Loss of myosin VI reduces secretion and the size of the Golgi in fibroblasts from Snell's waltzer mice

Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments showed that fully functional myosin VI was able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells.     

S-Nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.     

Membrane Transport Generated by the Osmotic and Hydrostatic Pressure. Correlation Relation for Parameters L p, σ, and ω

Standard approach to membrane transport generated by osmotic andhydrostatic pressures, developed by Kedem and Katchalsky, is based onprinciples of thermodynamics of irreversible processes. In this paper wepropose an alternative technique. We derive transport equations from fewfairly natural assumptions and a mechanistic interpretation of the flows.In particular we postulate that a sieve-type membrane permeability isdetermined by the pore sizes and these are random within certain range.Assuming that an individual pore is either permeable or impermeable tosolute molecules, the membrane reflection coefficient depends on the ratioof permeable and impermeable pores. Considering flows through permeableand impermeable pores separately, we derive equations for the total volumeflux, solute flux and the solvent flux across the membrane. Comparing themechanistic equations to the Kedem-Katchalsky equations we find the formereasier to interpret physically. Based on the mechanistic equations we alsoderive a correlation relation for the membrane transport parameters L _p,, and . This relation eliminates the need for experimentaldetermination of all three phenomenological parameters, which in somecases met with considerable difficulties.     

Interaction of Mouse Polycomb-Group (Pc-G) Proteins Enx1 and Enx2 with Eed: Indication for Separate Pc-G Complexes

The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development. Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood. To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method. Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.     

Efficient,Large Area,and Thick Junction Polymer Solar Cells with Balanced Mobilities and Low Defect Densities

The high power conversion efficiencies (PCEs) of laboratory‐scale polymer‐based organic solar cells are yet to translate to large area modules because of a number of factors including the relatively large sheet resistance of available transparent conducting electrodes (TCEs), and the high defect densities associated with thin organic semiconductor junctions. The TCE problem limits device architectures to narrow connected strips (<1 cm) causing serious fabrication difficulties and extra costs. Thin junctions are required because of poor charge transport (imbalanced mobilities) in the constituent organic semiconductors. These issues are addressed using a combination of approaches to create thick junctions conformally coated on low sheet resistance metal grid TCEs. An essential feature of these thick junctions is balanced carrier mobilities, which affords high fill factors and efficient carrier extraction. Conformal coating is achieved by promoting enhanced intermolecular interactions in the coating solution using a high molecular weight polymeric semiconductor and appropriate solvent system. This combination of balanced mobilities, conformal coating and metallic grid TCEs is a simple and generic approach to the fabrication of defect‐free large area organic solar cells (OSCs). The approach is demonstrated with 25 cm² monolithic devices possessing aperture‐corrected power conversion efficiencies of 5% and fill factors exceeding 0.5.     

Association of CMV-Specific T Cell-Mediated Immunity with CMV DNAemia and Development of CMV Disease in HIV-1–Infected Individuals

Background

Among HIV-1–infected individuals, cytomegalovirus (CMV) reactivation and disease occur in the setting of advanced immunosuppression. The value of a standardized assessment of CMV-specific T-cell mediated immunity by the CMV QuantiFERON assay (CMV-QFT) has not yet been thoroughly investigated in HIV-1–infected subjects.

Methods

Prospective, longitudinal study in 153 HIV-1–infected subjects with a CD4⁺ T cell count < 350/μL who simultaneously underwent CMV-QFT, CMV serology testing and CMV-DNA quantification. Factors associated with CMV-QFT were evaluated. Clinical screening for CMV manifestations was then performed every 3 months.

Results

Among the 141 CMV IgG-seropositive individuals the CMV-QFT assay yielded reactive results in 84% (118/141), negative results in 15% (21/141) and indeterminate (negative mitogen IFN-gamma response) results in 1% (2/141) of subjects. The mean actual CD4⁺ T cell count was significantly higher in CMV-QFT reactive subjects, when compared to CMV-QFT non-reactive individuals (183 ± 102 vs. 126 ± 104 cells/μL, P = 0.015). A significantly lower proportion of CMV-QFT reactive vs. non-reactive patients displayed CMV DNAemia > 100 copies/mL (23% (27/118) vs. 48% (11/23), P = 0.02). Furthermore, a statistically significant inverse association between mitogen IFN-gamma response and CMV-DNAemia > 1000 copies/mL was observed (P < 0.001). During the observational period, 5 CMV end-organ manifestations were observed. In three of the CMV cases the CMV-QFT yielded indeterminate results.

Conclusions

While CMV-QFT reactivity indicates CMV-specific immunity, indeterminate results due to negative mitogen IFN-gamma response might reflect HIV-1-induced immunodeficiency. Thus, dependency upon CD4⁺ T cell count should be considered when interpreting CMV-QFT results.