Orientational ordering and dynamics of the hydrate and exchangeable hydrogen atoms in crystalline crambin

Deuterium nuclear magnetic resonance studies of crambin crystals grown from deuterated solvent (2H2O/CH3CH2O2H or H2O/C2H3CH2OH) are reported. The extent to which the hydrate and exchangeable hydrogen atoms are dynamically disordered are then determined from the size of the residual deuterium quadrupole couplings, qcc. Rapid molecular reorientation (tau c-1 greater than 10(5) s-1) reduces the magnitude of the quadrupole coupling from its static value (216 kHz for solid water). We find that the room temperature spectrum of crambin is dominated by two features: a sharp line with very small residual quadrupolar coupling less than 3 kHz, and a broad pattern with a quadrupolar coupling in the range 185 to 195 kHz. The former is indicative of very nearly isotropically reorienting deuterons, whereas the latter is somewhat narrower than that observed for the amide deuterons of poly-gamma-benzyl-L-glutamate and thus indicative of deuterons that are almost but not completely stationary. By considering the nuclear magnetic resonance spectrum intensities along with the amino acid sequence, X-ray structure and the manner in which quadrupole couplings are reduced by dynamics, we conclude that the nuclear magnetic resonance signal from most of the water molecules of hydration are contained in the sharp line, i.e. reorient nearly isotropically in the crystalline protein. Unlike bulk water, which freezes abruptly in the manner of a phase transition, the water of hydration in crambin has a broad freezing range from 180 to 250K, as evidenced by the decreasing intensity of the sharp line that disappears at 180K. At temperatures between 150 and 200K, a typical hydrate molecule reorients at a rate comparable to the quadrupole coupling, 10(4) s-1 to 10(5) s-1, a process that occurs in hexagonal ice in the range of 240 to 270K. At 140K, the hydrate is stationary, tau c-1 less than 10(3) s-1. Studies of the protein crystallized from solvent deuterated only at the non-exchangeable methyl group of ethanol confirm that ethanol is in the lattice and show that this solvate behaves in much the same way as the hydrate. The refined X-ray structure has identified four ethanol solvate molecules. The deuterium spectrum at room temperature has a well-defined residual pattern with qcc = 2.2 kHz, i.e. a small-order parameter consistent with nearly isotropically reorienting molecules. The spectrum width broadens substantially only at temperatures below 200K and achieves the characteristic spectrum of a rotating methyl group with stationary C-C axis at 140K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of isovaleroyl lipids in channeling of sound in the porpoise melon

Physical properties (e.g. specific gravity, adiabatic compressibility and sound velocity) of lipids isolated from tissues from contiguous areas of the fatty melon of an echo-locating porpoise (Delphinus delphis) were determined to elucidate relations between lipid composition and structure, and sound transmission in the head. Lipid content varied greatly within the melon (13.6–77.6% of the tissue weight) and triacylglycerols (80–100%) were the major lipid components. This lipid class was composed of diisovaleroylglycerides (triacylglycerols containing two isovaleroyl moieties and a long-chain acyl moiety), monoisovaleroyldiacylglycerols and triacylglycerols consisting of long-chain acids. The lipid-rich (>45%) areas in the melon contained a high proportion (>45% of total triacylglycerols) of diisovaleroylglycerides. There were gradations of sound velocities within the melon; the lowest sound velocities were associated with high concentrations of diisovaleroylglycerides (<1400 m/s) and the highest with high concentrations of long-chain triacylglycerols. Assuming an average sound frequency of 75 kHz, and considering dimensions of melon (path length and width of 12–14 cm and 5 cm, respectively), a forward radiating lobe of 15–25 degrees is produced. Thus, the deposition of lipids of different acoustic properties in a three-dimensional matrix within the porpoise melon results in a lens for the projection of sound into the marine environment.     

Growth and thiophene accumulation by hairy root cultures of Tagetes patula in media of varying initial pH

Summary Hairy roots of Tagetes patula were grown for 24 days in modified Murashige and Skoog's liquid medium at different initial pH levels of 4.0, 5.0, 5.7, 6.0 and 7.0. Irrespective of the initial pH, after 12 days, the pH of the culture medium was approximately 4.5. However the final pH, after 24 days of growth, did depend weakly on the initial pH of the medium. The biomass yield was lowest at an initial pH of 4.0, possibly due to lower utilization of ammonium at this pH. Similar patterns of thiophene accumulation was observed at all pH levels tested. Maximum thiophene accumulation occurred in root cultures which were 12–16 days old.Abbreviations BBTOH 5-(4-hydroxy-1-butenyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butenyl)-2,2-bithienyl - BBT 5-(3-buten-1-ynyl)-2,2-bithienyl - MS Murashige and Skoog's nutrient medium - B5 Gamborg's B5 nutrient salts - HPLC High pressure liquid chromatography     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.     

Galanin-Like Immunoreactivity Is Unchanged in Alzheimer''s Disease and Parkinson''s Disease Dementia Cerebral Cortex

Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin-like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50-60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin-like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer-type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin-like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin-like immunoreactivity in the hippocampus. Galanin-like immunoreactivity in the human hypothalamus consisted of two molecular-weight species on gel-permeation chromatography, and two forms were resolved by reverse-phase HPLC. The paradoxical preservation of galanin-like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.     

Drosophila melanogaster Scramblases modulate synaptic transmission

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.     

Mitogenic response of rat lung and tracheal epithelial cells in monolayer primary cultures—Modulation of TNF-α expression

Epithelial cells isolated from rat lung and trachea were grown on monolayers and their response to a number of hormones and growth factors were studied. Maximum proliferative response in serum containing media was observed when insulin, cholera toxin and cortisol were present together. However, these additives when present independently showed a marginal response. The synergism, due to these factors in promoting growth was seen very early in culture (day 4) as shown by thymidine labelling studies, On examining the indices of early mitogenesis, such as the expression ofc-myc, our data suggests that these factors stimulate the expression ofc-myc within 4 h. With respect to expression of TNF-α mRNA, this study suggests a possible modulation of TNF-α expression in response to these mitogens that stimulate proliferation maximally. Whether this expression of TNF-α by these epithelial cells is due to a maximal proliferative stimulus and/or is an early step in the cascade of intracellular signalling events is to be investigated in detail.     

20-Hydroxyecdysone prevents oxidative stress damage in adult Pyrrhocoris apterus

Injections of 38 pmol paraquat (1,1'-dimethyl-4,4'-bypyridilium) into adult Pyrrhocoris apterus (average body weight 29.6 mg in males and 36.9 mg in females) caused a significant elevation of lipid peroxidation and protein carbonylation and a decline of membrane fluidity in the microsomal brain fraction. Another manifestation of oxidative stress was a depletion of the reduced glutathione pool and reduction of the gamma-glutamyl transpeptidase activity in the brain extracts. The damaging action of paraquat on the brain was counteracted by simultaneous injection of 1 pmol 20-hydroxyecdysone (20E). 20E restrained lipid peroxidation and the formation of protein carbonyls, ameliorated changes in microsomal membrane fluidity, enhanced the level of reduced glutathione, and upregulated the activity of gamma-glutamyl transpeptidase. At the organismic level, 20E curtailed three detrimental effects caused by paraquat injection: the disappearance of a blood protein, the suppression of fecundity and egg hatchability, and the shortening of adult life span. The data showed that 20E provided a systemic antioxidant protection but the significance of endogenous ecdysteroids in the management of oxidative stress remains to be shown.     

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.     

Alkaline lipase activity from the marine protists,thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.