Cardiovascular actions of rattlesnake bradykinin ([Val1,Thr6]bradykinin) in the anesthetized South American rattlesnake Crotalus durissus terrificus

Incubation of heat-denatured plasma from the rattlesnake Crotalus atrox with trypsin generated a bradykinin (BK) that contained two amino acid substitutions (Arg1 --> Val and Ser6 --> Thr) compared with mammalian BK. Bolus intra-arterial injections of synthetic rattlesnake BK (0.01-10 nmol/kg) into the anesthetized rattlesnake, Crotalus durissus terrificus, produced a pronounced and concentration-dependent increase in systemic vascular conductance (Gsys). This caused a fall in systemic arterial blood pressure (Psys) and an increase in blood flow. Heart rate and stroke volume also increased. This primary response was followed by a significant rise in Psys and pronounced tachycardia (secondary response). Pretreatment with N(G)-nitro-L-arginine methyl ester reduced the NK-induced systemic vasodilatation, indicating that the effect is mediated through increased NO synthesis. The tachycardia associated with the late primary and secondary response to BK was abolished with propranolol and the systemic vasodilatation produced in the primary phase was also significantly attenuated by pretreatment, indicating that the responses are caused, at least in part, by release of cathecholamines and subsequent stimulation of beta-adrenergic receptors. In contrast, the pulmonary circulation was relatively unresponsive to BK.     

Molecular mechanism of hTid-1, the human homolog of Drosophila tumor suppressor l(2)Tid, in the regulation of NF-kappaB activity and suppression of tumor growth

hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor kappaB (NF-kappaB), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-kappaB-IkappaB through direct interaction with IkappaBalpha/beta and the IKKalpha/beta subunits of the IkappaB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IkappaB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1DeltaN100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1DeltaN100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-kappaB through physical and functional interactions with the IKK complex and IkappaB and, in doing so, it modulates cell growth and death.     

Declining fertility in the lethal yellow mouse is related to progressive hyperleptinemia and leptin resistance

Mice possessing the lethal yellow mutation (C57BL/6J A(y)/a) become obese and develop hyperleptinemia and leptin resistance as they age. To determine the relationship between altered leptin physiology and reproductive function in these mice, we compared body weight (BW), serum leptin concentration, ovulation rate, and in vitro blastocyst development among 120- and 180-d-old lethal yellow and black non-mutant (a/a) mice. Estrous female yellow and black mice were mated with fertile black males. Oviducts were flushed approximately 36 h after mating and the recovered embryos were cultured for 96 h. BW, serum leptin levels, and the leptin:BW ratio differed among groups as follows: 180-d yellow > 120-d yellow > 180-d black = 120-d black. Ovulation rate was similar among 120-d yellow and black, and 180-d black mice. Among 180-d yellow mice, five of twelve mice failed to ovulate, but the other seven mice ovulated a similar number of oocytes as their black counterparts (8.4 +/- 0.9 versus 8.0 +/- 1.3). Non-ovulators had higher (P < 0.05) leptin levels (56.6 +/- 1.8 ng x mL(-1)) than ovulators (46.2 +/- 3.5), but BW did not differ significantly. Fewer embryos from 180-d yellow mice reached the blastocyst stage in culture than did the embryos from black mice (55% versus 83%, P < 0.05). Moreover, blastocyst development in 180-d old yellow mice negatively correlated with leptin levels (r = -0.797, P = 0.032) and leptin:BW ratio (r = -0.847, P = 0.016), but not with BW. Declining reproductive function in lethal yellow mice appears to be related to increasing levels of leptin and progression of leptin resistance.     

Plant genetic determinants of arthropod community structure and diversity

To test the hypothesis that genes have extended phenotypes on the community, we quantified how genetic differences among cottonwoods affect the diversity, abundance, and composition of the dependent arthropod community. Over two years, five major patterns were observed in both field and common-garden studies that focused on two species of cottonwoods and their naturally occurring F1 and backcross hybrids (collectively referred to as four different cross types). We did not find overall significant differences in arthropod species richness or abundance among cottonwood cross types. We found significant differences in arthropod community composition among all cross types except backcross and narrowleaf cottonwoods. Thus, even though we found similar richness among cross types, the species that composed the community were significantly different. Using vector analysis, we found that the shift in arthropod community composition was correlated with percent Fremont alleles in the host plant, which suggests that the arthropod community responds to the underlying genetic differences among trees. We found 13 arthropod species representing different trophic levels that were significant indicators of the four different cross types. Even though arthropod communities changed in species composition from one year to the next, the overall patterns of community differences remained remarkably stable, suggesting that the genetic differences among cross types exert a strong organizing influence on the arthropod community. Together, these results support the extended phenotype concept. Few studies have observationally and experimentally shown that entire arthropod communities can be structured by genetic differences in their host plants. These findings contribute to the developing field of community genetics and suggest a strategy for conserving arthropod diversity by promoting genetic diversity in their host plants.     

ICAT-MS-MS time course analysis of atrophying mouse skeletal muscle cytosolic subproteome

Skeletal muscle atrophy is a process in which protein degradation exceeds protein synthesis, resulting in a decrease of the muscle's physiological cross-sectional area and mass, and is often a serious consequence of numerous health problems. We used the isotope-coded affinity tag (ICAT) labelling approach and MS-MS to protein profile cytosolic subcellular fractions from mouse tibialis anterior skeletal muscle undergoing 0, 4, 8, or 16 days of immobilisation-induced atrophy. For the validation of peptide and protein identifications statistical algorithms were applied to the sequence database search results in order to obtain consistent sensitivity/error rates for protein and peptide identifications at each immobilisation time point. In this study, we identified and quantified a large number of mouse skeletal muscle proteins. At a protein probability (P) of P> or = 0.9 (corresponding to a false positive error rate of less than 1%) 807 proteins were identified (231, 226, 217 for 4, 8, 16 days of immobilisation and 133 for the control sample, respectively), from which 51 displayed altered protein abundance with atrophy. Due to randomness of data acquisition, a full time course could be generated only for 62 proteins, most of which displayed unchanged protein abundance. In spite of this, useful information about dataset characteristics and underlying biological processes could be obtained through gene over-representation analysis. 20 gene categories-mainly but not exclusively encoded by the subset of overlapping proteins--were consistently found to be significantly (p < 0.05) over-represented in all 4 sub-datasets.     

Mutants of FtsZ targeting the protofilament interface: effects on cell division and GTPase activity

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 microM, in contrast to the high GTPase activity of wild-type FtsZ above 1 muM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 microM K(d) for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.