Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

Inheritance and expression of tissue-specific catalase activity during development and aging in mice

The catalase activity in the liver, kidney, lung, and blood hemolysate was measured in newborn, 21-, 70-, 175-, and greater than 400-day-old mice from the strains BALB/c, Csb, C3H/HeSnJ, C3H/S, C57BL/6J, SW, and 129/ReJ. Catalase activity was found to be highest in the liver (approximately 0.33 U/mg protein) followed by the kidney (approximately 0.13 U/mg protein), lung (approximately 0.05 U/mg protein), and blood hemolysate (approximately 0.03 U/mg protein). ANOVA analysis indicated significant differences in enzyme activity among strains and age groups studied. The developmental profiles of enzyme activity were tissue and strain specific. Catalase activity in the blood, for example, was generally higher at birth and at old age, whereas the kidney catalase activity was low at birth and increased substantially with age. Strains could be classified as normal (129/ReJ, BALB/c, C3H/HeSnJ, C3H/S), hypocatalasemic (C57BL/6J, SW), and acatalasemic (Csb) with respect to enzyme activity and it was on this basis that the inheritance of the catalase phenotype was studied using appropriate crosses. The enzyme activity level in each tissue appears to be governed by a unique set of genetic regulators/modifiers that interact with a single structural gene (Cs) or its product to produce the catalase phenotype. Some of these (e.g., Ce-1 and Ce-2) have been previously described but based on the results of various crosses reported here, more must exist that remain still uncharacterized at the molecular level. Models proposed for the inheritance of the catalase phenotype vary in complexity from single allelic differences between strains (e.g., BALB/c x Csb; blood) to a system of multiple interacting genetic determinants (e.g., BALB/c x Csb; liver) each having dominant (e.g., C57BL/6J over BALB/c; liver) and recessive components (e.g., gene(s) conferring the acatalasemic phenotype in BALB/c x Csb; blood and kidney). Such results are important and offer an interesting model to further characterize aspects of eukaryotic gene regulation.     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

Low Temperature-Induced Decrease in trans-Delta-Hexadecenoic Acid Content Is Correlated with Freezing Tolerance in Cereals

The effect of growth at 5°C on the trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol was examined in a total of eight cultivars of rye (Secale cereale L.) and what (Triticum aestivum L.) of varying freezing tolerance. In these monocots, low temperature growth caused decreases in the trans-Δ³-hexadecenoic acid content of between 0 and 74% with concomitant increases in the palmitic acid content of phosphatidyl(d)glycerol. These trends were observed for whole leaf extracts as well as isolated thylakoids. The low growth temperature-induced decrease in the trans-Δ³-hexadecenoic acid content was shown to be a linear function (r² = 0.954) of freezing tolerance in these cultivars. Of the six cold tolerant dicotyledonous species examined, only Brassica and Arabidopsis thaliana L. cv Columbia exhibited a 42% and 65% decrease, respectively, in trans-Δ³-hexadecenoic acid content. Thus, the relationship between the change in trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol and freezing tolerance cannot be considered a general one for all cold tolerant plant species. However, species which exhibited a low growth temperature-induced decrease in trans-Δ³-hexadecenoic acid also exhibited a concomitant shift in the in vitro organization of the light harvesting complex II from a predominantly oligomeric form to the monomeric form. We conclude that the proposed role of phosphatidyl(d)glycerol in modulating the organization of light harvesting complex II as a function of growth temperature manifests itself to varying degrees in different plant species. A possible physiological role for this phenomenon with respect to low temperature acclimation and freezing tolerance in cereals is discussed.