Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis.

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication.     

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X_L, and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.     

In vitro CNS tissue analogues formed by self-organisation of reaggregated post-natal brain tissue

In this paper, we report the characterization of 'Hi-Spot' cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that 'Hi-Spots' are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.     

Networks and dominance hierarchies: does interspecific aggression explain flower partitioning among stingless bees?

1. The distribution of consumers among resources (trophic interaction network) may be shaped by asymmetric competition. Dominance hierarchy models predict that asymmetric interference competition leads to a domination of high quality resources by hierarchically superior species. 2. In order to determine the competitive dominance hierarchy and its effect on flower partitioning in a local stingless bee community in Borneo, interspecific aggressions were tested among eight species in arena experiments. 3. All species tested were strongly mutually aggressive in the arena, and the observed interactions were often lethal for one or both opponents. Aggression significantly increased with body size differences between fighting pairs and was asymmetric: larger aggressors were superior over smaller species. Additional aggression tests involved dummies with surface extracts, and results suggest that species‐ and colony‐specific surface profiles are important in triggering the aggressive behaviour. 4. Sixteen stingless bee species were observed foraging on 41 species of flowering plants. The resulting bee–flower interaction network showed a high degree of generalisation (network‐level specialisation H₂’ = 0.11), corresponding to a random, opportunistic distribution of bee species among available flower species. 5. Aggressions on flowers were rare and only occurred at a low level. The dominance hierarchy obtained in the arena experiments did not correlate significantly with plant quality, estimated as the number of flowers per plant or as total bee visitation rate. 6. Our findings suggest that asymmetries in interference competition do not necessarily translate into actual resource partitioning in the context of complex interacting communities.     

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States

Background

Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.

Methodology/Principal Findings

To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.

Conclusions/Significance

Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.     

Identification of XMRV infection-associated microRNAs in four cell types in culture

Introduction

XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.

Methods

Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.

Results

MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.

Discussion

The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.     

The measurement of water deficits in broadleaf plants

Summary A review of past work and a comparison of the well-known disk technique with Stocker's whole leaf method for determining water deficit of some hardwood trees reveals confusion in existing terms and methods. The water relations of leaf disks cut from broadleaf trees cannot be assumed to be the same as whole leaves, since the excised disk usually requires more water per unit weight to saturate. Stocker's term wasser defizit (WD) and the whole leaf method, when modified to allow shorter equilibration schedules, remain the best way to express and measure water deficits in forest trees.