Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering

A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors.     

WAX AND WANE OF MICROCYSTIS (CYANOPHYCEAE) AND MICROCYSTINS IN LAKE SEDIMENTS: A CASE STUDY IN QUITZDORF RESERVOIR (GERMANY)1

Benthic stages of the annual life cycle of the meroplanktonic cyanobacterium Microcystis spp. in relation to microcystin (MCYST) dynamics in sediments of a shallow lake (Quitzdorf Reservoir, Germany) were investigated. Based on changes in the absolute abundance of benthic Microcystis, the annual life cycle was subdivided into four phenological stages: reinvasion, pelagic growth, sedimentation, and overwintering. Habitat‐coupling processes, such as reinvasion of the pelagic zone in spring as well as autumnal sedimentation, were particularly triggered by changes in water temperature. During reinvasion substantial losses of Microcystis were detected. Only a minor part of benthic Microcystis (about 3%) formed the inoculum for pelagic growth. Between 65% and 85% of the benthic Microcystis stock disappeared during the reinvasion phase. Because these colonies were neither detected within the sediments nor in the pelagic inoculum, it was concluded that they were subjected to decay. The occurrence of extracellular MCYSTs in the pelagic zone during this period, which cannot solely originate from the pelagic Microcystis population, supports this conclusion. Dynamics of benthic Microcystis and MCYSTs were characterized by almost identical successions with a decrease during reinvasion, an increase during sedimentation, and remarkable invariability throughout pelagic growth and overwintering. It can be deduced that MCYSTs are preserved within benthic resting stages of Microcystis because they could play a role during overwintering or reinvasion.     

Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.     

Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.     

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.     

Characterization of the blastogenic and cytotoxic responses of normal mice to ecotropic C-type viral gp71.

Spleen cells from normal (B6C3)F1 mice demonstrated natural cytotoxic reactivity mediated by a "null" cell population which, in part, had immunologic specificity for the major envelope glycoprotein (gp71) of the endogenous ecotropic murine leukemia virus. In contrast, the cytotoxic reactivity reflected in spleen cells of NIH Swiss nude mice apparently had no immunologic specificity. A significant level of blastogenic response could be generated in vitro by using normal (B6C3)F1 and AKR spleen cells in the presence of gp71. This reactivity was highly type specific. Moreover, using normal spleen cells from (B6C3)F1, both secondary blastogenic responses and cytotoxic T cell responses could be induced in vitro with purified soluble viral gp71. These findings extend further our previous studies on the existence of a natural immune response in normal mice to their endogenous MuLV by providing in vitro evidence for the expression of cell-mediated response in addition to the humoral response and that at least two effector cell types are operable in the cell-mediated phase.     

Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses.

Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.