Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Quality prediction of cell substrate using gene expression profiling

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

General features in the stoichiometry and stability of ionophore A23187-cation complexes in homogeneous solution

Existing literature describing the stoichiometry and stability of complexes between A23187 and divalent cations in solution has been extended to include additional transition series cations, the heavy-metal cations Cd2+ and Pb2+, plus seven lanthanide series trivalent cations. Stability constants of 1:1 complexes between the ionophore and the divalent cations vary by 6.2 orders of magnitude between Cu2+ and Ba2+ which are the strongest and weakest complexes, respectively. Considering alkaline-earth and first-series transition cations together, the pattern of stability constants obeys the extended Irving-Williams series as is seen with many nonionophorous liganding agents. Cd2+ and Pb2+ are bound with an affinity similar to those of Mn2+ and Zn2+, whereas the lanthanides are bound with little selectivity and slightly higher stability. Titration of the ionophore in the 10(-5) M concentration range with di- and trivalent cations gives rise first to complexes of stoichiometry MA2 and subsequently to MA as the metal concentration is increased. The second stepwise stability constants for formation of the MA2 species exceeds the first constant by approximately 10-fold. With lanthanides, heavy metals, and transition-metal cations, OH-, at near physiological concentrations, competes significantly with free ionophore for binding to the 1:1 complexes. This competition is not apparent when Ca2+ or Mg2+ are the central cations. Possible implications of the 1:1 complex selectivity pattern, the ionophore-hydroxide competitive binding equilibria, and potential ternary complexes involving 1:1 ionophore:cation complexes and other anions present in biological systems are discussed with respect to the ionophore's transport selectivity and biological actions.     

Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskişehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform ınfrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), ınductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl₄ concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5–15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20–40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.     

Continuous monitoring of phytoplankton dynamics in Lake Balaton (Hungary) using on-line delayed fluorescence excitation spectroscopy

1. This study introduces delayed fluorescence (DF) excitation spectroscopy as an on‐line tool for in situ monitoring of the composition and biomass of various colour classes of phytoplankton when they are photosynthetically active (cyanobacteria, chlorophytes, chromophytes and cryptophytes). The DF data are validated by comparison with those from conventional methods (weekly microscopic counts and the measurement of chlorophyll concentration). 2. The composition of phytoplankton as assessed by DF agreed reasonably well with the results from microscopic counts, particularly when differences in chlorophyll‐specific DF integrals of the various colour classes were taken into account. 3. Integrals of DF spectra were converted into concentration of chlorophyll a using empirical factors derived from field data. The value of the conversion factor was nearly twice as high when the relative abundance of cyanobacteria was low (<15%) than when it was high. The converted DF‐chl time series agreed well with chlorophyll measurements particularly when blooms were developing. As the DF method is inherently free of the interference caused by pigment degradation products, the discrepancy between the two data sets increased during the collapse of blooms and when sediment resuspension was intense. 4. Fourier spectrum analysis of the time series of DF‐chl indicated that samples must be taken, at a minimum, every 2–3 days to capture the dynamics of phytoplankton. As a consequence, the dynamics of various algal blooms, including their timing, duration and net growth rate, could be estimated with greater confidence than by using conventional methods alone. 5. On‐line DF spectroscopy is an advanced technique for monitoring daily the biomass and composition of the photosynthetically active phytoplankton in aquatic environments, including turbid shallow lakes. At present, the detection limit is around 1 mg DF‐chl a m^?3 in terms of total biomass but confidence in estimates of phytoplankton composition declines sharply below about 5 mg chl a m^?3. 6. On‐line DF spectroscopy represents a promising approach for monitoring phytoplankton. It will be useful in water management where it can act as an early‐warning system of declines in water quality. In basic ecological research it can supplement manual methods. While default calibration spectra may be acceptable for routine monitoring, we suggest a careful individual calibration of the DF spectrometer for basic research. The statistical methods developed here help to assess the adequacy of various calibration sets.     

Restricted lateral mobility of plasma membrane CD4 impairs HIV-1 envelope glycoprotein mediated fusion

We investigated the effect of receptor mobility on HIV-1 envelope glycoprotein (Env)-triggered fusion using B16 mouse melanoma cells that are engineered to express CD4 and CXCR4 or CCR5. These engineered cells are resistant to fusion mediated CD4-dependent HIV-1 envelope glycoprotein. Receptor mobility was measured by fluorescence recovery after photobleaching (FRAP) using either fluorescently-labeled antibodies or transient expression of GFP-tagged receptors in the cells. No significant differences between B16 and NIH3T3 (fusion-permissive) cells were seen in lateral mobility of CCR5 or lipid probes. By contrast CD4 mobility in B16 cells was about seven-fold reduced compared to its mobility in fusion-permissive NIH3T3 cells. However, a CD4 mutant (RA5) that localizes to non-raft membrane microdomains exhibited a three-fold increased mobility in B16 cells as compared with WT-CD4. Interestingly, the B16 cells expressing the RA5 mutant (but not the wild type CD4) and coreceptors supported HIV-1 Env-mediated fusion. Our data demonstrate that the lateral mobility of CD4 is an important determinant of HIV-1 fusion/entry.