Citations

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca 2+ -binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (∼300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.

Abstract IGFBP-4 is an inhibitor of IGF-I in bone. We show that TGF-β regulates IGFBP-4 and enhances IGF-I-stimulated growth of cultured human bone cells through increased expression of an IGFBP-4 protease, PAPP-A. This effect of TGF-β on IGF-I bioavailability may promote local bone formation. Insulin-like growth factor binding protein (IGFBP-4) proteolysis is implicated in the regulation of local insulin-like growth factor (IGF)-I bioavailability during bone remodeling. The IGFBP-4 protease secreted by normal adult human osteoblastic (hOB) cells in culture is a novel metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). We have recently identified an inhibitor of PAPP-A, the precursor form of major basic protein (proMBP). Very little is known about the molecular regulation of this IGFBP-4 protease system. In the present study, we determined the effect of transforming growth factor (TGF)-β and IGF-II, the two most abundant growth factors in human bone, on PAPP-A and proMBP expression in primary cultures of hOB cells. Treatment with TGF-β resulted in time- and dose-dependent increases in PAPP-A mRNA expression, with a maximal 12-fold increase after 24 h of stimulation with 10 ng/ml TGF-β. Increased PAPP-A levels in hOB cell-conditioned medium paralleled PAPP-A gene expression. In addition, TGF-β completely suppressed proMBP expression. Treatment of hOB cells with IGF-II had no effect on PAPP-A or proMBP gene expression. However, IGFBP-4 proteolysis in cell-free assay was dependent on IGF-II, and there was increased IGF-II-dependent IGFBP-4 protease activity in conditioned medium from hOB cells that were treated with TGF-β. IGF-I stimulation of hOB cell proliferation was markedly enhanced by pretreatment with TGF-β and [Leu27]IGF-II, and this enhancement was prevented with protease-resistant IGFBP-4. In summary, TGF-β regulates IGFBP-4 proteolysis in hOB cells through increased expression of the protease, PAPP-A, and decreased expression of the inhibitor, proMBP. However, functional activation of the IGFBP-4 protease system is dependent on IGF-II, which acts at a post-translational level. These data support a model whereby local TGF-β and IGF-II in the bone microenvironment coordinately amplify IGF-I bioavailability through controlled IGFBP-4 proteolysis, which may be a means to promote bone formation.

ABSTRACTThe objectives of this study were to evaluate soybean [Glycine max (L.) Merr.] agronomic and quality traits and to examine correlations among traits in an interspecific soybean population. The agronomic traits included maturity, plant canopy height, canopy spread, leaflet length, leaflet width, seed yield, and seed size. The seed quality traits were sucrose, raffinose, and stachyose content. A total of 308 recombinant inbred lines (RIL) were developed from the cross between a large‐seeded Glycine max breeding line (V71‐370) and a small‐seeded Glycine soja Sieb. & Zucc. accession (PI 407162). Normal distribution was observed for most of the traits with a slight skewness towards the G. soja parent. Most correlation coefficients were highly significant, such as the positive correlation between yield and sucrose and between seed size and sucrose. Genotype × environment interaction was significant for all of the agronomic traits but not for sugar contents. Maturity, seed size, and sucrose content were highly heritable, whereas plant height, canopy spread, leaflet length, leaflet width, yield, stachyose content, and raffinose content had relatively low broad‐sense heritabilities. Our findings suggest that the lack of genotype × location effects on sugar content favors progress through selection, but the strong correlation among the sugars may hinder progress in selecting lines that are high in sucrose but low in raffinose and stachyose over different environmental conditions. The information on heritabililty and interrelationships among the agronomic and seed quality traits will be helpful to breeders in constructing their breeding populations and implementing selection strategies.

Abstract Background BNP is a cardiac peptide with vasodilating, lusitropic and natriuretic properties mediated by the second messenger cGMP. We have previously shown that chronic subcutaneous (SQ) administration of BNP in experimental CHF resulted in improved haemodynamics and unloading of the heart. However, it is unknown if this will lead to the development of tolerance to exogenous BNP. Methods The current study extends our previous study and compares the cardiorenal effects of acute administration of SQ BNP (5 μg/kg) in a group of dogs (n = 5) with rapid ventricular pacing induced CHF (180 bpm for 10 days) to a separate group of CHF dogs (n = 6), who received chronic SQ BNP (5 μg/kg) three times a day for 10 days. Results Acute administration of SQ BNP resulted in similar increases in both plasma cGMP (35 ± 5 vs. 29 + 2 pmol/ml) and urinary cGMP excretion (UcGMPV) (6000 ±1000 vs. 4000 ± 600 pmol/min) in both the Chronic SQ BNP treated and the Untreated CHF groups (P>0.05). These were associated with decreased cardiac filling pressures and increased urine flow, which were also similar in both groups. Conclusion In experimental CHF, chronic SQ BNP administration did not result in the development of tolerance as demonstrated by increases in both plasma cGMP and UcGMPV following acute administration of SQ BNP. This may have important clinical implications, suggesting that chronic BNP administration does not lead to the development of tolerance to acute BNP administration.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G 2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of γ-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.

Pulse pressure, the difference between systolic and diastolic blood pressure, is an independent risk factor for cardiovascular disease. Increased pulse pressure reflects reduced compliance of arteries and is a marker of atherosclerosis. To locate genes that affect pulse pressure, a genome-wide linkage scan for quantitative trait loci influencing pulse pressure was performed using variance components methods as implemented in sequential oligogenic linkage analysis routines. The analysis sample included 10 798 participants in 3320 families who were recruited as part of the Family Blood Pressure Program and were phenotyped with an oscillometric blood pressure measurement device using a consistent protocol across centers. Pulse pressure was adjusted for the effects of sex, age, age 2 , age-by-sex interaction, age 2 -by-sex interaction, body mass index, and field center to remove sources of variation other than the genetic effects related to pulse pressure. Significant linkage was observed on chromosome 18 (logarithm of odds [LOD]=3.2) in a combined racial sample, chromosome 20 (LOD=4.4), and 17 (LOD=3.6) in Hispanics, chromosome 21 (LOD=4.3) in whites, chromosome 19 (LOD=3.1) in a combined sample of blacks and whites, and chromosome 7 (logarithm of odds [LOD]=3.1) in blacks from the GenNet Network. Our genome scan shows significant evidence for linkage for pulse pressure in multiple areas of the genome, supporting previous published linkage studies. The identification of these loci for pulse pressure and the apparent congruence with other blood pressure phenotypes provide increased support that these regions contain genes influencing blood pressure phenotypes.