Virulence of pre-CTXphi-carrying Vibrio cholerae: genotypic and phenotypic characteristics

AIM: Complex assessment of virulence of cholera vibrios carrying the truncated CTX element (pre-CTXphi prophage). MATERIALS AND METHODS: Twenty-two strainsof Vibriocholerae O1 and non-O1/non-O139 were studied by PCR and laboratory models. RESULTS: Genomes of all strains, besides pre-CTXphi genes, contained genes hapA (hemagglutinin/proteases), cef (CHO cell elongating factor), rtxA (high-molecular cytotoxin), and rtxC (its activator). Nucleotide sequences of rtxA and vgrG genes from ACD domains, genes VPI and VPI-2 from islands of pathogenicity, mshA (mannose-sensitive pili) gene were presented in different combinations. None strains contained shiga-like toxin (slt1) aswell as thermostable direct (tdh) and thermostable direct-related (trh) hemolysin genes of V. parahaemoliticus. On the model of infant rabbits almost all strains caused a significant enteropathogenic effect sometimes resembling cholera effect and in a number of cases dissemination of bacteria into various organs and tissues took place. Cultural supernatants of the majority of strains stipulated cell rounding in CHO cultures (one of them caused cell destruction) and disconnection of cells in McCoy and L-929 dense monolayers as well as increase of skin permeability in Craig's test. Conclusion. Apparently, diarrhea of different severity observed in patients from whom these strains were isolated as well as signs of virulence revealed in the laboratory models were determined by the expression of genes of accessory pathogenicity factors including those detected in the present study.     

Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I

Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.5-50 μg/kg) and DNase I (0.02-2.3 mg/kg) inhibits the development of metastasis to 60-90% and RNase A exerts 30% retardation of tumor growth. Remarkably, the increase in RNase A dose from 50 μg/kg to 10 mg/kg leads to a disappearance of antitumor and antimetastatic effects. Simultaneous treatment of tumor-bearing animals with RNase A and DNase I leads to an additive effect and results in almost total absence of metastases. The use of RNase A as an adjuvant in conjunction with conventional cytostatic cyclophosphamide results in a reliable enhancement of antitumor and antimetastatic effect of the therapy compared with the use of these agents individually. The search for possible molecular mechanism of antimetastatic effect of nucleases showed that daily administration of the enzymes reduced the pathologically increased level of extracellular nucleic acids and increased nuclease activity of the blood plasma of tumor-bearing mice back to the level of healthy animals. Thus, we unequivocally show that the proposed protocol of treatment of tumor-bearing animals with RNase A and DNase I has a general systemic and immunomodulatory effect, leads to a drastic suppression of metastasis development, and in perspective may become an effective component of intensive complex therapy of cancer.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Synthetic conformational antigen which simulates the extracellular part of the M2-muscarinic receptor: interaction with blood sera of patients suffering from idiopathic arrhythmias

Fragments corresponding to the 83–98 sequence of the first extracellular loop and to the 168–192 and 171–182 sequences of the second extracellular loop of the M2-muscarinic receptor (antibodies to this receptor could be markers of early symptoms of heart disorders) were synthesized by solid phase method using the Fmoc-SPPS strategy. A new conformational antigen with the natural location of the disulfide bridge was prepared by selective formation of disulfide bond between the corresponding cysteine residues in the synthe-sized peptides and characterized. The comparative analysis of reactivity of the synthesized peptides towards sera from patients which had no organic heart disease was performed. A new conformational antigen was effectively bound to the sera from patients with idiopathic arrhythmias, but without symptoms of organic heart disease.     

基于网络药理学的谷糠结合态多酚抗乳腺癌机制研究

本研究旨在探讨谷糠结合态多酚(bound phenol of inner shell,BPIS)发挥抗乳腺癌细胞活性的作用机制。首先采用细胞计数法检测BPIS对乳腺癌细胞以及正常乳腺细胞活性的影响;然后综合运用SEA、SIB以及GeneCards等数据库获得BPIS和乳腺癌的相关靶点,并分析活性成分与作用靶点的互作网络以及通路。本研究筛选得到BPIS抗乳腺癌相关靶点39个,主要涉及糖脂代谢和细胞自噬等生物过程以及MAPK、PI3K/AKT、FoxO等多条信号通,表明BPIS抗乳腺癌是多成分、多靶点、多通路协同作用的过程,而与细胞死亡相关的细胞自噬很可能在BPIS抑制乳腺癌过程中发挥主要作用。     

Dynamics of hypothalamic CRH immune reactivity in active and passive rats in the course of development of behavioural depression

A possible relation between activity of the main CRH-producing centers of hypothalamus and depressive-like behavior of animals was studied. We used genetically selected strains--KHA (Koltushi High Avoidance) and KLA (Koltushi Low Avoidance) rats, demonstrating active and passive strategy of adaptive behavior in novelty situaltions, respectively. Rats were exposed to inescapable stress to develop a "learned helplessness". We observed considerable differences between two strains of animals in CRH-expression in parvo-, magno-cellular parts of the paraventricular nucleus and in the supraoptic nucleus in the course of behavioral depression development. Significant differences between control groups were seen only in paraventricular nucleus. On the 1st post-stress day in hypothalamus of KLA rats, we detected decreased CRH immune reactivity that remained unchanged up to the 10th day. In KHA rats, there were no notable changes of CRH expression in all studied nuclei. These findings, including previous results on different dynamics of behavioral changes and different hypothalamo-pituitary-adrenocortical system activity during development of depression in KLA and KHA rats, indicate that "learned helplessness" in these two groups of animals provides the model analogues of different types of depression. Besides, these findings indicate different implication of hypothalamus CRH-system in the behavioral depression development in rats with divergent strategy of adaptive behavior.