Control of Ras cycling by Ca2+

Ras GTPases are binary switches, cycling between an inactive GDP-bound form and an active GTP-bound form at the membrane. They transduce signals into the cytoplasm via effector pathways that regulate cell growth, differentiation and apoptosis. Ras activation is enhanced by guanine nucleotide exchange factors (GEFs); deactivation is accelerated by GTPase-activating proteins (GAPs). Recently, new roles for Ca(2+) and diacylglycerol (DAG) in the control of Ras cycling have emerged with the discovery of a series of novel GEFs and GAPs. These regulators of Ras cycling are likely to play a key role in the information processing of Ca(2+) and DAG signals.     

A new entry route for HIV

Identification of HIV-1 variants capable of entering T cells via the CD8 receptor suggests a new mode of viral pathogenesis. But are these variants rare, aberrant viruses or a real problem?     

Normobaric hyperoxic stress in budgerigars: non-enzymic antioxidants

The effects of oxygen exposure on pulmonary and blood non-enzymic antioxidant concentrations was evaluated in budgerigars (Melopsittacus undulatus). Budgerigars were exposed to acute (3 h), repeated acute (3 exposures each of 3 h) or chronic (72 h) normobaric hyperoxic environments and the pulmonary and plasma concentrations of selected non-enzymic antioxidants, namely glutathione, uric acid, alpha- and gamma-tocopherol and carotenoids were assayed. With increasing duration of oxygen exposure, the ratio of oxidised to reduced glutathione was significantly increased, while the concentrations of uric acid, alpha- and gamma-tocopherol and carotenoids were significantly reduced, especially following chronic oxygen exposure. Following acute and repeated acute exposure, alteration in glutathione concentrations and reduction in alpha-tocopherol concentrations indicated oxygen stress. Following chronic exposure, depletion of non-enzymic antioxidants indicated exhaustion of these protective mechanisms and progression from oxygen stress to oxygen toxicity.     

Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis

Inositol 1,3,4,5-tetrakisphosphate (IP(4)) has been linked to a potential role in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP(4) remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP(4) receptor, GAP1(IP4BP), affects [Ca(2+)](i) following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1(IP4BP) appear to have no effect on Ca(2+) mobilization or Ca(2+) entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1(IP4BP) production on [Ca(2+)](i). In HeLa cells in which the endogenous level of GAP1(IP4BP) has been suppressed by approximately 95%, we failed to observe any effect on Ca(2+) mobilization or Ca(2+) entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1(IP4BP) in intact HeLa cells, we have been unable to observe any detectable effect of GAP1(IP4BP) on [Ca(2+)](i).     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.