nm attributed to the primary electron donor, P-680+; the inhibition of photoreduction of the acceptor Qa; and the elimination of the ESR Signal 2s and Signal 2f. These observations suggest the critical participation of specific arginine residues on both the oxidizing and reducing sides of Photosystem II and also implicate phenylglyoxal as a quinone-binding site inhibitor (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271).     

Observation on a 65-kilodalton protein isolated from kanagawa positive strains of Vibrio parahaemolyticus

Crude hemolysin from four KP+ strains of Vibrio parahaemolyticus belonging to serotype 02:K3 exhibited a major protein band (molecular weight, 65 kilodaltons (kDa] in addition to a previously known thermostable direct hemolysin band (molecular weight, 21 kDa) in SDS - polyacrylamide slab gel electrophoresis. These strains showed maximum virulence leading to 100% mouse lethality within 2-6 h. It is hypothesized that this 65-kDa protein may play a vital role in the pathogenesis of the disease caused by V. parahaemolyticus.     

Gibberellic acid by solid state fermentation: Consistent and improved yields

Five strains each of Gibberella fujikuroi and Fusarium monoliforme were screened to select G. fujikuroi P-3, a strain capable of giving consistent production of gibberellic acid (GA(3)) by solid state fermentation (SSF). The comparative production of GA(3) by SSF and submerged fermentation (SmF) indicated better productivity with the former technique. The accumulation of GA(3) was 1.626 times higher in the case of SSF. On the basis of available carbohydrates in the media, the percent conversions were 0.096 and 0.156 in SmF and SSF, respectively. The use of coarse wheat bran of the particle size of 0.3-0.4 cm resulted in an increase of 2.5 times in the yield of GA(3). The enrichment of commercial wheat bran with soluble starch gave enhanced accumulation to an extent of 3.5 times. The relation between GA(3) production and cell growth in SSF was similar to that encountered in SmF. The consistent and improved yields to a tune of 1.22 g GA(3) per kilogram dry moldy bran (DMB) establish the potential and feasibility of SSF for the production of GA(3) by G. fujikuroi P-3. On preliminary cost analysis, a net savings of about 60% and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SmF technique was evident.     

Forest vegetation of the Himalaya

This review deals with the forest vegetation of the Himalaya with emphasis on: paleoecological, phytogeographical, and phytosociological aspects of vegetation; structural and functional features of forest ecosystem; and relationship between man and forests. The Himalayan mountains are the youngest, and among the most unstable. The rainfall pattern is determined by the summer monsoon which deposits a considerable amount of rain (often above 2500 mm annually) on the outer ranges. The amount of annual rainfall decreases from east to west, but the contribution of the winter season to the total precipitation increases. Mountains of these dimensions separate the monsoon climate of south Asia from the cold and dry climate of central Asia. In general, a rise of 270 m in elevation corresponds to a fall of 1°C in the mean annual temperature up to 1500 m, above which the fall is relatively rapid. Large scale surface removals and cyclic climatic changes influenced the course of vegetational changes through geological time. The Himalayan ranges, which started developing in the beginning of the Cenozoic, earlier supported tropical wet evergreen forests throughout the entire area (presently confined to the eastern part). The Miocene orogeny caused drastic changes in the vegetation, so much so that the existing flora was almost entirely replaced by the modern flora. Almost all the dominant forest species of the Pleistocene continue to maintain their dominant status to the present. Presently the Himalayan ranges encompass Austro-Polynesian, Malayo-Burman, Sino-Tibetan, Euro-Mediterranean, and African elements. While the Euro-Mediterranean affinities are well represented in the western Himalayan region (west of 77°E long.), the Chinese and Malesian affinities are evident in the eastern region (east of 84°E long.). However, the proportion of endemic taxa is substantial in the entire region. A representation of formation types in relation to climatic factors, viz., rainfall and temperature, indicates that boundaries between the types are not sharp. Formation types often integrate continuously, showing broad overlaps. Climate does not entirely determine the formation type, and the influence of soil, fire, etc., is also substantial. The ombrophilous broad leaf forests located in the submontane belt (< 1000 m) of the eastern region are comparable to the typical tropical rain forests. On the other extreme, communities above 3000 m elevation are similar to sub-alpine and alpine types. From favorable to less favorable environments, as observed with decreasing moisture from east to west, or with decreasing temperature from low to high elevations, the forests become increasingly open, shortstatured and simpler, with little vertical stratification. Ordination of forest stands distributed within 300–2500 m elevations of the central Himalaya, by and large indicates a continuity of communities, with scattered centers of species importance values in the ordination field. Within the above elevational transect, sal (Shorea robusta) and oak (Quercus spp.) forests may be designated as the climax communities, respectively, of warmer and cooler climates. The flora of a part of the central Himalayan region is categorized as therohemigeophytic and that of a part of the western Himalayan region as geochamaephytic. An analysis of population structure over large areas in the central Himalaya, based on density-diameter distribution of trees, suggests that oldgrowth forests are being replaced by even-aged successional forests, dominated by a few species, such asPinus roxburghii. Paucity of seedlings of climax species, namelyShorea robusta andQuercus spp. over large areas is evident. The Himalayan catchments are subsurface-flow systems and, therefore, are particularly susceptible to landslips and landslides. Loss of water and soil in terms of overflow is insignificant. Studies on recovery processes of forest ecosystems damaged due to shifting cultivation or landslides indicate that the ecosystems can recover quite rapidly, at least in elevations below 2500 m. For example, on a damaged forest site, seedlings of climax species (Quercus leucotrichophora) appeared only 21 years after the landslide. In the central Himalaya, the biomass of a majority of forests (163-787 t ha^?1) falls within the range (200-600 t ha^?1) given for many mature forests of the world, and the net primary productivity (found in the range of 11.0–27.4 t ha^?1 yr^?1) is comparable with the range of 20–30 t ha^?1 yr^?1 given for highly productive communities of favorable environments. In most of the forests of this region, the litter fall values (2.1-3.8 t C ha^?1 yr^?1) are higher than the mean reported for warm temperate forests (2.7 t C ha^?1 yr^?1). Of the total litter, the tree leaves account for 54–82% in the Himalayan forests. The rate of decomposition of leaves in some broadleaf species of submontane belt (0.253-0.274% day^?1) are comparable with those reported for some tropical rain forest species. Because of the paucity of microorganisms and microarthropods in the forest litter and soil, high initial C:N ratio and high initial lignin content in leaves, the rate of leaf litter decomposition inPinus roxburghii is markedly slower than in other species of the central Himalaya. The fungal species composition of the leaf litterof Pinus roxburghii is also distinct from those of other species. A greater proportion of nutrients is accumulated in the biomass component of the Himalayan forests than in the temperate forests. Although litter fall is the major route through which nutrients return from biomass to the soil pool, a substantial proportion of the total return is in the form of throughfall and stemflow. Among the dominant species of the central Himalaya, retranslocation of nutrients from the senescing leaves was markedly greater inPinus roxburghii than inQuercus spp. andShorea robusta. Consequently, the C:N ratio of leaf litter is markedly higher inPinus roxburghii than in the other species. Immobilization of nutrients by the decomposers of the litter with high C:N ratio is one of the principal strategies through whichPinus roxburghii invades other forests and holds the site against possible reinvasion by oaks. Observations on the seasonality of various ecosystem functions suggest that Himalayan ecosystems are geared to take maximum advantages of the monsoon period (rainy season). Most of the human population depends on shifting-agriculture in the eastern region and on settled agriculture in the central and western regions. Either of these is essentially a forest-dependent cultivation. Each unit of agronomic energy produced in the settled agriculture entails about seven units of energy from forests. Consequently, forests with reasonable crown cover account for insignificant percentage of the land. Tea plantations and felling of trees for timber, paper pulp, etc., are some of the major commercial activities which adversely affected the Himalayan forests.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila melanogaster. III. Variations in Genetic Structure and Their Causes between Drosophila melanogaster and Its Sibling Species Drosophila simulans

The natural populations of Drosophila melanogaster and Drosophila simulans were compared for their genetic structure. A total of 114 gene-protein loci were studied in four mainland (from Europe and Africa) and an island (Seychelle) populations of D. simulans and the results were compared with those obtained on the same set of homologous loci in fifteen worldwide populations of D. melanogaster. The main results are as follows: (1) D. melanogaster shows a significantly higher proportion of loci polymorphic than D. simulans (52% vs. 39%, P<0.05), (2) both species have similar mean heterozygosity and mean number of alleles per locus, (3) the two species share some highly polymorphic loci but they do not share loci that show high geographic differentiation, and (4) D. simulans shows significantly less geographic differentiation than D. melanogaster. The differences in genetic differentiation between the two species are limited to loci located on the X and second chromosomes only; loci on the third chromosome show similar level of geographic differentiation in both species. These two species have previously been shown to differ in their pattern of variation for chromosomal polymorphisms, quantitative and physiological characters, two-dimensional electrophoretic (2DE) proteins, middle repetitive DNA and mitochondrial DNA. Variation in niche-widths and/or genetic "strategies" of adaptation appear to be the main causes of differences in the genetic structure of these two species.     

Effect of phosphate supplementation on oxygen delivery at high altitude

In the present communication, effect of low doses of phosphate supplementation on short-term high altitude adaptation has been examined. Studies were carried out in 36 healthy, male, sea-level residents divided in a double blind fashion into drug and placebo treated groups. 3.2 mmol of phosphate were given orally to each subject of the drug treated group once a day for 4 days on arrival at an altitude of 3,500 m. Sequential studies were done in the subjects in both groups on the 3rd, 7th, 14th and 21st day of their altitude stay. Haemoglobin, haematocrit, erythrocyte and reticulocyte counts increased to the similar extent in both groups. Blood pH, pO₂ and adenosine tri-phosphate (ATP) did not differ between the two groups. On 3rd day of the altitude stay, inorganic phosphate and 2,3-diphosphoglycerate (2,3 DPG) levels in the drug treated group increased significantly as compared to the placebo group. No significant difference in inorganic phosphate and 2,3 DPG was observed later on in the two groups. Psychological and clinical tests also indicated that the drug treated subjects felt better as compared to the placebo treated subjects. The present study suggests that low doses of phosphate increases circulating 2,3-DPG concentration which in turn brings about beneficial effect towards short term high altitude adaptation.     

Effect of cyanophage N-1 infection on the synthesis and stability ofNostoc muscorum nitrate reductase

The control operative on the nitrate reductase enzyme system of host cyanobacteriumNostoc muscorum was studied after being infected with the cyanophage N-1. Phage infection lifted the host nitrate reductase activity level via accelerating the enzyme synthesis. It was found that the phage-mediated increase in the molybdenum cofactor synthesis was a major contributing factor for apparent elevated nitrate reductase level of the host. This process was inhibited in the presence of erythromycin and tungsten, the inhibitors of protein synthesis and new nitrate reductase synthesis respectively. While the preformed nitrate reductase of healthy cyanobacterium was inhibited by hydrogen peroxide, an oxidizing photosynthetic product, the same enzyme of infected cells remained virtually insensitive to this inhibitor. These data suggest involvement of new nitrate reductase synthesis and its resistance to oxidative inactivation as joint factors controlling the characteristic high enzyme level of host cyanobacterium.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.