Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.     

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.     

A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome

Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.     

Effect of reduced bronchial circulation on lung fluid flux after smoke inhalation in sheep

We determined the effect of reduced bronchialblood flow on lung fluid flux through changes in lung lymph flow, lungwet weight-to-dry weight (wet/dry) ratios, and pulmonary microvascularreflection coefficient (). In the first of two surgical procedures,Merino ewes (n = 21) were surgicallyprepared for chronic study. Five to seven days later, in a secondoperation, the bronchial artery of the injection group(n = 7) was ligated, and 4 ml of 70%ethanol were injected into the bronchial artery to cause sclerosis of the airway circulation. In the ligation group(n = 7), only the bronchial artery wasligated. In the sham group (n = 7),the bronchial artery was surgically exposed but left intact withoutligation or ethanol injection. One day after these operations theanimals received a tracheotomy and 48 breaths of cotton smoke. Thevalue of  was determined at two points: 24 h before the secondsurgical procedure and 24 h after smoke inhalation. Lung lymph flow,blood-gas parameters, and hemodynamic data were measured every 4 hafter injury. At the end of investigation, samples of lung were taken for determination of blood-free wet/dry ratio. In the sham group, inhalation injury induced a gradual increase in pulmonary vascular resistance and lung lymph flow, which was associated with deterioration of oxygenation. Reduction of the bronchial blood flow attenuated thesepathophysiological changes, and the degree of this attenuation wasgreater in the injection group than in the ligation group. The value of was significantly higher after smoke inhalation in the injectiongroup compared with the sham group (0.77 ± 0.04 vs. 0.61 ± 0.03, means ± SE) at 24 h. The mean wet/dry ratio value of theinjection group animals was 30% less than that of the sham group. Ourdata show that the bronchial circulation contributes to edema formationin the lung occurring after acute lung injury with smoke inhalation.

     

Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

Will krill fare well under Southern Ocean acidification?

Antarctic krill embryos and larvae were experimentally exposed to 380 (control), 1000 and 2000 µatm pCO₂ in order to assess the possible impact of ocean acidification on early development of krill. No significant effects were detected on embryonic development or larval behaviour at 1000 µatm pCO₂; however, at 2000 µatm pCO₂ development was disrupted before gastrulation in 90 per cent of embryos, and no larvae hatched successfully. Our model projections demonstrated that Southern Ocean sea water pCO₂ could rise up to 1400 µatm in krill''s depth range under the IPCC IS92a scenario by the year 2100 (atmospheric pCO₂ 788 µatm). These results point out the urgent need for understanding the pCO₂-response relationship for krill developmental and later stages, in order to predict the possible fate of this key species in the Southern Ocean.     

Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.     

Daily Rhythm of Mutualistic Pollinator Activity and Scent Emission in Ficus septica: Ecological Differentiation between Co-Occurring Pollinators and Potential Consequences for Chemical Communication and Facilitation of Host Speciation

The mutualistic interaction between Ficus and their pollinating agaonid wasps constitutes an extreme example of plant-insect co-diversification. Most Ficus species are locally associated with a single specific agaonid wasp species. Specificity is ensured by each fig species emitting a distinctive attractive scent. However, cases of widespread coexistence of two agaonid wasp species on the same Ficus species are documented. Here we document the coexistence of two agaonid wasp species in Ficus septica: one yellow-colored and one black-colored. Our results suggest that their coexistence is facilitated by divergent ecological traits. The black species is longer-lived (a few more hours) and is hence active until later in the afternoon. Some traits of the yellow species must compensate for this advantage for their coexistence to be stable. In addition, we show that the composition of the scent emitted by receptive figs changes between sunrise and noon. The two species may therefore be exposed to somewhat different ranges of receptive fig scent composition and may consequently diverge in the way they perceive and/or respond to scents. Whether such situations may lead to host plant speciation is an open question.     

Modeling the Role of Negative Cooperativity in Metabolic Regulation and Homeostasis

A significant proportion of enzymes display cooperativity in binding ligand molecules, and such effects have an important impact on metabolic regulation. This is easiest to understand in the case of positive cooperativity. Sharp responses to changes in metabolite concentrations can allow organisms to better respond to environmental changes and maintain metabolic homeostasis. However, despite the fact that negative cooperativity is almost as common as positive, it has been harder to imagine what advantages it provides. Here we use computational models to explore the utility of negative cooperativity in one particular context: that of an inhibitor binding to an enzyme. We identify several factors which may contribute, and show that acting together they can make negative cooperativity advantageous.     

Erythropoiesis in cultures of dispersed cell preparations from early chick blastodiscs

Optimal conditions were determined for differentiation of erythroid cells in liquid cultures of dispersed cell preparations from chick blastodiscs at the primitive streak and head-fold stages of development. Most of the unattached erythroid cells free in the medium were mature erythrocytes.