Variation of mini- and microsatellite DNA repeats in parthenogenetic lizard Darevskia armeniaca as revealed by DNA fingerprinting analysis

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of pathogenetic lizard Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GATA)n and (TCC)n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli. D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to the variability level observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi (0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC)n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.     

Genetic variation in parthenogenetic Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca, L. unisexualis) analyzed by DNA fingerprinting

Multilocus DNA fingerprinting has been used to study the variability of some mini- and microsatellite sequences in parthenogenetic species of Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca and L. unisexualis). We demonstrate that these clonally reproducing lizards possess species-specific DNA fingerprints with a low degree of intra- and interpopulation variation. Mean indices of similarity obtained using M13 DNA, (GACA)4 and (TCC)50 as probes were 0.962 and 0.966 in L. dahli and L. armeniaca, respectively. The mean index of similarity obtained using M 13 and GATA probes in L. unisexualis was estimated to be 0.95. However, despite the high degree of band-sharing, variable DNA fragments were revealed in all populations with the microsatellite probes. An particularly high level of variability was observed for (TCC)n microsatellites in populations of L. unisexualis. In fact TCC-derived DNA fingerprints were close to being individual-specific, with a mean index of similarity of 0.824. Fingerprint analysis of parthenogenetic families of L. armeniaca showed that all maternal fragments were inherited together by the progeny, and no differences in fingerprint patterns were observed. On the other hand, while identical DNA fingerprints were obtained from L. unisexualis families with M13 and (GATA)4 probes, use of the (TCC)50 probe revealed remarkable intrafamily variation in this species. It is assumed that the genetic heterogeneity observed in parthenogenetic populations may be explained, at least in part, by the existence of genetically unstable microsatellite loci. Our data serve to illustrate processes of spontaneous mutagenesis and the initial stages of clonal differentiation in natural populations of the lizard species studied.     

Formation of DNA triple helix containing N(4)-(6-aminopyridin-2-yl)-2'-deoxycytidine.

A cytidinyl derivative, N(4)-(6-aminopyridin-2-yl)- 2'-deoxycytidine ((p)C), could interact with a CG base pair to support the triple-helix (triplex) formation of oligodeoxyribonucleotides. Characteristics of (p)C in the formation of both intramolecular triplex, i.e., a "paper clip type" triplex ((P)CT) and intermolecular triplex, i.e., a "linear type" triplex (LT) was monitored by optical methods and isothermal titration calorimetric measurements. Experimental results revealed that the LT with (p)C*CG internally was independent of the solution pH. Only single substitution of (p)C, situated internally but not terminally, facilitated the (P)CT formation by the UV thermal melting study at the neutral pH. However, the best stabilization of the PCT in acidic conditions occurred when (p)C at the end of the triplex rather than internally. In addition, an LT, but not a (P)CT, containing an alternating (p)CT(p)CT(p)C sequence, could be formed in the conditions of 20 mM MgCl(2) and/or 5 mM spermine. Thus, the presence of several nucleotides of (p)C in proximity along the Hoogsteen strand may lead to structural distortion such that the more flexible LT with multiple substitutions is formed in favor of the more rigid PCT.     

Morphometry of Macaca mulatta forelimb. II. Fiber-type composition in shoulder and elbow muscles

The present study examined the fiber-type proportions of 22 muscles spanning the shoulder and/or elbow joints of three Macaca mulatta. Fibers were classified as one of three types: fast-glycolytic (FG), fast-oxidative-glycolytic (FOG), or slow-oxidative (SO). In most muscles, the FG fibers predominated, but proportions ranged from 25-67% in different muscles. SO fibers were less abundant except in a few deep, small muscles where they comprised as much as 56% of the fibers. Cross-sectional area (CSA) of the three fiber types was measured in six different muscles. FG fibers tended to be the largest, whereas SO fibers were the smallest. While fiber-type size was not always consistent between muscles, the relative size of FG fibers was generally larger than FOG and SO fibers within the same muscle. When fiber CSA was taken into consideration, FG fibers were found to comprise over 50% of the muscle's CSA in almost all muscles.     

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.     

Assessment of DNA strand breakage by the alkaline COMET assay in dialysis patients and the role of Vitamin E supplementation

Although the role of reactive oxygen species (ROS) in chronic renal failure (CRF) is not definitely demonstrated, a consistent number of observations has provided evidence for the presence of oxidative stress in uremic patients undergoing maintenance dialysis. In order to investigate this hypothesis further and to understand the role of antioxidant supplementation, peripheral blood lymphocytes were taken from 36 dialysis patients before and after Vitamin E supplementation in a dosage of 600 mg per day (2x300 mg) for 14 weeks and examined in the alkaline Comet assay for DNA strand breakage. The results were also compared with those of 36 controls with comparable age, sex, and smoking habits, and with no history of renal disease. The DNA breakage observed in the lymphocytes of patients before Vitamin E supplementation was significantly higher than in the controls (P<0.001) but a clear protective effect of Vitamin E supplementation were observed after 14 weeks of therapy.     

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (approximately 31 and 3.4 microM, respectively), whereas the other four peptides bind weakly (>100 microM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly.     

Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1-E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2- and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3- and quasi 3-fold axes. We show here, using cryo- electron microscopy, that low pH, as occurs in the endosomes during virus uptake, results in the relaxation of protomer interactions around the 2- and the 5-fold axes in the shell, and movement of protomers towards 3- and quasi 3-fold axes in a way that reciprocally relocates their putative E1 and E2 domains. This seemed to be facilitated by a trimerization of transmembrane segments at the same axes. The alterations observed help to explain several key features of the spike-mediated membrane fusion reaction, including shell dissolution, heterodimer dissociation, fusion peptide exposure and E1 homotrimerization.     

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.