Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.     

Overview of Voltage-Dependent Calcium Channels

Voltage-dependent calcium channels couple electrical signals to cellular responses in excitable cells. Calcium channels are crucial for excitation–secretion coupling in neurons and endocrine cells, and excitation–contraction coupling in muscle. Several pharmacologically and kinetically distinct calcium channel types have been identified at the electrophysiological and molecular levels. This review summarizes the basic properties of voltage-dependent calcium channels, including mechanisms of ion permeation and block, gating kinetics, and modulation by G proteins and second messengers.     

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.     

Morganella morganii urease: purification, characterization, and isolation of gene sequences

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.     

Functional and phenotypic changes associated with the in vitro development of human monocytes into activated macrophages

Freshly isolated human peripheral blood monocytes had minimal cytotoxic effect in vitro on the schistosomula of Schistosoma mansoni. However, stimulation of the cells with either interferon gamma (IFN) or specific anti-parasite antiserum caused an increase in cytotoxicity. Additionally, the normal development of monocytes into macrophages over 7 days was associated with a sharp increase in cytotoxicity. The non-cytotoxic monocytes were compared with activated macrophages to assess whether cytotoxicity was associated with changes in immunophenotype. As monocytes developed into macrophages there were marked increases in transferrin receptors (HB21), macrophage cellular integrin (3.9), and Fc receptors (KB61). A further three markers showed increased expression in 7-day-old macrophages stimulated by IFN, namely a high affinity Fc gamma receptor (10.1), MHC Class II (1B5) and tumour necrosis factor (TNF).     

Vertical distribution of marine fungi onRhizophora apiculata at Morib mangrove, Selangor, Malaysia

Studies on the vertical distribution of marine fungi in aRhizophora apiculata mangrove stand in Morib, Selangor were carried out in June 1993 and June to November 1997. Prop roots, subterranean roots and overhanging branches ofR. apiculata were collected from three intertidal levels namely upper (high water mark), middle and lower. Fifty-three species were recorded including 39 ascomycetes, 13 deuteromycetes and one basidiomycete. The most common fungi wereHalocyphina villosa (frequency occurrence 21%),Kallichroma tethys (20%),Lulworthia grandispora (18%),Leptosphaeria australiensis (16%),Julella avicenniae (15%) andMassarina ramunculicola (13%). The fungi were found to be vertically zoned, some were limited to the upper level such asPyrenographa xylographoides, Julella avicenniae andAigialus grandis or lower level such asTrichocladium achrasporum andT. alopallonellum, while only five species showed a broader distribution, being present at all levels:Leptosphaeria australiensis, Halocyphina villosa, Cryptovalsa sp.,Lulworthia grandispora andLulworthia sp. The greatest diversity of marine fungi were collected from the middle level with a Shannon Diversity Index of 5.9 while the Jaccard Similarity Index of 2.25 indicated that the upper and middle levels were the most similar in terms of species composition. Fungi with certain characteristics were also limited to particular levels, for example, carbonaceous and superficial ascomata were confined above mean tide while membranous walls and immersed ascomata were common below mean tide level.