Volume and compressibility changes accompanying thermally-induced native-to-unfolded and molten globule-to-unfolded transitions of cytochrome c: a high pressure study

We have measured the transition temperatures, T(M), and van't Hoff enthalpies, DeltaH(M), of the thermally induced native-to-unfolded (N-to-U) and molten globule-to-unfolded (MG-to-U) transitions of cytochrome c at pressures between 50 and 2200 bar. We have used the pressure dependence of T(M) to evaluate the changes in volume, Delta(v), accompanying each protein transition event as a function of temperature and pressure. From analysis of the temperature and pressure dependences of Delta(v), we have additionally calculated the changes in expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), associated with the thermally induced conformational transitions of cytochrome c. Specifically, if extrapolated to 25 degrees C, the native-to-unfolded (N-to-U) transition is accompanied by changes in volume, Delta(v), expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), of -(5 +/- 3) x 10(-3) cm(3) g(-1), (1.8 +/- 0.3) x 10(-4) cm(3) g(-1) K(-1), and approximately 0 cm(3) g(-1) bar(-1), respectively. The molten globule-to-unfolded (MG-to-U) transition is accompanied by changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), of -(2.9 +/- 0.3) x 10(-3) cm(3) g(-1) at 40 degrees C and -(1.9 +/- 0.3) x 10(-6) cm(3) g(-1) bar(-1) at 35 degrees C, respectively. By comparing the volumetric properties of the N-to-U and N-to-MG transitions of cytochrome c, we have estimated the properties of the native-to-molten globule (N-to-MG) transition. For the latter transition, the changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), are approximately 0 cm(3) g(-1) at 40 degrees C and 1.9 cm(3) g(-1) bar(-1) at 35 degrees C, respectively. Our estimate for the change in expansibility, Delta(e), upon the N-to-MG is negative and equal to -(5 +/- 3) x 10(-4) cm(3) g(-1) K(-1). This finding contrasts with the results of previous studies all of which report positive changes in expansibility associated with protein denaturation. In general, our volumetric data permit us to assess the combined effect of temperature and pressure on the stability of various conformational states of cytochrome c.     

Volumetric and spectroscopic characterizations of glucose-hexokinase association

The binding of D-glucose to hexokinase PII at 25 degrees C and pH 8.7 has been investigated by a combination of ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. The binding of glucose to the enzyme results in significant dehydration of the two interacting molecules, while the intrinsic coefficient of adiabatic compressibility of hexokinase slightly decreases. Glucose-hexokinase association is an entropy-driven process. The favorable change in entropy results from compensation between two large contributions. The binding-induced increase in hydrational entropy slightly prevails over the decrease in the configurational entropy of the enzyme. Taken together, our results emphasize the crucial role of water in modulating the energetics of protein recognition.     

Arachidonic and eicosapentaenoic acids in tissues of the firefly, Photinus pyralis (Insecta: Coleoptera)

We report on the presence of high proportions of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) in the tissue lipids of adult fireflies, Photinus pyralis. Arachidonic acid typically occurs in very small proportions in phospholipids (PLs) of terrestrial insects, ranging from no more than traces to less than 1% of PL fatty acids, while 20:5n-3 is often missing entirely from insect lipids. Contrarily, 20:4n-6 made up approximately 21% of the PL fatty acids prepared from whole males and females, and from heads and thoraces prepared from males. Proportions of 20:4n-6 associated with PLs varied among tissues, including approximately 8% for male gut epithelia, 13% for testes, and approximately 25% for light organs and body fat from males. Substantial proportions of 20:5n-3 were also associated with PLs prepared from male firefly tissues, including 5% for body fat and 8% for light organs. Because 20:4n-6 and 20:5n-3 are precursors for biosynthesis of prostaglandins and other eicosanoids, we considered the possibility that firefly tissues might produce eicosanoids at exceptionally high rates. Preliminary experiments indicated otherwise. Hence, fireflies are peculiar among terrestrial insects with respect to maintaining high proportions of PL 20:4n-6 and 20:5n-3.     

Molecular Cloning and Characterization of <Emphasis Type="Italic">nodD</Emphasis> Genes from <Emphasis Type="Italic">Rhizobium</Emphasis> sp. SIN-1, a Nitrogen-Fixing Symbiont of <Emphasis Type="Italic">Sesbania</Emphasis> and Other Tropical Legumes

Rhizobium sp. SIN-1, a nitrogen-fixing symbiont of Sesbania aculeata and other tropical legumes, carries two copies of nodD, both on a sym plasmid. We have isolated these two nodD genes by screening a genomic library of Rhizobium sp. SIN-1 with a nodD probe from Sinorhizobium meliloti. Nucleotide sequence and the deduced amino acid sequence analysis indicated that the nodD genes of Rhizobium sp. SIN-1 are most closely related to those of R. tropici and Azorhziobium caulinodans. Rhizobium sp. SIN-1 nodD1 complemented a S. meliloti nodD1D2D3 negative mutant for nodulation on alfalfa, but failed to complement a nodD1 mutant of S. fredii USDA191 for soybean nodulation. A hybrid nodD gene, containing the N-terminus of S. fredii USDA191 nodD1 and the C-terminus of Rhizobium sp. SIN-1 nodD1, complemented the nodD1 negative mutant of USDA191 for nodulation on soybean. Received: 17 January 2002 / Accepted: 18 February 2002     

Role of nuclear PKC delta in mediating caspase-3-upregulation in Jurkat T leukemic cells exposed to ionizing radiation

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.     

Host plant nodule parameters associated with nitrogen fixation efficiency in French bean (Phaseolus vulgaris L) cultivars

Two cultivars of French bean (Phaseolus vulgaris L.) viz. contender and arka komal were planted in polythene bags containing sand and grown under glasshouse conditions. The nodulation status, shoot/root biomass, activities of several nodule enzymes, total soluble protein and leghaemoglobin contents were monitored over the entire growth period. Allantoinase activity in leaves was measured to monitor the ureide degrading capacity. Significant genotype difference was observed in both the cultivars. All the parameters showed a decline after flowering except uricase, which declined before flowering. Malate dehydrogenase and isocitrate dehydrogenase showed a constant decline throughout the growth period. Degree of decline varied with the genotype for all the parameters. Leghaemoglobin content, PEP carboxylase activity and ureide degrading capacity of leaves did not show an appreciable decline in contender and were significantly higher than in arka komal. These factors can be used to increase nitrogen fixation in French bean.     

Does shoot water status limit leaf expansion of nitrogen-deprived barley?

The role of shoot water status in mediating the decline in leaf elongation rate of nitrogen (N)-deprived barley plants was assessed. Plants were grown at two levels of N supply, with or without the application of pneumatic pressure to the roots. Applying enough pressure (balancing pressure) to keep xylem sap continuously bleeding from the cut surface of a leaf allowed the plants to remain at full turgor throughout the experiments. Plants from which N was withheld required a greater balancing pressure during both day and night. This difference in balancing pressure was greater at high (2.0 kPa) than low (1.2 kPa) atmospheric vapour pressure deficit (VPD). Pressurizing the roots did not prevent the decline in leaf elongation rate induced by withholding N at either high or low VPD. Thus low shoot water status did not limit leaf growth of N-deprived plants.     

Syntheses of novel substituted-boranophosphate nucleosides

A number of substituted (borano) nucleic acids, 3'-[diethylphosphite(cyano, carboxy, or carbamoyl) borano] deoxynucleosides (3a-4c) and 5'-[diethylphosphite(cyano or carboxy) borano] deoxynucleosides (6a-7d) were prepared by a variety of synthetic procedures. The syntheses of the pyrophosphates (2a-2c), as precursors for 3a-4c, are also described.     

Ecology of dermatophytes and other keratinophilic fungi in swimming pools and polluted and unpolluted streams

The biodiversity and richness of keratinophilic fungal communities including dermatophytes were assessed in three stream sites and three swimming pools in the Nablus district in Palestine, using hair baiting (HBT) and surface dilution plate (SDP) techniques, over 8- and 6-month periods, respectively. The effect of wastewater effluent and selected ecological factors on these fungi in relation to species diversity and population densities were also considered. Fifty keratinophilic fungal species were recovered from the aquatic habitats studied, of which 42 were recovered from stream sites and 22 from swimming pools. Of these fungi 6 were either dermatophytes (Microsporum gypseum, and Trichophyton mentagrophytes) or dermatophyte related species (Chrysosporium merdarium, Ch. tropicum, Ch. keratinophilum and T. terrestre). The most frequently isolated species in the three pools were Acremonium strictum and Cladosporium cladosporioides, using Sabouraud dextrose agar medium (SDA). The most abundant species were Acr. strictum, and Aspergillus flavus. However, only 4 species were isolated using the SDA medium amended with 5-flurocytosine (5-FC). The most frequent and abundant species in the three stream sites using SDA medium were Geotricum candidum, and Penicillium chrysogenum. The most frequent species in the three sites using the 5-FC medium, was Paecilomyces lilacinus. Using HBT, the most abundant and frequent species in the three stream sites were G. candidum, and Pa. lilacinus, on SDA medium, and Pa. lilacinus, and Gliocladium nigrovirens on the 5-FC medium. The 5-FC medium was more suitable for the isolation of dermatophytes and closely related species than the SDA medium; 6 were recovered on 5-FC, whereas only one on the SDA medium. Variation in the levels of keratinophilic fungal populations from the three stream sites sampled 5 times over an 8-month period, followed comparable fluctuation patterns. Wastewater affected fungal population densities with the highest levels in the un-polluted stream sites, and lowest in the heavily polluted sites. Swimming pools, polluted and un-polluted stream sites were found to be rich in pathogenic and potentially pathogenic fungi.