Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.     

The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assessed by the colony-forming assay

The radiosensitivity of human lymphocytes was investigated by the method of colony formation in the absence of interleukin-2 (IL2) and feeder cells, both of which enhance growth of T-cell colonies. The shape of the survival curve and the radiosensitivity was shown to depend upon the ability of lymphocytes to produce IL2: the survival curve for lymphocytes that were the most competent producers of IL2 is the closest to linearity; the lymphocytes that were poor producers show biphasic survival curves. The radiosensitivity of the lymphocytes from the first group is less than that of the latter, when the comparison is based on the first part of the biphasic survival curve. This is more easily seen when cultures are irradiated 24 h after stimulation by phytohemagglutinin (the time of the peak IL2 production) than when cultures are irradiated 2 h before stimulation. This study demonstrates that growth conditions influence the response of lymphocytes to irradiation and that optimal growth conditions result in a linear survival curve.     

Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol

Summary Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.     

Novel features of retroviruses associated with human diseases.

The last decade has witnessed a new chapter in the history of retrovirology. As of now, four human retroviruses have been identified and molecularly characterized. They are associated with a wide spectrum of human diseases including cancer, immunodeficiency and neurological disorders. By virtue of their clinical relevance, their novel genes and regulatory mechanisms these viruses have become the focal point of research in retrovirology. The study of these viruses is of fundamental importance in understanding the mechanisms leading to transformation of human cells and distortion of the immunological state.     

Data bank of three-dimensional structures of disaccharides,a tool to build 3-D structures of oligosaccharides

This work presents the first part of a database of conformations for all the disaccharide fragments that are found inN-glycans. The conformational study of the five disaccharides found in the oligo-mannose type are presented here. For each disaccharide, several possible conformations are described. A method is presented to obtain realistic models of oligosaccharides using molecular mechanic methods. Analysis of some possible conformations of the oligo-mannose type glycan Man₆-GlcNAc₂ is given as an illustration of the possibilities.On post-doctoral leave from CERMAV-CNRS, Grenoble, France     

Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells

Recombinant human insulin-like growth factor II (IGF-II) was produced in NIH-3T3 cells transfected with a plasmid containing a construct encoding the signal peptide and the sequence of mature human IGF II. Successfully transfected clones secreted correctly processed recombinant human IGF II at rates of about 100ng per 24 hours per 10(6) cells. The biological activity of the purified recombinant human IGF II exhibited similar potencies as standard human IGF II isolated from serum in radio-receptorassays as well as in thymidine incorporation assays.     

Changes in the Activity of the Chloroplastic and Cytosolic Forms of Dihydroxyacetone Phosphate Reductase during Maturation of Leaves

Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or overnight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were returned to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during in vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform.     

Mitochondrial metabolism of guanine nucleotides. Possible role of guanosine

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.     

Mutational analysis of conserved nucleotides in a self-splicing group I intron.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.     

Investigating the role of impoundment and forest structure on the resistance and resilience of mangrove forests to hurricanes

The present study evaluates how a prolonged artificial flooding regime in impounded mangrove forests influences the regeneration after medium-sized forest destruction created by two hurricanes in 2004 in the Indian River Lagoon, Florida, USA. Disturbance patterns, forest structure, and regeneration were investigated. We found disturbed areas, characterized by uprooted and snapped trees, and intact forests in close proximity. The canopy turnover was greater in forests in higher succession stages with median tree diameter at breast height of 7.6 ± 5.7 cm compared to the intact forest with 3.7 ± 1.2 cm. Larger trees with lower densities were more susceptible to hurricane damage. We observed that regeneration of the open patches was dominated by the flood-tolerant species Rhizophora mangle (89.9%) instead of the faster-growing pioneer species Laguncularia racemosa (7.0%). Some of the disturbed areas created by the hurricanes were not recolonized. The regeneration rate of the disturbed areas expressed by vegetation closure >50% differed between almost zero to 100% in three different impoundments. We concluded that the artificial flooding regime imposed by impoundment is the predominant selective force in the successive process according to the species composition. However, we were not able to detect the driving factors that prevented mangrove establishment in disturbed areas.