Improved gas chromatographic-mass spectrometric determination of the N-methylcarbamoyl adduct at the N-terminal valine of globin,a metabolic product of the solvent N,N-dimethylformamide

A sensitive method for determination of the N-methylcarbamoyl adduct at the N-terminal valine of globin, a new metabolic product of the industrial solvent N,N-dimethylformamide (DMF), has been developed and validated. The method includes conversion of the adduct by the Edman degradation to 3-methyl-5-isopropylhydantoin (MVH), which is followed by optimized gas chromatographic analysis with mass spectrometric detection at m/z 114. The recovery of MVH from terminal N-methylcarbamoylvaline was determined using a model dipeptide to be 90%. Calibration of the method is done with MVH, employing 3-methyl-5-isobutylhydantoin as the internal standard. The limit of detection is 0.2 nmol MVH/g globin when a 100-mg sample is used. Within- and between-day precision is 4-10%. The method has been used to determine the background levels of MVH in unexposed subjects. Further, toxicokinetic studies in volunteers laid the grounds for setting the reference value for biological monitoring of occupational exposure to DMF.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

N-terminal truncations of manganese stabilizing protein identify two amino acid sequences required for binding of the eukaryotic protein to photosystem II and reveal the absence of one binding-related sequence in cyanobacteria

Manganese stabilizing protein (MSP) is an intrinsically disordered extrinsic subunit of photosystem II that regulates the stability and kinetic performance of the tetranuclear manganese cluster that oxidizes water to oxygen. An earlier study showed that deletion of the (1)E-(3)G domain of MSP caused no loss of activity reconstitution, whereas deletion of the (4)K-(10)E domain reduced binding of the protein from 2 to 1 mol of MSP/mol of photosystem II and lowered activity reconstitution to about 50% of the control value [Popelkova et al. (2002) Biochemistry 41, 2702-2711]. In this work we present evidence that deletion of 13 or 14 amino acid residues from the MSP N-terminus (mutants DeltaS13M and DeltaK14M) does not interfere either with functional binding of one copy of MSP to photosystem II or with reconstitution of oxygen evolution activity to 50% of the control level. Both of these mutants exhibit nonspecific binding to photosystem II at higher protein concentrations. Truncation of the MSP sequence by 18 amino acids (mutant DeltaE18M), however, causes a loss of protein binding and activity reconstitution. This result demonstrates that the N-terminal domain (15)T-(18)E is required for binding of at least one copy of MSP to photosystem II. Analyses of CD spectra reveal changes in the structure of DeltaE18M (loss of beta-sheet, gain of unordered structure). Use of the information gained from these experiments in analyses of N-terminal sequences of MSP from a number of species indicates that higher plants and algae possess two recognition domains that are required for MSP binding to PSII, whereas cyanobacteria lack the first N-terminal domain found in eukaryotes. This may explain the absence of a second copy of MSP in the crystal structure of PSII from Synechococcus elongatus [Zouni et al. (2001) Nature 409, 739-743].     

Vibrational circular dichroism spectroscopy study of paroxetine and femoxetine precursors

The solution structures of (3R,4S)- and (3S,4R)- 4-(4-fluorophenyl)-3-hydroxylmethyl- 1-methylpiperidine, which are intermediates in the synthesis of the two pharmaceuticals paroxetine and femoxetine, were studied by vibrational circular dichroism (VCD) spectroscopy. In addition, six derivatives with different substituents attached to the C3 atom were prepared and their VCD and absorption spectra discussed with the aid of ab initio simulations. The VCD spectra were found to be sensitive to the geometry changes. In addition, a subtle variation caused by intermolecular aggregation was apparent in the spectra. The VCD technique can be applied for structural analysis of chiral pharmaceuticals in solutions.     

Socio-economic Predictors of Life Style. A pilot study

In epidemiological studies there exists the evidence of relationship between socio-economic status (SES) and health. The suspicious cause of these differences is life-style. The aim of the presented study was to identify the relationship between SES, demographic characteristics and the life-style in an industrial region. A structured questionnaire was distributed to a random sample of some 3,000 aged 25-70 and collected by postal delivery. The SES and demographic factors being analysed were sex, age, education, marital status and economic situation of the family in the relationship with a wide range of information on life style. The data was analysed using the chi-square test and logistic regression. The total number of analysed questionnaires was 634. The lowest response-rate was in the group with the basic education. Significant differences were found by sex in education (less women with university degree) and household income (more women in the lowest income category, men in the highest one). A detail analysis of the aggregated variables (adjusted for all SES variables) found: passivity and contentment were in adverse correlation with the level of education and economic situation; psychical well-being was correlated with family status and economic situation, risk behaviour was correlated with all SES factors except sex and education. Among the analysed factors economical situation of respondents, family and education were the most significant determinants of the differences in life-style.     

Biochemical regulation of breast cancer cell expression of S1P2 (Edg-5) and S1P3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.     

Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation

A rapid and simple electroporation method to transform osmotolerant yeast Zygosaccharomyces rouxii has been developed and conditions for efficient transformation of mutants derived from different Z. rouxii wild-type strains optimized.     

Analysis of state-specific phosphorylation of proteins by two-dimensional gel electrophoresis approach

In this paper we focus on the detection of specific state of protein phosphorylation within a complex protein mixture separated by two-dimensional gel electrophoresis followed by immunoblotting. The availability of antibodies that specifically recognize the phosphorylated residue(s) of proteins make this approach feasible as exemplified by the study of the regulatory mechanisms of the cell cycle. The major advantage of the presented approach is its relative simplicity and sensitivity that allows specific detection of protein phosphorylation and distinguishes different phosphorylation sites of target protein. Current findings demonstrate that this method represents a reasonable alternative to the use of other tools to study protein phosphorylation.     

Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.