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. 相似文献
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.
Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.
Results
KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr161 (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr15 (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.
Conclusions
The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs. 相似文献
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. 相似文献
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 SiaLeX-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. 相似文献
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. 相似文献
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. 相似文献