Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel-Lindau protein

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL-associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its beta-domain, and naturally occurring beta-domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild-type pVHL into human kidney tumor cell lines carrying endogenous mutant non-functional pVHL facilitates ubiquitylation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7-induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.     

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.     

Molecular Evolution of the GapC Gene Family in Amsinckia spectabilis Populations That Differ in Outcrossing Rate

Molecular evolutionary analysis of the glyceraldehyde 3-phosphate dehydrogenase (GapC) gene family was conducted in the plant genus Amsinckia (Boraginaceae), a group that exhibits marked variation in the mating system. GapC genes in this group differ from those of Arabidopsis thaliana in terms of both intron size and number. Phylogenetic and Southern hybridization analyses suggest the presence of multiple GapC loci, each defined by a set of base substitutions that are in strong linkage disequilibrium. One species of Amsinckia, A. spectabilis, was studied in some detail. This species consists of selfing (A. s. spectabilis) and outcrossing (A. s. microcarpa) varieties. Two selfing populations and one outcrossing population sample were analyzed in detail for variation at one of the members of this gene family, GapC3. A reduction in number of GapC3 haplotypes and level of genetic diversity was observed in the selfing populations of A. spectabilis. GapC3 in the outcrossing population (but not the two selfing populations) exhibited a significant departure from neutrality in the direction of an excess of singletons. These results are discussed in the context of forces acting on sequence evolution in populations with different mating systems.[Reviewing Editor: Dr. Martin Kreitman]     

Genomic mutation in lines of Arabidopsis thaliana exposed to ultraviolet-B radiation

Studies that have attempted to estimate the rate of deleterious mutation have typically been conducted under low levels of ultraviolet-B (UV-B) radiation, a naturally occurring mutagen. We conducted experiments to test whether the inclusion of natural levels of UV-B radiation in mutation-accumulation (MA) experiments influences the rate and effects of mildly deleterious mutation in the plant Arabidopsis thaliana. Ten generations of MA proved insufficient to observe significant changes in means or among-line variances in experimental lines maintained either with or without supplemental UV-B radiation. Maximum-likelihood estimates of mutation rate for total flower number revealed a small but significant rate of mutation for MA lines propagated under supplemental UV-B exposure, but not for those in which supplemental UV-B was omitted. A fraction of the flower number mutations under UV-B (approximately 25-30%) are estimated to increase flower number. Results from the application of transposon display to plant materials obtained after MA, in both the presence and absence of supplemental UV-B, suggest that the average rate of transposition for the class I and II transposable elements (TEs) surveyed was no more than 10(-4). Overall, the estimates of mutation parameters are qualitatively similar to what has been observed in other MA experiments with this species in which supplemental UV-B levels have not been used. As well, it appears that naturally occurring levels of UV-B do not lead to detectable increases in levels of transposable element activity.     

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Minimal activity in both proliferation and apoptosis of interstitial cells indicates seasonally persisting Leydig cell population in roe deer

Seasonally regulated breeding is associated with significant changes in testis mass, structure and function. This includes the variation in size, structure and function of the Leydig cells. Recently, interstitial cells have been characterised as a numerically constant population in roe deer. However, no consistent data are available regarding changes in the number of Leydig cells, their differentiation or turnover in seasonally breeding mammals. This study has quantified the numbers of both proliferating and apoptotic cells in roe deer testis bimonthly during a complete annual cycle. Proliferation was detected by immunolocalisation of PCNA and Ki-67 in tissue sections, whereas apoptosis was localised by the TUNEL technique and an antibody to caspase-3. The labelled cells were counted by using a computer-aided image-analysing system. The number of proliferating spermatogenic cells per tubule cross section showed seasonal changes with a maximum in April (14.9±0.6) and a subsequent decline up to December (1.6±0.3). Percentages of positive cells per square millimetre of interstitial area were below 1% throughout the year. The average number of apoptotic cells per tubule cross section was low and varied only between 0.5 and 1.4 (caspase-3) or 0.1 and 2.1 (TUNEL). In the interstitial compartment, only a few apoptotic cells (0.7%) were found sporadically scattered within the intertubular region during all studied seasonal periods. The results suggest that a constant total number of interstitial cells arise from a conserved cell population of changing functional state rather than from a steady-state population with a definite turnover of cells during seasonal changes in testicular activity.The study was supported by a grant from the Deutsche Forschungsgemeinschaft (BL 319/6-2).     

Hereditäre Chondrocalcinosis Articularis

Zusammenfassung Die Chondrocalcinosis articularis ist eine genetisch determinierte Krankheit, die mit charakteristischen Verkalkungen der hyalinen und fibrösen Knorpel einhergeht. Es wird über eine Untersuchung von fünf Personen einer Familie aus zwei Generationen berichtet, von denen vier Personen erkrankt sind.

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A family is reported with inherited Chondrocalcinosis articularis. Of the 5 family members studied, 4 had this disease. Three of these cases belonged to the first generation, one to the second. The disease is clinically manifested by episodic inflammatory involvement, acute or subacute, of one or more joints. The pathognomonic criterion for the diagnosis is the radiographic evidence of calcified hyalin and fibrous cartilage particularly in the large joints. In the articular cartilage a dense narrow band, continuous or discontinuous, followed the contur of the epiphysis. In the fibrocartilage, as seen in menisci or symphysis pubis, the calcification is more diffuse and of granular nature. The pathophysiology is unknown but it should be noted, that most cases of articular chondrocalcinosis give no evidence of a disturbance of the calcium or phosphorus metabolism. The chondrocalcinosis is an hereditary disease, but up to date it is impossible to define the type of hereditary transmission exactly. An autosomal dominant gene is the most polable explanation to date.

     

Mountain goat survival in coastal Alaska: Effects of age,sex, and climate

Ecological theory predicts that individual survival should vary between sex and age categories due to differences in allocation of nutritional resources for growth and reproductive activities. During periods of environmental stress, such relationships may be exacerbated, and affect sex and age classes differently. We evaluated support for hypotheses about the relative roles of sex, age, and winter and summer climate on the probability of mountain goat (Oreamnos americanus) survival in coastal Alaska. Specifically, we used known-fates analyses (Program MARK) to model the effects of age, sex, and climatic variation on survival using data collected from 279 radio-marked mountain goats (118 M, 161 F) in 9 separate study areas during 1977–2008. Models including age, sex, winter snowfall, and average daily summer temperature (during Jul–Aug) best explained variation in survival probability of mountain goats. Specifically, our findings revealed that old animals (9+ yr) have lower survival than younger animals. In addition, males tended to have lower survival than females, though differences only existed among prime-aged adult (5–8 yr) and old (9+ yr) age classes. Winter climate exerted the strongest effects on mountain goat survival; summer climate, however, was significant and principally influenced survival during the following winter via indirect effects. Furthermore, old animals were more sensitive to the effects of winter conditions than young or prime-aged animals. These findings detail how climate interacts with sex and age characteristics to affect mountain goat survival. Critically, we provide baseline survival rate statistics across various age, sex, and climate scenarios. These data will assist conservation and management of mountain goats by enabling detailed, model-based demographic forecasting of human and/or climate-based population impacts. © 2011 The Wildlife Society.