Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.     

Reduced representation bisulfite sequencing design for assessing the methylation of human CpG islands in large samples

The reduced representation bisulfite sequencing (RRBS) method has been developed for the high-throughput analysis of DNA methylation based on the sequencing of genomic libraries treated with sodium bisulfite by next-generation approaches. In contrast to whole-genome sequencing, the RRBS approach elaborates specific endonucleases to prepare libraries in order to produce pools of CpG-rich DNA fragments. The original RRBS technology based on the use of the MspI libraries allows one to increase the relative number of CpG islands in the pools of genomic fragments compared to whole-genome bisulfite sequencing. Nevertheless, this technology is rarely used due to the high cost compared with bisulfite methylation analysis with hybridization microarrays and significant residual amount of data represented by the sequences of genomic repeats that complicates the alignment and is not of particular interest for developing DNA methylation markers, which is often the main goal of biomedical research. We have developed an algorithm for estimating the likelihood that recognition sites of restriction endonucleases will be represented in CpG islands and present a method of reducing the effective size of the RRBS library without a significant loss of the CpG islands based on the use of the XmaI endonuclease for library preparation. In silico analysis demonstrates that the optimum range of the XmaI-RRBS fragment lengths is 110–200 base pairs. The sequencing of this library allows one to assess the methylation status of over 125000 CpG dinucleotides, of which over 90000 belong to CpG islands.

     

Genetic diversity of wild reindeer (<Emphasis Type="Italic">Rangifer tarandus</Emphasis>) of Taimyr: Analysis of polymorphism of the control region of mitochondrial DNA

Based on an analysis of the polymorphism of nucleotide sequences (n = 111) of the mtDNA control region (left domain), the genetic diversity of the largest population of wild reindeer Rangifer tarandus in Eurasia, which inhabits Taimyr peninsula, was studied. High levels of haplotype (H) and nucleotide (π) diversity (0.987 and 0.018, respectively) were revealed, which indicate the long existence and the sufficiently stable growth of this population. The absence of long periods of abrupt decrease in the number of the Taimyr wild reindeer population and/or facts of formation of its genetic diversity as a result of mixing of genetically distant conspecific populations is supported by the data on the pattern of mismatch distributions and the topology of the phylogenetic tree. The low level of genetic differences between reindeer from the western, central, and eastern groups reflects their common origin and close relationship.     

Liposome formulations of combretastatin A4 and its 4-arylcoumarin analogue prodrugs: The antitumor effect in the mouse model of breast cancer

The antimitotic agent combretastatin A-4 (CA-4) has been recently proposed as an antivascular agent for anticancer therapy. In order to reduce systemic toxicity by means of administration in liposome formulations, new lipophilic prodrugs, oleic derivatives of CA-4 and its 4-arylcoumarin analogue (CA4-Ole and ArC-Ole, respectively), have been synthesized in this study. Liposomes with mean diameter of 100 nm prepared on the basis of egg phosphatidylcholine and baker’s yeast phosphatidylinositol quantitatively included up to 15 mol% of CA4-Ole, or 7 mol% of ArC-Ole. To achieve targeting to neovascular endothelium prodrug bearing liposomes decorated with the tetrasaccharide selectin ligand Sialyl Lewis X (SiaLeX) have been also prepared. The antitumor activity was studied in vivo using the model of slow-growing mouse breast cancer. Under the dose used (22 mg/kg) and the administration protocol (four injections, one per a week, starting from the appearance of palpable tumors) cytostatic CA-4 did not reveal any anticancer effect; moreover, it even stimulated tumor growth. The liposome formulations of CA4-Ole did not demonstrate such stimulation. However, to achieve a pronounced antitumor effect, the number of injections of liposomes should be apparently increased. The cytotoxic activity of a novel antimitotic agent ArC was one order of magnitude lower in the human breast carcinoma cell culture in vitro. Nevertheless, in vivo in the mouse model of breast cancer the antitumor effect of this compound corresponded to the double equivalent dose of CA-4. The results demonstrate perspectives of SiaLe^X-liposomes loaded with ArC-Ole: the preparation partially inhibited tumor growth already after the second injection. Thus, subsequent optimization of doses and regimens of administration both for ArC and liposomal ArC-Ole formulations are needed.     

Survival of civilian and prisoner drug-sensitive, multi- and extensive drug- resistant tuberculosis cohorts prospectively followed in Russia

Objective and Methods

A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.

Results

MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).

Conclusions

Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.     

Two sequential phosphates 3' adjacent to the 8-oxoguanosine are crucial for lesion excision by E. coli Fpg protein and human 8-oxoguanine-DNA glycosylase

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are base excision repair enzymes involved in the 8-oxoguanine (oxoG) repair pathway. Specific contacts between these enzymes and DNA phosphate groups play a significant role in DNA-protein interactions. To reveal the phosphates crucial for lesion excision by Fpg and hOGG1, modified DNA duplexes containing pyrophosphate and OEt-substituted pyrophosphate internucleotide (SPI) groups near the oxoG were tested as substrate analogues for both proteins. We have shown that Fpg and hOGG1 recognize and specifically bind the DNA duplexes tested. We have found that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the 8-oxoguanosine (oxodG) and one nucleotide 3' away from it. In contrast, they efficiently incised DNA duplexes bearing the same phosphate modifications 5' to the oxodG and two nucleotides 3' away from the lesion. The effect of these phosphate modifications on the substrate properties of oxoG-containing DNA duplexes is discussed. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or SPI groups immediately 3' to the oxodG or one nucleotide 3' away from it are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a wild-type enzymes bound to oxoG-containing DNA.     

Cations in the human blood serum

The review summarizes data (more than 450 references) on concentration of human serum cations (Na+, K+, Ca2+, and Mg2+) and human blood serum osmolality depending on age, diverse physiological and pathological states, and action of physiologically active substances. There are summarized data of many thousand measurements of physicochemical parameters of the blood serum, the mean values of osmolality and cation concentrations in healthy people are calculated. The values are kept at a stable level throughtout the entire life since the moment of birth; in many cases they are maintained by regulatory systems within the normal limits and during various physiological and pathological states. There are formulated the main types of the states characterized by deviations from norm of physicochemical parameters of the internal medium fluids.