The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Specific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples

An important issue in the management of zebra mussel (Dreissena polymorpha) populations is early, rapid, and accurate detection of the planktonic larvae (veliger) of the zebra mussel. The goal of this study was to explore the feasibility of developing a molecular approach for the detection of zebra mussel larvae in diverse environments. In this study a Dreissena polymorpha-specific 18S ribosomal RNA gene targeted oligonucleotide primer (ZEB-715a) and Polymerase Chain Reaction (PCR) assay was developed and compared with cross-polarized microscopy as a means to detect zebra mussel veligers in plankton samples. The design of the zebra mussel-specific primer was facilitated by sequencing nearly the complete 18S rRNA gene from the zebra mussel and three other closely related freshwater Veneroids including the quagga mussel (D. bugensis), the dark false mussel (Mytilopsis leucophaeata), and the Asian freshwater clam (Corbicula fluminea). The specificity of the primer for the zebra mussel was empirically tested by using the primer as a direct probe in a blot hybridization format. A single veliger in a plankton sample could be detected by PCR using this approach. Veliger detection sensitivity using the PCR approach was estimated to be over 300 times more sensitive than cross-polarized light microscopy based techniques. Cross-polarized light microscopy and the PCR technique were used to identify the presence of zebra mussel larvae in plankton samples that were collected from a variety of natural and industrial water sources. Detection results (presence or absence) were generally consistent between the two methods. Although additional studies will be required before routine application of molecular based veliger detection technology is available, a long-term goal of this work is the application of molecular technology to the development of a field device for the routine detection and quantification of zebra mussel veligers.     

Vocal identification of individual African Wood Owls Strix woodfordii: a technique to monitor long-term adult turnover and residency

The hoot-calls of Wood Owls Strix woodfordii , recorded in the Kruger National Park, South Africa, from 1986 to 1998, are sufficiently different to distinguish individuals. Thirteen hoot-call variables, measured from recordings over this period, show that male and female Wood Owls have such temporal stability that these calls can be used reliably as a long-term census technique. manova , based on ordinations from principal component analysis, was used to identify individuals statistically between sampling periods. A forward stepwise discriminant function analysis achieved 100% classification success of individual male ( n  = 3) and female ( n  = 4) owls from a single sampling period. For all the individuals recorded over the whole study period we achieved a classification success of 80.9% ( n  = 9) for male hoot calls and of 96.3% ( n  = 13) for female calls. We found mean occupation periods of 82.25 and 65 months and annual turnover rates of 19.3% and 13.65% for males and females, respectively. Our survey, using vocalizations, is unique since we use data collected over a 12-year period to derive estimates of population turnover in Wood Owls, and consider what questions can be addressed in similar studies.     

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.     

Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts

Translocations of chloroplasts induced by blue light were investigated in both leaves and protoplasts isolated from leaf mesophyll of Nicotiana tabacum. In the leaf tissue, the responses of chloroplasts were similar to those observed in other, higher and lower plant species. Weak and strong light induced movements of chloroplasts towards cell walls perpendicular and parallel to the light direction, respectively. Treatment with cytochalasin D, an actin-disturbing agent, blocked the movements. This shows that actin is involved in the motile system of chloroplast translocation in tobacco. By monitoring the response of chloroplasts to light in isolated protoplasts, we addressed the question whether the presence of the cell wall is necessary for the translocations of chloroplasts to occur. In control protoplasts (isolated at room temperature from unstressed leaves), no clear light intensity-dependent changes were observed in chloroplast distribution pattern. In contrast, in protoplasts obtained from plants treated with 4 °C for 8 h the chloroplasts maintained their responsiveness to light. Atomic Force Microscopy was used to measure elastic properties of the protoplasts. Young’s modulus, which reflects rigidity of the material, was 10 times higher for protoplasts of the coldstressed plants as compared to those isolated from the control plants. The rigidity of protoplasts isolated from the plants treated with low temperature was reduced four-fold by exposure to cytochalasin D. It appears that the status of protoplast actin is a factor responsible for elasticity of protoplasts. We speculate that unknown, cold stress-induced factors, maintain the orientational movements due to anchorage of the actin cytoskeleton in the plasma membrane despite the cell wall removal.