Amino acid active transport and stimulation by substrates in the absence of a Na+ electrochemical potential gradient

Summary Uptake of -aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 g/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na⁺ and K⁺ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of 9.0. When all but 40mm Na⁺ in the medium was replaced by choline, nigericin resulted in K⁺ loss and Na⁺ gain and both cation distribution ratios approached 2.8–3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of –28 to –33 mV. This conclusion was supported by the observation that the addition of 5×10^–7 m valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na⁺ and K⁺, AIB was accumulated to a distribution ratio of 5.4 in the low-Na⁺ medium. Addition of 0.1mm oubain or 50 m vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na⁺+K⁺)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na⁺ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na⁺ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na⁺ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.     

Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus

HIV-1 Vpr is a protein with multiple functions. It has been suggested that such pleiotropic effects by a viral protein may be mediated by its association with viral and cellular proteins or through modulation of expression of specific cellular genes. To address this, we used cDNA microarray techniques to analyze the regulation of a panel of host cellular genes by HIV-1 Vpr using isogenic HIV-1 either with or without Vpr expression. Results indicate that Vpr downregulated the expression of genes involved in cell cycle/proliferation regulation, DNA repair, tumor antigens, and immune activation factors, and upregulated many ribosomal and structural proteins. These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule.     

Phospholipid hydroperoxide glutathione peroxidase induces a delay in G1 of the cell cycle

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an antioxidant enzyme that reduces cellular phospholipid hydroperoxides (PLOOHs) to alcohols. Cellular peroxide tone has been implicated in cell growth and differentiation. By reducing the PLOOH level in the cell membrane, PhGPx regulates the peroxide tone and thereby might be involved in cell growth. We hypothesized that overexpression of PhGPx in human breast cancer cells would decrease their growth rate. We stably transfected MCF-7 cells (Wt) with L-PhGPx and measured cell doubling time, plating efficiency, and cell cycle phase transit times. P-4 cells (8-fold increase in PhGPx activity) showed a 2-fold increase in doubling time; doubling time increased directly with PhGPx activity (r = 0.95). The higher the PhGPx activity, the lower the plating efficiency (r = -0.86). The profile of other antioxidant enzymes was unchanged. Overexpression of PhGPx lowered the steady-state level of PLOOH (by > 60%). Results from bromodeoxyuridine pulse-chase experiments and flow cytometry indicate that PhGPx induced a delay in MCF-7 proliferation that was primarily due to a slower progression from G1 to S. These results support the hypothesis that PhGPx plays a regulatory role in the progression of MCF-7 cells from G1 to S possibly by regulating the steady-state levels of PLOOH. These data suggest that PhGPx can lower the peroxide tone, which might change the cellular redox environment resulting in a delay in G1 transit. Thus, PhGPx could be an important factor in cell growth.     

A mechanism of drug resistance to tamoxifen in breast cancer

Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam.     

Kinetics of small molecule inhibitor binding to p38 kinase.

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.     

Features of the extracellular domain of the S-locus receptor kinase from Brassica

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5′ end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.     

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57⁺) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57⁺ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.Human immunodeficiency virus type I (HIV-1)-infected individuals who control viremia spontaneously without antiviral therapy have been termed HIV controllers (3, 18, 21, 48, 52). Unraveling the mechanisms associated with this phenotype should provide important insights regarding HIV pathogenesis and could contribute to vaccine development.Host and viral genetics, as well as host innate and adaptive immune responses, influence the rate of disease progression in HIV-1 infection (reviewed in reference 18). Several studies have reported the correlation between in vitro HIV replication capacity and level of plasma virus loads or disease progression in individuals with chronic infection (6, 13, 35, 45, 50, 55). Studies of HIV-1 elite controllers (EC), who control viremia to below the limit of detection in commercial assays, have revealed the presence of replication-competent viruses in these individuals (7), although these viruses appear to be less fit based on studies of envelope (35) and Gag-protease (45). This fitness defect in the chronic phase of infection is due at least in part to fitness-impairing mutations induced by cytotoxic-T-lymphocyte (CTL) responses restricted by “protective” HLA class I alleles (46).In contrast, little is known about viruses obtained from the acute/early phase of infection in persons who subsequently become HIV-1 controllers, largely due to the difficulty in enrolling such people during the acute/early phase of infection. The characterization of acute/early-phase viruses in individuals who subsequently achieve low set-point virus loads is of paramount importance to our understanding of the mechanisms of HIV-1 control.In the present study, we analyzed acute/early-phase plasma HIV RNA sequences from 18 untreated individuals who were diagnosed during the acute/early phase and subsequently became controllers (<2,000 RNA copies/ml). We compared these to sequences from a group of HIV-1 noncontrollers enrolled similarly during acute/early infection. We also generated chimeric viruses carrying patient-derived gag-protease sequences from acute/early-phase infection and compared the viral replication capacities of the chimeric viruses from controllers and from noncontrollers.We observed that the chimeric viruses derived from controllers have significantly reduced replicative capacities compared to those from noncontrollers. Moreover, we observed that at least 80% of these individuals who go on to become controllers featured transmission of attenuated drug-resistant viruses, transmission of HLA-B57-restricted CTL escape variants to HLA-mismatched recipients, selection of attenuated CTL escape variants in HLA-B57-positive (B57⁺) recipients, or combinations of these factors. Taken together, these results indicate that the initial viral dynamics have a major influence on the subsequent course of disease.