Prevalence of alleles of polymorphic variants Leu33Pro and Leu66Arg gene ITGB3 among inhabitants of Siberia

The frequency of the polymorphic variant T196C (Leu33Pro, rs5918) of ITGB3 gene was studied in several groups of inhabitants of Siberia, including women with reproductive disorders (n = 186), patients with acute coronary syndrome (n = 330), and population control (n = 858). The frequency of the rare PLA2 allele among residents of Tomsk and Kemerovo was 14.7% and 15.0% respectively. There were no differences in the allele and genotype frequencies of polymorphic variant between patients with acute coronary syndrome and the control group (p = 0.925, p = 0.622). The highest frequency of abnormal PLA2 allele (22.1%) and the PLA2/PLA2 genotype (8.8%) was observed among women who had miscarried, which was significantly different from the frequency of this allele and genotype in the control group (14.7%, p = 0.017; 2.1%, p = 0.0009). Sequencing showed that all samples with the nonspecific band had the polymorphic rs5918 variant and rs36080296 mutations (T216G, Leu66Arg). The frequency of the rs36080296 mutation among the residents of Siberia was 0.51%. Among the women with reproductive disorders, the frequency of rs36080296 was 2.7%, while in the group who suffered from miscarriages, it was 4.4%; this was different from the frequency in the control group (0.08%, p = 0.2 × 10^?6). The accumulation of mutations was also observed among men with acute coronary syndrome (0.6%), but the differences from the control group (0%) had no statistical significance. Thus, the rs36080296 mutation may be a factor in predisposition to miscarriage, especially in combination with the PLAII allele. In addition, the rs36080296 variant among men may be associated with acute cor onary syndrome, which requires further study.     

Influence of renal denervation on renal effects of acute nitric oxide and ETA/ETB receptor inhibition in conscious normotensive rats.

The role of renal nerves in the effects of concomitant NO synthase and non-selective ET(A/)ET(B) receptor inhibition on renal function was investigated in conscious normotensive Wistar rats. NO synthase inhibition alone (10 mg/kg b. w. i.v. L-NAME) in sham-operated rats with intact renal nerves induced an increase in systolic, diastolic and mean arterial pressure, urine flow rate, sodium, chloride and calcium excretion (p<0.05). The effect of L-NAME was markedly reduced by bosentan (10 mg/kg b.w. i.v.) and the values of urine flow rate, sodium, chloride and calcium excretions returned to control level (p<0.05). L-NAME administration one week after a bilateral renal denervation increased blood pressure to a similar extent as in sham-operated rats but decreased urine flow rate (p<0.05) and did not change electrolyte excretion. ET(A/)ET(B) receptor inhibition with bosentan during NO synthase inhibition in the renal denervated rats did not produce changes in urine flow rate or electrolyte excretion. NO synthase inhibition as well as concurrent NO synthase and ET(A/)ET(B) receptor inhibition did not change clearance of inulin or paraaminohippuric acid in sham-operated or renal denervated rats. These results indicate that renal sympathetic nerves play an important modulatory role in NO and endothelin induced effects on renal excretory function.     

Violet bioluminescence and fast kinetics from W92F obelin: structure-based proposals for the bioluminescence triggering and the identification of the emitting species

Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation of the bound substrate, coelenterazine. The W92F obelin mutant is noteworthy because of the unusually high speed with which it responds to sudden changes of [Ca(2+)] and because it emits violet light rather than blue due to a prominent band with lambda(max) = 405 nm. Increase of pH in the range from 5.5 to 8.5 and using D(2)O both diminish the contribution of the 405 nm band, indicating that excited state proton transfer is involved. Fluorescence model studies have suggested the origin of the 485 nm emission as the excited state of an anion of coelenteramide, the bioluminescence reaction product, and 405 nm from the excited neutral state. Assuming that the dimensions of the substrate binding cavity do not change during the excited state formation, a His22 residue within hydrogen bonding distance to the 6-(p-hydroxy)-phenyl group of the excited coelenteramide is a likely candidate for accepting the phenol proton to produce an ion-pair excited state, in support of recent suggestions for the bioluminescence emitting state. The proton transfer could be impeded by removal of the Trp92 H-bond, resulting in strong enhancement of a 405 nm band giving the violet color of bioluminescence. Comparative analysis of 3D structures of the wild-type (WT) and W92F obelins reveals that there are structural displacements of certain key Ca(2+)-ligating residues in the loops of the two C-terminal EF hands as well as clear differences in hydrogen bond networks in W92F. For instance, the hydrogen bond between the side-chain oxygen atom of Asp169 and the main-chain nitrogen of Arg112 binds together the incoming alpha-helix of loop III and the exiting alpha-helix of loop IV in WT, providing probably concerted changes in these EF hands on calcium binding. But this linkage is not found in W92F obelin. These differences apparently do not change the overall affinity to calcium of W92F obelin but may account for the kinetic differences between the WT and mutant obelins. From analysis of the hydrogen bond network in the coelenterazine binding cavity, it is proposed that the trigger for bioluminescence reaction in these Ca(2+)-regulated photoproteins may be a shift of the hydrogen bond donor-acceptor separations around the coelenterazine-2-hydroperoxy substrate, initiated by small spatial adjustment of the exiting alpha-helix of loop IV.     

Exhibition of persistent and drug-tolerant L-form habit of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> during infection in rats

A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.     

Spontaneous aneuploidy level in blood cells of fertile females

In this study, we presented results of molecular cytogenetic assay of blood cells of fertile women of reproductive age. Cultivated and uncultivated cells and two sets of FISH probes with direct and indirect labeling were used. The middle level of aneuploidy for four chromosomes and statistical limits for aneuploidy detection were estimated. Aneuploidy determined with direct multicolor FISH in cultivated lymphocytes varied from 0.1 to 1.3%. The middle level of aneuploidy for all four chromosomes (13, 18, 21, and X) was 1.39%. The limit of mutation detection was 3.4%. We found differences in results with direct and indirect labeling. It was shown that the cultivation process influenced the level of aneuploidy. The absolute error in FISH technique was 0.13% and the relative error was 4.08%.     

Characteristics of morphological changes in the myocardium of rats with different degrees of hereditary resistance to hypoxia during the development of adrenaline-induced myocardial dystrophy

By means of polarizational microscopy method morphological changes of the cardiac muscle have been studied in highly-(HS) and poorly-stable (PS) rats to hypoxia 1 h and 1 day after injection of cardiotoxic dose of adrenaline (1.5 mg/kg of body mass). One hour after injection of adrenaline in PS animals the II-III stages of contractive myofibrillar changes are revealed, while in HS rats--the I-II stages. In 1 day in the myocardium of PS animals contractures of the III-IV degree are observed, more foci of clumping decay and myocytolysis than in HS rats. Severity of the morphological changes of the cardiac muscle after cardiotoxic dose of adrenaline essentially depends on the organism's reactivity.     

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.     

Activity and isoenzyme composition of peroxidase and esterase in fertile and male-sterile lines of tomatoes (Lycopersicon esculentum Mill.)

A comparative study of the isoenzyme patterns of esterase and peroxidase and overall peroxidase activity in stamens of male-sterile (MS) lines of Pearson ms-35 and P ms-35aa and of the respective male-fertile (MF) tomato plants have been conducted. The study has been made at two stages of stamens development — tetrad and pollen. Higher activities of the esterase isoenzymes in the MF stamens than that of MS in both ontogeny stages have been found. The slow moving esterase isoenzymes both of the MF and the MS stamens are the major isoenzymes in the early stage and are connected with tapetum development while the fast moving esterase isoenzymes are connected with pollen formation in the later ontogeny stage. Overall peroxidase levels in the MS stamens were higher than those of MF. The peroxidase patterns of the MS lines are also characterized by the greater number of isoenzymes and also the presence of specific isoenzymes, the contrast between the MF and the MS stamens being more strongly expressed at the later stage of development. A strong similitude between esterase and peroxidase patterns behaviour in both MS lines has been found.     

Eligibility and Safety of Triple Therapy for Hepatitis C: Lessons Learned from the First Experience in a Real World Setting

Background

HCV protease inhibitors (PIs) boceprevir and telaprevir in combination with PEG-Interferon alfa and Ribavirin (P/R) is the new standard of care in the treatment of chronic HCV genotype 1 (GT1) infection. However, not every HCV GT1 infected patient is eligible for P/R/PI therapy. Furthermore phase III studies did not necessarily reflect real world as patients with advanced liver disease or comorbidities were underrepresented. The aim of our study was to analyze the eligibility and safety of P/R/PI treatment in a real world setting of a tertiary referral center.

Methods

All consecutive HCV GT1 infected patients who were referred to our hepatitis treatment unit between June and November 2011 were included. Patients were evaluated for P/R/PI according to their individual risk/benefit ratio based on 4 factors: Treatment-associated safety concerns, chance for SVR, treatment urgency and nonmedical patient related reasons. On treatment data were analyzed until week 12.

Results

208 patients were included (F3/F4 64%, mean platelet count 169/nl, 40% treatment-naïve). Treatment was not initiated in 103 patients most frequently due to safety concerns. 19 patients were treated in phase II/III trials or by local centers and a triple therapy concept was initiated at our unit in 86 patients. Hospitalization was required in 16 patients; one patient died due to a gastrointestinal infection possibly related to treatment. A platelet count of <110/nl was associated with hospitalization as well as treatment failure. Overall, 128 patients were either not eligible for therapy or experienced a treatment failure at week 12.

Conclusions

P/R/PI therapies are complex, time-consuming and sometimes dangerous in a real world setting, especially in patients with advanced liver disease. A careful patient selection plays a crucial role to improve safety of PI based therapies. A significant number of patients are not eligible for P/R/PI, emphasizing the need for alternative therapeutic options.