Complete genome sequence of a new circular DNA virus from grapevine

A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae, but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.     

A2A adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis

Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime mediator of adenosine's anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induction experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte infiltration. However, A2AAR(-/-) mice developed a more severe acute EAE phenotype characterized by more proinflammatory lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR(-/-) donor hematopoietic cells potentiated severe EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in the suppressive ability of A2AAR(-/-) regulatory T cells was observed, A2AAR(-/-) lymphocytes were shown to proliferate more and produced more IFN-γ following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment still protected against EAE when A2AAR(-/-) lymphocytes were adoptively transferred to T cell-deficient A2AAR(+/+) mice. These results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.     

The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.     

Analysis of myosin heavy chain functionality in the heart

Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.     

A new type of mutation in phytochrome A causes enhanced light sensitivity and alters the degradation and subcellular partitioning of the photoreceptor

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction pathways in Arabidopsis. Because of their phenotype, the mutants were called eid for empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light. One of the isolated mutants, eid4, is a novel semi-dominant allele of the phytochrome A gene that carries a missense mutation in the chromophore-binding domain. The mutation did not change the photochemical properties of the photoreceptor, but it leads to an increased stability under light conditions that induce its rapid degradation. Fusion proteins with the green fluorescent protein exhibited clear alterations in subcellular localization of the mutated photoreceptor: The fusion protein was impaired in the formation of sequestered areas of phytochrome in the cytosol, which can explain its reduced light-dependent degradation. In contrast, the mutation stabilizes nuclear speckles (NUS) that appear late under continuous far-red light, whereas the formation of early, transiently appearing NUS remained more or less unaltered.     

The adaptation of enzymes to temperature: catalytic characterization of glucosephosphate isomerase homologues isolated from Mytilus edulis and Isognomon alatus, bivalve molluscs inhabiting different thermal environments

Homologues of glucosephosphate isomerase (GPI, EC 5.3.1.9) were purified to homogeneity and kinetically characterized from Mytilus edulis and Isognomon alatus, two bivalve molluscs experiencing contrasting thermal environments. The enzyme isolated from I. alatus functions at warmer temperatures (25-35 C) than GPI from M. edulis, a species that inhabits colder marine littoral habitats (5-20 C). The former exhibits apparent first-order (with respect to substrate) catalytic rate constants (Vmax/KM) in vitro that become progressively greater than the mussel enzyme as the assay temperature is raised. Apparent zero-order catalytic rate constants (Vmax) are relatively less differentiated. Catalytic efficiency, defined as the rate at which a catalytic event occurs in either reaction direction for reference standard states (substrate concentrations), is greater for the enzyme from the tropical species (I. alatus) at all realistic combinations of temperature and substrate concentration except for the lowest temperatures and highest substrate concentrations, where the GPI from the boreal/temperate M. edulis is more efficient. This pattern of catalytic divergence appears to be due primarily to differentiation in Vmax/KM. These results and other published data are reviewed and shown to be inconsistent with claims that adaptation of enzymes to higher cell temperatures requires a loss in catalytic efficiency.      

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Effect of vasoconstriction on longitudinal distribution of pulmonary vascular pressure and volume

We used an improved version of the low-viscosity bolus method to evaluate longitudinal (arterial-to-venous) differences in the sensitivity of the dog lung lobe vasculature to selected vasoconstrictor stimuli, including hypoxia, and serotonin, histamine, and norepinephrine infusions. This method revealed a bimodal distribution of local vascular resistance vs. cumulative vascular volume under the zone 3 conditions studied. Our interpretation of the two modes of relatively high resistance is that they correspond to high resistance per unit volume segments of the arteries and veins upstream and downstream from the relatively low resistance per unit volume capillary bed. Thus an increase in the height of the upstream and downstream modes of the resistance distribution suggests constriction in small arteries and veins, respectively. Horizontal displacement of the modes along the cumulative volume axis suggests changes in the distribution of volume among the arteries, veins, and capillary bed. By use of these criteria, the results are consistent with the concept that each of the vasoconstrictor stimuli studied had a different longitudinal response pattern. Hypoxia constricted mainly small arteries, whereas serotonin constricted small and large arteries. Histamine constricted large and small veins, and norepinephrine constricted large and small veins and arteries.     

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.