Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.     

Bone density parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women.

OBJECTIVE--To examine the relation between bone density and indices of calcium metabolism including parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women. DESIGN--A cross sectional study. SETTING AND SUBJECTS--138 women volunteers aged 45-65 with no known osteoporosis and unselected for disease status recruited for a dietary assessment study from the community using general practice registers. Volunteer rate was 20%. MAIN OUTCOME MEASURE--Bone mineral density measured with dual energy x ray absorptiometry. RESULTS--Bone density at the lumbar spine and neck and trochanteric regions of the femur was inversely related to serum intact parathyroid hormone concentrations and positively related to serum 25-hydroxyvitamin D concentrations. These associations were independent of possible confounding factors, including age, body mass index, cigarette smoking habit, menopausal status, and use of diuretics and postmenopausal hormone replacement therapy. These associations were apparent throughout the whole distribution of bone density and 25-hydroxyvitamin D and parathyroid hormone concentrations within the normal range, suggesting a physiological relation. CONCLUSIONS--The findings are consistent with the hypothesis that parathyroid hormone and 25-hydroxyvitamin D concentrations influence bone density in middle aged women. Findings from this study together with other work suggest that the role of vitamin D in osteoporosis should not be neglected. The associations with parathyroid hormone also indicate plausible biological mechanisms. The roughly 5-10% difference in bone density between top and bottom tertiles of serum 25-hydroxyvitamin D concentrations, though not large in magnitude, may have considerable public health implications in terms of prevention of osteoporosis and its sequelae, fractures.     

Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation.

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.     

Vertical distribution of zooplankton in the frontal zone of the Gulf Stream and Labrador Current

Zooplankton data collected during September 1995 in the NorthWest Atlantic at 4139'N, 4958'W (the location of the siteof the ‘Titanic’ wreck) were analysed. The regioninvestigated was characterized by a very sharp frontal zonebetween the Gulf Stream and the main stream of the LabradorCurrent. The total plankton biomass in the water column wasvery high. The macroplankton biomass values below the 600 mlayer were significantly higher as compared with the similarvalues measured before in other productive boreal regions ofthe Atlantic and Pacific oceans. A lot of dead mesoplanktonanimals occurred in the deep layers. The reason was that thecold-water mesoplankton advected by the Labrador Current diedoff intensively within the deep layers of the frontal zone andwere used as a food resource by the macroplankton carnivoresand scavengers that were very abundant there.     

A finite element based method to determine the properties of planar soft tissue.

A finite element based method to determine the incremental elastic material properties of planar membranes was developed and evaluated. The method is applicable to tissues that exhibit inhomogeneity, geometric and material nonlinearity, and anisotropy. Markers are placed on the tissue to form a four-node quadrilateral element. The specimen is loaded to an initial reference state, then three incremental loading sets are applied and the nodal displacements recorded. One of these loadings must include shear. These data are used to solve an over-determined system of equations for the tangent stiffness matrix. The method was first verified using analytical data. Next, data obtained from a latex rubber sheet were used to evaluate experimental procedures. Finally, experiments conducted on preconditioned rat skin revealed nonlinear orthotropic behavior. The vector norm comparing the applied and calculated nodal force vectors was used to evaluate the accuracy of the solutions.     

An implantable transducer for measuring tension in an anterior cruciate ligament graft.

The goal of this study was to develop a new implantable transducer for measuring anterior cruciate ligament (ACL) graft tension postoperatively in patients who have undergone ACL reconstructive surgery. A unique approach was taken of integrating the transducer into a femoral fixation device. To devise a practical in vivo calibration protocol for the fixation device transducer (FDT), several hypotheses were investigated: (1) The use of a cable versus the actual graft as the means for applying load to the FDT during calibration has no significant effect on the accuracy of the FDT tension measurements; (2) the number of flexion angles at which the device is calibrated has no significant effect on the accuracy of the FDT measurements; (3) the friction between the graft and femoral tunnel has no significant effect on measurement accuracy. To provide data for testing these hypotheses, the FDT was first calibrated with both a cable and a graft over the full range of flexion. Then graft tension was measured simultaneously with both the FDT on the femoral side and load cells, which were connected to the graft on the tibial side, as five cadaver knees were loaded externally. Measurements were made with both standard and overdrilled tunnels. The error in the FDT tension measurements was the difference between the graft tension measured by the FDT and the load cells. Results of the statistical analyses showed that neither the means of applying the calibration load, the number of flexion angles used for calibration, nor the tunnel size had a significant effect on the accuracy of the FDT. Thus a cable may be used instead of the graft to transmit loads to the FDT during calibration, thus simplifying the procedure. Accurate calibration requires data from just three flexion angles of 0, 45, and 90 deg and a curve fit to obtain a calibration curve over a continuous range of flexion within the limits of this angle group. Since friction did not adversely affect the measurement accuracy of the FDT, the femoral tunnel can be drilled to match the diameter of the graft and does not need to be overdrilled. Following these procedures, the error in measuring graft tension with the FDT averages less than 10 percent relative to a full-scale load of 257 N.