Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides

Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.     

Convenient preparation of L-arabino-hexos-5-ulose derivatives from lactose

2,6-di-O-benzyl- (9), 2-O-benzyl-3,4-O-isopropylidene- (19), and 2-O-benzyl-6-O-m-chlorobenzoyl-L-arabino-hexos-5-ulose (20) have been prepared using 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose dimethyl acetal derivatives 7 and 14 as key intermediates. The synthesis of enol ethers 7 and 14 has been performed with good yields by base-promoted elimination of acetone or p-toluenesulfonic acid from 2',6'-di-O-benzyl-, and 6'-O-p-toluenesulfonyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal, respectively. The epoxidation with MCPBA of 7 and 14 in methanol or dichloromethane furnishes C-5'-methoxy and C-5'-m-chlorobenzoyloxy derivatives, easily transformed with good yields into L-arabino 5-ketoaldohexoses 9, 19 and 20.     

Genetic variation and population structure in Scandinavian wolverine (Gulo gulo) populations

Wolverine (Gulo gulo) numbers in Scandinavia were significantly reduced during the early part of the century as a result of predator removal programmes and hunting. Protective legislation in both Sweden and Norway in the 1960s and 1970s has now resulted in increased wolverine densities in Scandinavia. We report here the development of 15 polymorphic microsatellite markers in wolverine and their use to examine the population sub-structure and genetic variability in free-ranging Scandinavian wolverine populations as well as in a sample of individuals collected before 1970. Significant subdivision between extant populations was discovered, in particular for the small and isolated population of southern Norway, which represents a recent recolonization. Overall genetic variability was found to be lower than previously reported for other mustelids, with only two to five alleles per locus and observed heterozygosities (H(O)) ranging from 0.269 to 0.376 across the examined populations, being lowest in southern Norway. Analysis of the mitochondrial DNA control region revealed no variation throughout the surveyed populations. As the historical sample did not show higher levels of genetic variability, our results are consistent with a reduction in the genetic variation in Scandinavian wolverines that pre-dates the demographic bottleneck observed during the last century. The observed subdivision between populations calls for management caution when issuing harvest quotas, especially for the geographically isolated south Norwegian population.     

Tripartite genetic subdivisions in the ornate shrew (Sorex ornatus)

We examined cytochrome b sequence variation in 251 ornate shrews (Sorex ornatus) from 20 localities distributed throughout their geographical range. Additionally, vagrant (S. vagrans) and montane (S. monticolus) shrews from four localities were used as outgroups. We found 24 haplotypes in ornate shrews from California (USA) and Baja California (Mexico) that differed by 1-31 substitutions in 392 bp of mitochondrial DNA (mtDNA) sequence. In a subset of individuals, we sequenced 699 bp of cytochrome b to better resolve the phylogeographic relationships of populations. The ornate shrew is phylogeographically structured into three haplotype clades representing southern, central and northern localities. Analysis of allozyme variation reveals a similar pattern of variation. Several other small California vertebrates have a similar tripartite pattern of genetic subdivision. We suggest that topographic barriers and expansion and contraction of wetland habitats in the central valley during Pleistocene glacial cycles account for these patterns of genetic variation. Remarkably, the northern ornate shrew clade is phylogenetically clustered with another species of shrew suggesting that it may be a unique lowland form of the vagrant shrew that evolved in parallel to their southern California counterparts.     

Comparison of three fungal laccases from Rigidoporus lignosus and Pleurotus ostreatus: correlation between conformation changes and catalytic activity

The conformation changes in solution of three fungal laccases in different environmental conditions were examined by circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy. CD measurements indicate that the secondary structure of proteins depends slightly on the pH or ionic strength, though the presence of salt could interfere in the molecular recognition process between substrates and enzymes. The enzymes, however, are highly destabilized by prolonged exposure to low pH or high temperature. The observed unfolding of the proteins coincides with their inactivation and, in some cases, with precipitation. On the other hand, these conditions do not determine the disruption of the geometric arrangement of their metal centres, and this fact suggests that these centres represent the more stable core of the proteins.     

Adsorption step in the biological degradation of a textile dye

This research documents the removal of the dye Gris Lanaset G from aqueous solutions by fungal pellets. Adsorption of the dye by dead biomass pellets of Trametes versicolor was determined and compared with dye removal by enzymatic degradation. Six kinetic equations were fitted to the experimental adsorption data obtained. The results indicate that kinetics such as the Elovich equation, which considers that the rate-controlling step is the diffusion of the dye molecules, show the best fit. Nonlinear Langmuir and Freundlich equations were also fitted into the adsorption data, and it can be concluded that the adsorption equilibrium can be interpreted by the Langmuir isotherm. Adsorption plays an important role in the process of the elimination of color from textile wastewater, although not all of the elimination is due to this physical process when the microorganism is active. The removal of color (around 90%) with active microorganisms is greater than that obtained with the adsorption process.     

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells

Summary. GABA_A receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABA_A receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABA_A receptors mediating the tonic form of inhibition in cerebellar granules. Received December 12, 2000 Accepted February 12, 2001