Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.     

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan(HA), a large polysaccharide abundant in the extracellular matrixof vertebrate tissues. Six genes have been predicted to encodehyaluronidases in humans, but the protein products of only SPAM1,HYAL1, and HYAL2 have been characterized. We have now expressedthe mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in BabyHamster Kidney (BHK) cells and demonstrated the presence ofmultiple forms of Hyal3 ranging from 45 to 56 kDa in expressionlysates. Complete and partial digestions of the expressed proteinwith PNGase F showed three N-linked oligosaccharides accountedfor all forms of Hyal3 detected in expression lysates. Mostof these oligosaccharides were Endo H sensitive, indicatingthat they were high mannose or hybrid N-linked oligosaccharides.Subcellular fractionation of Hyal3-expressing BHK cells by densitygradient centrifugation revealed most Hyal3 in a low-densityvesicular population. Low levels of Hyal3 were detected in higherdensity vesicles, but no colocalization with the late endosomal/lysosomalmarker Lamp1 was found by immunofluorescence microscopy. BHKcells stably expressing Hyal3 had increased acid-active hyaluronidaseactivity, but no such activity was detected when Hyal3 was transfectedinto Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpressionof Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels,suggesting that the increased hyaluronidase activity in thesecells was due to Hyal1 rather than Hyal3. The results indicatethat Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidaseactivity and that Hyal3 may contribute to HA metabolism by augmentingthe activity of Hyal1.     

Metabolism of 1α-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1α,20-dihydroxyvitamin D3

Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1α-hydroxyvitamin D3 and whether products were biologically active. The major product of 1α-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1α,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1α,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The K_m for 1α-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The k_cat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1α,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1α,25-dihydroxyvitamin D3. 1α,20-Dihydroxyvitamin D3 (10 μM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1α,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1α,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically.     

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.     

Bilateral Functional Connectivity of the Basal Ganglia in Patients with Parkinson’s Disease and Its Modulation by Dopaminergic Treatment

Parkinson’s disease is characterised by excessive subcortical beta oscillations. However, little is known about the functional connectivity of the two basal ganglia across hemispheres and specifically the role beta plays in this. We recorded local field potentials from the subthalamic nucleus bilaterally in 23 subjects with Parkinson’s disease at rest, on and off medication. We found suppression of low beta power in response to levodopa (t₂₂ = −4.4, p<0.001). There was significant coherence between the two sides in the beta range in 19 of the subjects. Coherence was selectively attenuated in the low beta range following levodopa (t₂₂ = −2.7; p = 0.01). We also separately analysed amplitude co-modulation and phase synchronisation in the beta band and found significant amplitude co-modulation and phase locking values in 17 and 16 subjects respectively, off medication. There was a dissociable effect of levodopa on these measures, with a significant suppression only in low beta phase locking value (t₂₂ = −2.8, p = 0.01) and not amplitude co-modulation. The absolute mean values of amplitude co-modulation (0.40±0.03) and phase synchronisation (0.29±0.02) off medication were, however, relatively low, suggesting that the two basal ganglia networks may have to be approached separately with independent sensing and stimulation during adaptive deep brain stimulation. In addition, our findings highlight the functional distinction between the lower and upper beta frequency ranges and between amplitude co-modulation and phase synchronization across subthalamic nuclei.     

Molecular mechanism of tyrosinase regulation by L-dopa in hamster melanoma cells

Exposure of hamster amelanotic melanoma cells to L-dihydroxyphenylalanine (L-DOPA) resulted in a time dependent increase of cell pigmentation, tyrosinase concentration and activity with peak after 24 hours. Northern blot analysis showed a small but reproducible increase of tyrosinase mRNA after 3 hours and a decrease below the control level after 9 hours. After 24 and 48 hours tyrosinase mRNA was undetectable. It is suggested that L-DOPA or its oxidation products can stimulate intracellular tyrosinase concentration and regulate tyrosinase mRNA level both in positive and negative fashion.     

Serotonin metabolism in rat skin: characterization by liquid chromatography-mass spectrometry

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.     

Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.     

Serum Vitamin D Concentrations in Baboons (Papio spp.) during Pregnancy and Obesity

Obesity is associated with vitamin D deficiency, which can lead to serious problems during pregnancy. However, the mechanisms of the deficiency and guidelines for vitamin D supplementation during pregnancy are not established yet, and variations in environmental exposures combined with the difficulties of performing research in pregnant women are obstacles in the evaluation of vitamin D metabolism. Baboons (Papio spp.) are an excellent, well-established model for reproductive research and represent a unique opportunity to study vitamin D metabolism in a controlled environment. This study used secondary data and specimen analysis as well as a novel experimental design to evaluate pregnant and nonpregnant baboons that were or were not exposed to sunlight while they were obese and after weight reduction. Daily D₃ intake was 71% higher in nonpregnant obese baboons than in their nonobese counterparts, but serum vitamin D concentrations did not differ between these populations. In addition, serum 25-hydroxyvitamin D concentrations correlated negatively with the obesity index. This report is the first to show the effect of obesity and pregnancy on vitamin D concentrations in a NHP population. These data underline the importance of adequate vitamin D supplementation in obese animals.Vitamin D deficiency during pregnancy is a major public health problem³⁰ that is associated with increased maternal and neonatal morbidity.^5,6,22,31 Even though vitamin D is actively involved in placental function and fetal growth,^5,11,43 the dosage of vitamin D supplementation during pregnancy has not been established.³⁰ Maternal serum concentrations of 25-hydroxyvitamin D are directly related to vitamin D intake and sunlight exposure in humans.²⁷ Obesity is an important risk factor for vitamin D deficiency for which the mechanisms remain unknown, and the effect of weight loss on vitamin D status is unclear.^14,15,33,34 In addition, obesity itself is a significant public health problem.^18,51 Several mechanisms linking obesity and vitamin D deficiency have suggested, including decreased exposure to sunlight, sequestration of vitamin D in the adipose tissue,⁵⁰ and other factors related to obesity.^34,49 In pregnancy an additional factor involved in vitamin D activation and metabolism is the placenta^46-48—a temporary endocrine organ whose function is linked to the metabolism of the adipose tissue.⁷An understanding of the relationship between vitamin D, obesity, and pregnancy is critical for reducing maternal and neonatal morbidity. Baboons (Papio spp.) have been used extensively in pregnancy-related research.^17,40 The advantages of this model include the similarity of its placentation to that in humans and the ability to create uniform exposures to environmental factors (for example, sunlight, dietary composition) and thus study underlying metabolic pathways, which are impossible to study in the heterogeneous human population. However, only a very few studies related to the vitamin D requirements in these NHP species are available,^35,39 and none of them addresses the effect of obesity and pregnancy on vitamin D metabolism. The objective of the current study was to evaluate the effects of obesity, weight reduction, and pregnancy on the systemic vitamin D status of baboons, by using data collected during prior studies as well as a novel study design.     

The role of polymorphic variants of gene of inducible NO-synthase NOS2 in brain infarction in patients with acute ischemic stroke

Cerebrovascular diseases including stroke are an important problem of public health. Stroke development depends on external factors and individual genetic specificity of patient. Excessive NO production by inducible NO-synthase (iNOS) damages brain tissue at various stages of the disease. The goal of this work was to study the role of 4 polymorphic variants of gene of inducible NO-synthase iNOS (-2447C/G, -1659C/T, -0,7(OTTA)n I/D, S608L (150C/T)) in brain infarction in patients with acute ischemic stroke. A statistically significant correlation between S608L (150C/T) polymorphism and infarction dynamics was observed during days 1-3 and 7-21 after infarction. These parameters correlated with neurological status estimated using the Orgogozo scale during days 1-7 of the disease development. It was demonstrated that genotype N150N was associated with ischemic focus propagation regardless of its volume and neurological status by Orgogozo scale in patients with acute stroke. It was also observed that genotype N150N had effect on ischemic damage during days 1-3 in case of low initial volume.