Salvia (Lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of Salvia and tribe Mentheae

Salvia, with over 900 species from both the Old and New World, is the largest genus in the Lamiaceae. Unlike most members of the subfamily Nepetoideae to which it belongs, only two stamens are expressed in Salvia. Although the structure of these stamens is remarkably variable across the genus, generally each stamen has an elongate connective and divergent anther thecae, which form a lever mechanism important in pollination. In a preliminary investigation of infrageneric relationships within Salvia, the monophyly of the genus and its relationship to other members of the tribe Mentheae were investigated using the chloroplast DNA regions rbcL and trnL-F. Significant conclusions drawn from the data include: Salvia is not monophyletic, Rosmarinus and Perovskia together are sister to an Old World clade of Salvia, the section Audibertia is sister to subgenus Calosphace or the monotypic Asian genus Dorystaechas, and the New World members of section Heterosphace are sister to section Salviastrum. Owing to the non-monophyly of Salvia, relationships at the next clearly monophyletic level, tribe Mentheae, were investigated.     

Pulsing Cl- channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation

Seed coat cells in the developing seeds of grain legumes release nutrients to the developing embryo. This occurs into an apoplastic space that separates the maternal (seed coat) and filial (embryo) generations. Protoplasts of seed coat cells from coats of Phaseolus vulgaris L. seeds were isolated and whole-cell current across their plasma membranes was characterized using the patch-clamp technique. A pulsing inward current that displayed a spontaneous activation and voltage-dependent inactivation was observed. The frequency and magnitude of the current pulses were positively dependent on cytoplasmic Cl(-) concentrations and independent of external cations. The pulse current was inhibited by DIDS and La(3+), but not by Gd(3+). Single channel events (conductance=18 pS) could be identified with the inactivating phase of the pulses. Together, these findings are consistent with the current being carried by a burst of Cl(-) efflux through Cl(-)-permeable channels that activate almost simultaneously. Neomycin caused a reversible inhibition of the pulsed current, suggesting that its activation is likely to be modulated by an IP(3)-dependent intracellular Ca(2+) release. The pharmacological profiles of Cl(-) efflux from excised seed coats were comparable with those of the Cl(-) channels in the whole cell configuration, suggesting that the Cl(-) channels may underpin Cl(-) efflux from the seed coats. Efflux of Cl(-) from the seed coats was also stimulated by hypo-osmotic treatment as was the frequency and magnitude of Cl(-) channel in whole-cell patch clamp experiments. This implies that the Cl(-) channels responsible for the pulsed Cl(-) currents are likely to be a component of the turgor-regulatory mechanism in developing bean seeds.     

CD4+CD25+ cells controlling a pathogenic CD4 response inhibit cytokine differentiation, CXCR-3 expression, and tissue invasion

It is well established that CD4(+)CD25(+) regulatory T cells (Tregs) inhibit autoimmune pathology. However, precisely how the behavior of disease-inducing T cells is altered by Tregs remains unclear. In this study we use a TCR transgenic model of diabetes to pinpoint how pathogenic CD4 T cells are modified by Tregs in vivo. We show that although Tregs only modestly inhibit CD4 cell expansion, they potently suppress tissue infiltration. This is associated with a failure of CD4 cells to differentiate into effector cells and to up-regulate the IFN-gamma-dependent chemokine receptor CXCR-3, which confers the ability to respond to pancreatic islet-derived CXCL10. Our data support a model in which Tregs permit T cell activation, yet prohibit T cell differentiation and migration into Ag-bearing tissues.     

Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections

Control of hepatitis C virus (HCV) infection could be influenced by the timing and magnitude of CD4+ T cell responses against individual epitopes. We characterized CD4+ T cells targeting seven Pan troglodytes (Patr) class II-restricted epitopes during primary and secondary HCV infections of a chimpanzee. All Patr-DR-restricted HCV epitopes bound multiple human HLA-DR molecules, indicating the potential for overlap in epitopes targeted by both species. Some human MHC class II molecules efficiently stimulated IL-2 production by chimpanzee virus-specific T cell clones. Moreover, one conserved epitope designated NS3(1248) (GYKVLVLNPSV) overlapped a helper epitope that is presented by multiple HLA-DR molecules in humans who spontaneously resolved HCV infection. Resolution of primary infection in the chimpanzee was associated with an initial wave of CD4+ T cells targeting a limited set of dominant epitopes including NS3(1248.) A second wave of low-frequency CD4+ T cells targeting other subdominant epitopes appeared in blood several weeks later after virus replication was mostly contained. During a second infection 7 years later, CD4+ T cells against all epitopes appeared in blood sooner and at higher frequencies but the pattern of dominance was conserved. In summary, primary HCV infection in this individual was characterized by T cell populations targeting two groups of MHC class II-restricted epitopes that differed in frequency and kinetics of appearance in blood. The hierarchial nature of the CD4+ T cell response, if broadly applicable to other HCV-infected chimpanzees and humans, could be a factor governing the outcome of HCV infection.     

Overexpression of torsinA in PC12 cells protects against toxicity

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.     

Migration of antigen-specific T cells away from CXCR4-binding human immunodeficiency virus type 1 gp120

Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.     

Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load = 142,000 copies/ml; median CD4 count = 71/ micro l) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/10(6) peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P = 0.032) and magnitude (P = 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferon-positive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.     

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.     

Modeling the temperature kinetics of aerobic solid-state biodegradation

This study evaluated three models of microbial temperature kinetics using CO2 respiration data from aerobic solid-state biodegradation experiments. The models included those of Andrews and Kambhu/Haug, Ratkowsky et al., and the Cardinal Temperature Model with Inflection (CTMI) of Rosso et al. A parameter estimation routine implemented the Complex-Box search method for each model on 48 data sets collected during the composting of synthetic food waste or sewage-sludge (biosolids) mixed with maple wood chips at different oxygen concentrations and extents of decomposition. Each of the three nonlinear temperature kinetic functions proved capable of modeling a wide range of experimental data sets. However, the models differed widely in the consistency of their parameters. Parameters in the CTMI model were more stable over the course of the degradation process, and that variability which did arise was directly related to changes in the microbial process. Additional benefits of the CTMI model include the ease of parameter determinations, which can be approximated directly from laboratory experiments or full-scale system analysis, and the direct value of its parameters in engineering design and process control under a wide range of biodegradation conditions.