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A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies seroconversion due to true HIV infections. A study examined the reactivity of plasma samples from >3000 infections with diverse HIV subtypes worldwide and it is felt that HIV-SELECTEST can be an important differential diagnostic tool for HIV vaccine trials, population screening all over the world and blood banks.

Brown fat cells were tested for ATP responses to show that cytosolic calcium levels were increased by ATP through release from intracellular stores and through influx. Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes. ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells. The myriad responses to extracellular ATP indicate that P2 receptor-mediated signaling is important in brown adipocyte physiology.

The exposure of brown adipocytes to extracellular ATP leads to exocytosis of intracellular membranes and increases the purinergic receptor mediated membrane trafficking. There is also a purinergic stimulated increase in the cell surface due to adipocyte vesicle-mediated secretion. The ATP exposure also increases the total cell membrane capacitance and the cytosolic calcium (Ca) levels. Adrenergic activation mobilizes calcium without influencing the amount of membrane trafficking. This indicates that the increase in Ca content is not solely responsible for the ATP-induced cellular responses.

The adrenergic depolarization of conductance in brown adipocytes is due to an increase in the intracellular calcium which stimulates a chloride-sensitive conductance. This conductance is different from the volume-sensitive chloride conductance of the cell. Cytoplasmic factors and intracellular calcium affect this chloride conductance. Cells cultured for short periods show depolarization of conductance more than those cultured for longer periods.

Fluorescence imaging studies of brown tissues of rats and hamsters reveal that alpha-adrenergic stimulation of the neurotransmitter, norepinephrine (NE), results in increased intracellular pH and subsequent activation of uncoupling protein through reduced purine inhibition leading to oxidation of fatty acids and heat dissipation. Removal of NE results in the reverse process due to beta-adrenoreceptor stimulation and downregulation of thermogenesis due to slower acidification. The sympathetic activity of NE provides favorable conditions for thermogenesis regulation.

The effect of adrenergic stimulation on cultured brown fat cells was investigated. The study protocol involved the measurement during adrenergic stimulation of the intracellular calcium (Ca2+) concentration using fura-2 ratio imaging of individual cells. The results showed that brown fat cells respond to adrenergic stimulation by a rapid increase in intracellular Ca2+ which is maintained for the duration of the stimulus. The maintenance of the rise in calcium, but not theninitial rapid rise, depends on extracellular Ca2+ and on the plasma membrane electrochemical gradient.

The most common channel observed in primary human peripheral T lymphocytes was voltage-gated and inactivating. This potassium channel comprise a majority of the outward current in lymphocytes. Another channel less frequently observed was transient and has a unitary conductance of nearly 10 pS. This channel is voltage-independent, lacks inactivation and has distinct kinetic parameters.

Abstract Background XMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors. Methods To study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR. Results Infection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP. Conclusion XMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.

Abstract Background The HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients. Methodology Blood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database. Results Phylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies. Conclusions Our results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.

Abstract Background With the advent of entry inhibitors, monitoring of viral tropism in the clinical setting is important. Conventional methods are cell-based and lengthy, therefore V3 sequence based prediction algorithms are becoming increasingly attractive as monitoring tools. Here we report a comparative analysis of viral tropism of strains circulating in Cameroon where diverse and emerging variant strains are prevalent. Methods Viruses were isolated from 17 HIV positive individuals from three cities in Cameroon. Ghost cell lines expressing either CCR5 or CXCR4 with CD4 or CD4 alone (NIH AIDS Reagent Program) were used to determine co-receptor usage. HIV replication was determined by measuring p24 antigen levels. Plasma viral load (VL) was determined using the Versant bDNA assay. Nucleotide sequencing was performed on the V3 region and sequences were edited, aligned and translated into amino acids as described in the algorithm. Bio-informatics tools based on the 11/25 and charge rule were used to predict co-receptor usage. Results The majority of patient isolates in our study were CRF02_AG or CRF02_AG containing recombinants. Tropism of these complex viruses based on the cell culture assay was determined to be R5 in 15/17 (88.2%) patients. However, two patient isolates were dual tropic R5X4 and had drug-specific mutations. Of these two patients, one was on antiretroviral treatment with a VL of 20,899 copies/ml and the other was drug-naïve with 141,198 copies/ml. Genotype based prediction was overall in good agreement with phenotype for R5 viruses, where 93% (14/15) of results were comparable, dual tropic viruses being reported as X4 viruses by prediction. Conclusion Our results indicate that most HIV strains in Cameroon were R5 tropic and some harbored drug-resistant mutations. V3 sequence based prediction compared well with cell based assays for R5 strains and may be useful even in settings where highly diverse strains are prevalent.

We used patch clamp electrophysiology and concurrent imaging with the [Ca.sup.2+]-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) during mouse egg fertilization. We found an ~2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [[[Ca.sup.2+]].sub.i] at sperm fusion. This was followed ~1 min later by a rise in [[[Ca.sup.2+]].sub.i] that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-[micro]M peak [[[Ca.sup.2+]].sub.i] favored capacitance decrease, while higher [[[Ca.sup.2+]].sub.i] favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [[[Ca.sup.2+]].sub.i]. The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [[[Ca.sup.2+]].sub.i] dependence. Raising [[[Ca.sup.2+]].sub.i] by the intracellular introduction of [IP.sub.3] initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [[[Ca.sup.2+]].sub.i] was increased modestly by activation of an endogenous [Ca.sup.2+] current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [[Ca.sup.2+]]i and that sperm and the active component of sperm factor may be doing more than initiating the [IP.sup.3]-mediated release of intracellular [Ca.sup.2+]. [C] 2001 Academic Press Key Words: patch clamp; exocytosis; endocytosis; fura-2; calcium imaging; calcium channel; ionomycin.

Wilson, Sean M., Sherwin C. Lee, Sheryl Shook, and Pamela A. Pappone. ATP and [Beta]-adrenergic stimulation enhance voltage-gated K current inactivation in brown adipocytes. Am J Physiol Cell Physiol 279: C1847-C1858, 2000.--Sympathetic activation of brown fat thermogenesis stimulates adrenergic and purinergic receptors. We examined the effects of extracellular ATP and [Beta]-adrenergic agonists on voltage-activated K currents (IKv) in voltage-clamped rat brown adipocytes. ATP or the [Beta]-adrenergic agonist isoproterenol increased the development of IKv inactivation during depolarizing voltage steps in perforated patch-clamped cells. The effects on inactivation developed slowly in the presence of agonist and continued to increase for long times following agonist washout. 8-bromo-cAMP or forskolin had similar effects on IKv inactivation. Development of IKv inactivation during depolarizations was consistently enhanced by ATP or [Beta]-adrenergic stimulation in perforated-patch voltage-clamped cells but was not altered by these agents in whole cell recordings, suggesting that cytosolic factors are necessary for inactivation modulation. In either recording configuration, ATP or isoproterenol shifted the activation voltage dependence of IKv to more negative potentials, indicating the activation effect is mediated by a different pathway. Since both P2 purinergic and [Beta]-adrenergic signaling pathways generate fatty acids, we tested whether fatty acids could reproduce these modulations of IKv. Linoleic or arachidonic acid applied in whole cell recordings had effects similar to those of ATP or isoproterenol in perforated-patch experiments. These results are consistent with the possibility that [Beta]-adrenergic and P2 receptor stimulation modulate IKv through generation of fatty acids. adenosine triphosphate/pharmacology; brown fat/physiology; electrophysiology; potassium channels/physiology; receptors; purinergic/agonists/metabolism

Converting oil palm empty fruit bunches into biochar is an alternative waste management method and has strong potential to improve N fertiliser use efficiency in agriculture. The aim of this study was to determine the effectiveness of oil palm empty fruit bunch biochar (EFBB) in improving recovery of15N-labelled nitrogen fertiliser by maize (Zea maysL.) and leaching of mineral N and K. An experiment was conducted in a mini-lysimeter system with randomised complete block design layout and six replications under controlled environment in a rain shelter. Each mini-lysimeter was filled with 20 kg of sandy loam soil before adding EFBB (0, 5, 10 and 20 Mg ha−1). The N source used was (15NH4)2SO4at 80 kg N ha−1(2 at%15N excess). Maize was irrigated to induce leaching every 4 days. Maize plant and soils were sampled 58 days after sowing (tasselling stage). Application of EFBB significantly reduced cumulative leachate volume and mineral N leaching. Soils applied with EFBB significantly improved15N fertiliser recovery in maize and dry matter weight. This study shows that EFBB has the potential to be applied on highly weathered acidic soil as an amendment to improve fertiliser efficiency and crop growth. [ABSTRACT FROM AUTHOR]