Molecular systematics of Batrachoseps (Caudata, Plethodontidae) in southern California and Baja California: mitochondrial-nuclear DNA discordance and the evolutionary history of B. major

Inferences about species boundaries and evolutionary history are often complicated by discordance between datasets. In recent times, considerable effort has been devoted to understanding the causes of discordance between the patterns of genetic variation and structure shown by different unlinked molecular markers. The genus Batrachoseps (Caudata, Plethodontidae), the most diverse group of salamanders in western North America, is characterized by limited morphological variation and discordance between molecular datasets, making it a challenging group for taxonomists but also a good model to test newly developed analytical methods to sort out possible sources of discordance. In this study, we present a comprehensive assessment of the evolutionary history of B. major, one of the most widespread species in the genus, based on extensive sampling and phylogenetic and coalescent analyses of data from mitochondrial and nuclear markers. We found non-monophyly of mtDNA in B. major, with two lineages (northern and southern) that are more closely related to other species in the genus than to each other, but this division was not apparent in nuclear DNA. Despite non-monophyly in gene trees, species tree analyses recovered a sister group relationship between the two lineages of B. major, and coalescent simulations suggested that there is no need to invoke gene flow to account for the discordance across gene trees. The possibility that these two lineages represent sister, cryptic taxa, is discussed in the context of Bayesian methods of species/lineage delineation. Contrary to prior expectations, B. major has experienced extensive diversification on the Baja California Peninsula, where four endemic lineages have persisted for at least 4 million years.     

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.     

Induction of astrocytic cyclooxygenase-2 in epileptic patients with hippocampal sclerosis

Induction of cyclooxygenase-2 (COX-2) has been described in a wide range of neurological diseases including animal models of epilepsy. The present study was undertaken to assess COX-2 expression in hippocampal biopsies from patients with therapy-refractive temporal lobe epilepsy (TLE). For this purpose, hippocampal CA1 subfield was dissected from epileptic patients with (n=5) or without (n=2) hippocampal sclerosis (HS). COX-2 expression was investigated using immunohistochemistry and semi-quantitative RT-PCR. COX-2 immunoreactivity in TLE patient material in the absence of HS was restricted to a few neurons of the hippocampus. In the presence of HS, on the other hand, a significant induction of astrocytic COX-2 immunoreactivity associated with a concomitant increase in the steady-state level of COX-2 mRNA was observed in the CA1 subfield. These findings suggest that induction of astrocytic COX-2 is implicated in the pathogenesis of HS in TLE and is consistent with the previous findings of increased concentrations of prostaglandins in the cerebrospinal fluid of these patients.     

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.     

Dynamic regulation of the inducible nitric-oxide synthase by NO: comparison with the endothelial isoform

We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (Slama-Schwok, A., Négrerie, M., Berka, V., Lambry, J.-C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825).     

Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity

Adiponectin is an adipocyte-specific secretory protein that circulates in serum as a hexamer of relatively low molecular weight (LMW) and a larger multimeric structure of high molecular weight (HMW). Serum levels of the protein correlate with systemic insulin sensitivity. The full-length protein affects hepatic gluconeogenesis through improved insulin sensitivity, and a proteolytic fragment of adiponectin stimulates beta oxidation in muscle. Here, we show that the ratio, and not the absolute amounts, between these two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity. We define a new index, S(A), that can be calculated as the ratio of HMW/(HMW + LMW). db/db mice, despite similar total adiponectin levels, display decreased S(A) values compared with wild type littermates, as do type II diabetic patients compared with insulin-sensitive individuals. Furthermore, S(A) improves with peroxisome proliferator-activated receptor-gamma agonist treatment (thiazolidinedione; TZD) in mice and humans. We demonstrate that changes in S(A) in a number of type 2 diabetic cohorts serve as a quantitative indicator of improvements in insulin sensitivity obtained during TZD treatment, whereas changes in total serum adiponectin levels do not correlate well at the individual level. Acute alterations in S(A) (DeltaS(A)) are strongly correlated with improvements in hepatic insulin sensitivity and are less relevant as an indicator of improved muscle insulin sensitivity in response to TZD treatment, further underscoring the conclusions from previous clamp studies that suggested that the liver is the primary site of action for the full-length protein. These observations suggest that the HMW adiponectin complex is the active form of this protein, which we directly demonstrate in vivo by its ability to depress serum glucose levels in a dose-dependent manner.     

GeneBlocs are powerful tools to study and delineate signal transduction processes that regulate cell growth and transformation

The study of signal transduction processes using antisense oligonucleotides is often complicated by low intracellular stability of the antisense reagents or by nonspecific effects that cause toxicity. Here, we introduce a new class of antisense molecules, so-called GeneBlocs, which are characterized by improved stability, high target RNA specificity, and low toxicity. GeneBlocs allow for efficient downregulation of mRNA expression at nanomolar concentrations, and they do not interfere with cell proliferation. We demonstrate these beneficial properties using a positive readout system. GeneBloc-mediated inhibition of tumor suppressor PTEN (phosphatase and tension homologue detected on chromosome 10) expression leads to hyperactivation of the phosphatidylinositol (PI) 3-kinase pathway, thereby mimicking the loss of PTEN function and its early consequences observed in mammalian cancer cells. Specifically, cells treated with PTEN GeneBlocs show functional activation of Akt, a downstream effector of PI 3-kinase signaling, and exhibit enhanced proliferation when seeded on a basement membrane matrix. In addition, GeneBlocs targeting the catalytic subunit of PI 3-kinase, p110, specifically inhibit signal transduction of endogenous or recombinant PI 3-kinase. This demonstrates that GeneBlocs are powerful tools to analyze and to modulate signal transduction processes and, therefore, represent alternative reagents for the validation of gene function.