The efficient synthesis of isotopically labeled peptide-derived Amadori products and their characterization

Protein glycation is often a cause of diabetes-associated complications. The isotopically labeled peptide-derived Amadori products may serve as standards for quantitative determination of the glycated proteins. In this paper, we discussed various approaches to the synthesis of Amadori products labeled selectively with stable isotopes ²H, ¹³C and ¹⁸O.     

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.     

Dominance-related changes in spatial memory are associated with changes in hippocampal cell proliferation rates in mountain chickadees

It is well established that spatial memory is dependent on the hippocampus in both mammals and birds. As memory capacity can fluctuate on a temporal basis, it is important to understand the mechanisms mediating such changes. It is known that early memory-dependent experiences in young animals result in hippocampal enlargement and in increased neurogenesis, including cell proliferation and neuron survival. It is less clear, however, whether temporal changes in spatial memory are also associated with changes in hippocampal anatomy and cell proliferation in fully grown and experienced adult animals. In a previous study, we experimentally demonstrated that socially subordinate mountain chickadees (Poecile gambeli) showed inferior spatial memory performance compared to their dominant group mates, in the absence of significant differences in baseline corticosterone levels. Here we investigated whether these differences in memory between dominant and subordinate birds were associated with changes in the hippocampus. Following memory tests, chickadees were injected with 5-bromo-2'-deoxyuridine to label dividing cells and sacrificed 2 days after the injections. We found no significant differences in volume or the total number of neurons in the hippocampal formation between dominant and subordinate chickadees, but subordinate birds had significantly lower cell proliferation rates in the ventricular zone adjacent to both the hippocampus and mesopallium compared to the dominants. Individuals, which performed better on spatial memory tests tended to have higher levels of cell proliferation. These results suggest that social status can affect cell proliferation rates in the ventricular zone and support the hypothesis that neurogenesis might be involved in memory function in adult animals.     

Insulin resistance and serum concentrations of ovarian and adrenal androgen in obese women without additional disease and with policystic ovary syndrome

THE AIM: of the present study was to evaluate serum concentrations of adrenal and ovarian androgens and sex hormone-binding globulin in obese women without additional diseases and in obese women with polycystic ovary syndrome with and without insulin resistance. MATERIAL AND METHODS: The study group involved 73 obese women (39 with PCOS--A and 34 obese without additional diseases--B). The serum concentration of glucose and insulin were measured and the study group was divided on the basis of HOMA index into two subgroups: A I-PCO without insulin resistance (n=18, mean age 27.2+/-5.9 yr; BMI 33.2+/-3.5 kg/m2); AII-PCO with insulin resistance (n=21, mean age 27.5+/-7.1 yr; BMI 37.6+/-6.5 kg/m2); B I-obese without insulin resistance (n=8, age 33.5+/-7.5 yr; BMI 35.2+/-4.8 kg/m2); B II-obese with insulin resistance (n=24, age 30.3+/-5.2 yr; BMI 36.4+/-5.8 kg/m2). Body mass and height were measured and body mass index was calculated with formula. Body composition was measured using bioimpedance method. The serum concentrations of FSH, LH, total and free testosterone, androstendione, DHEAS, SHBG and insulin were determined by RIA method and glucose was determined by enzymatic procedure. RESULTS: We observed significantly higher body mass, fat mass and BMI in AII subgroup when compared to AI, BI and BII subgroups. Only serum concentration of free testosterone was significantly higher in AII subgroup when compared to AI subgroup. We observed a positive correlation between serum concentrations of insulin and free testosterone in both groups A and B, moreover we observed positive correlations between serum concentrations of insulin and both DHEAS and LH in group B. CONCLUSIONS: It seems that insulin resistance plays a key role in the development of hyperandrogenism in obese women. However mechanisms leading to hyperandrogenism in PCOS are still unrevealed and seem to be more complex.     

Strains of genus Salmonella isolated from extraintestinal infections

Every year in Poland from tens to more than hundred bacteriologically verified extraintestinal infections caused by Salmonella species have been registered. These unusually located infections have substantially heavy course and in many cases hospitalisation and antibiotic therapy have to be involved. Cases of extraintestinal infections with these Gram-negative rods, which were described in the literature, concerned: pneumonias, lung abscesses and thoracic empyemas, and infections of: blood, bones and joints, wounds, fistulas and urinary tract. The aim of this study was to set extraintestinal Salmonella infections and to analyze a susceptibility of isolated strains to antimicrobial agents. Between 01.07.2002 and 31.12.2004 (30 months) 13 strains of Salmonella genus have been isolated, including 11 S. enteritidis and 2 S. Hadar. In general, with one exception, isolated strains were susceptible to tested antibiotics/chemotherapeutics. ESBL - positive strains were not detected. The tendency of Salmonella strains to cause extraintestinal infections has been noticed. The problem is still escalating, especially in group of patients chronically treated, with immunodeficiency and immunosuppression, after complicated medical procedures, also in the group of small children and aged persons. Therefore, it is necessary to evaluate a susceptibility to antibiotics/chemotherapeutics of every strain from confirmed case of Salmonella extraintestinal infection and it is important to apply a guided therapy.     

A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene

A simple bacterial model for studying effects of human mutations in vivo, when homologous genes exist in bacterial and human cells, is presented. We have constructed Escherichia coli strains bearing different alleles of the metF gene, an ortologue of human MTHFR gene, coding for 5,10-methylenetetrahydrofolate reductase. These strains bear a null mutation in the chromosomal metF gene and different metF alleles on plasmid(s), and thus there are merozygotes mimicking wild-type homozygotes, heterozygotes and recessive mutant homozygotes. The A177V mutantion in metF corresponds to one of the most common MTHFR polymorphism, A222V, which has been shown to be associated with increased levels of homocysteine in plasma that, in turn, causes many serious medical problems. Results of relatively simple and quick experiments with these strains are compatible with previously published reports on effects of the A222V substitution in the product of MTHFR gene. In addition, these results suggest either impairment of formation of heterodimers and/or heterotetramers by wild-type and A177V metF variants or dominance of the wild-type polypepides in such structures. Moreover, positive effects of folic acid and vitamins B2 and B12 on physiology of the mutant cells, suggested on the basis of clinical studies, is confirmed. Therefore, we conclude that the bacterial model described in this report may be a useful tool in studies on human mutations.     

Melatonin increases tissue accumulation and toxicity of cadmium in the bank vole (<Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>)

Recent study has shown that a short photoperiod increases the accumulation and toxicity of cadmium (Cd) in the bank vole as compared to a long photoperiod. Since many of the effects of photoperiod on physiological processes in small mammals are transduced by the pineal gland and its hormone melatonin, in this study the effect of subchronic melatonin injection (7 mol/kg/day for 6 weeks) on the hepatic, renal and intestinal Cd accumulation in the bank voles raised under a long photoperiod and exposed to dietary Cd (0.9 mol/g) was examined. Simultaneously, histological examinations of the liver and kidneys, and analyses of metallothionein (MT) and lipid peroxidation were carried out. Melatonin co-treatment brought about a significant increase in the hepatic (61%), renal (79%) and intestinal (77%) Cd concentrations as compared to those in the Cd alone group. However, the concentrations of MT in the liver and kidneys of the Cd + melatonin co-treated bank voles did not differ from those in the Cd alone group. Also, histopathological changes in the liver (infiltration of leukocytes) and kidneys (glomerular swelling and a focal tubular cell degeneration) as well as an increase (2-fold) in the renal lipid peroxidation occurred only in animals from the Cd + melatonin group. These data indicate that (1) subchronic melatonin injection has similar effect on the tissue accumulation and toxicity of Cd to that produced by a short photoperiod and (2) the Cd-induced toxicity in the liver and kidneys of melatonin co-treated bank voles is probably due to increased Cd accumulation and decreased synthesis of MT.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Proteomic analysis of complexes formed by human topoisomerase I

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.