The application of nanocrystaline diamond coatings in medicine.

The search for an endoprosthesis with the longest possible durability has been in progress for many years. In spite of three decades of intensive development of implant materials, contemporary endoprosthesis are still far from satisfactory. In an environment as corrosive as the human body, even the best metal alloys release ions: Co, Cr and Mo all dissolve at a rate of approximately 50 ng per year. The biocompatibility of an implants is directly by the extent of corrosion. Tests in vivo have shown that Ni, Co and Cr ions are bound to the body's own proteins, predominantly albumins, which can then transfer them to other ergans, thus disturbing their functions.
Facing this challenge, a new, entirely cementless endoprosthesis for the coxofemoral joint, comprised of a pin, a head and two bushings, has been constructed in a cooperative project involving three research centers: ECAM in Lyons, the Technical University of Bratislava, and the Thin Film Division of the Technical University of Lodz. Both the pin and the outer bushing are made of a titanium alloy, coated with a nanocrystalline diamond film. The heads is made of corundum ceramics, and the inner bushing is made if high molecular weight polyethylene. The shape of the pin has been designed in compliance with all biomechanical requirements.
As of this writing these endoprostheses have been implanted in test animals, and remain under continous biological monitoring. To this point all the test results have been postitive.

High-efficiency transient transfection of endothelial cells for functional analysis.

The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5-fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl-terminal domain of the beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.

Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis.

Sphingosylphosphorylcholine (SPC) is one of the biologically active phospholipids that may act as extracellular messengers. Particularly important is the role of these lipids in the angiogenic response, a complex process involving endothelial cell migration, proliferation, and morphologic differentiation. Here we demonstrate that SPC and its hydrolytic product, sphingosine, induce chemotactic migration of human and bovine endothelial cells. The response is approximately equal to that elicited by vascular endothelial cell growth factor. The effect of SPC and sphingosine was associated with a rapid down-regulation of Edg1, a sphingosine 1-phosphate (SPP)-specific receptor involved in endothelial cell chemotaxis. Both SPC and sphingosine induced differentiation of endothelial cells into capillary-like structures in vitro. Thus, SPC and sphingosine join SPP among the biologically active lipids with angiogenic potential. Since neuronal abnormalities accompany pathological accumulation of SPC in brain tissue, it is possible that SPC is a modulator of angiogenesis in neural tissue upon its release from brain cells following trauma or neoplastic growth.

Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKA-dependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms.

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.

Phylogenetic analysis of the Chlamydia trachomatis major outer membrane protein and examination of potential pathogenic determinants.

Phylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains of Chlamydia trachomatis. Nucleotide sequences of omp1, the gene encoding the major outer membrane protein (MOMP) of C. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with available omp1 sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.

Saccharomyces cerevisiae homologs of mammalian B and B' subunits of protein phosphatase 2A direct the enzyme to distinct cellular functions.

Protein phosphatase 2A (PP2A) is a major cellular serine/threonine protein phosphatase, present in the cell in a variety of heterotrimeric forms that differ in their associated regulatory B-subunit. Cloning of the mammalian B' subunit has allowed the identification of a highly homologous Saccharomyces cerevisiae gene, RTS1. Disruption of the gene results in a temperature-sensitive growth defect that can be suppressed by expression of rabbit B'alpha or B'gamma isoforms. The B'alpha subunit is much more effective in restoring normal growth at 37 degrees C than B'gamma. Immunoprecipitated Rts1p was found associated with type 2A-specific protein phosphatase activity that is sensitive to 2 nM okadaic acid, but not to 100 nM phosphatase inhibitor-2, and to be phosphorylated in vivo. However, overexpression of RTS1 was unable to suppress the cold sensitivity, defective cytokinesis, and abnormal cell morphology resulting from defects in the CDC55 gene, which encodes the yeast homolog of a different B subunit of another form of 2A phosphatase, PP2A1. These results indicate that Rts1p is a yeast homolog of the mammalian B' subunit and that the various regulatory B-subunits of PP2A are not functionally redundant but direct the enzyme to distinct cellular functions.

PBS2, a yeast gene encoding a putative protein kinase, interacts with the RAS2 pathway and affects osmotic sensitivity of Saccharomyces cerevisiae.

The yeast gene PBS2 encodes a presumed protein kinase. The gene is essential for manifestation of resistance to the antibiotic polymyxin B. Deletion of PBS2 enables a ras2-530 null mutant to grow on nonfermentable carbon sources; overexpression of PBS2+ enhances viability of a RAS2Val19 mutant. Overexpression of PBS2+ also diminishes cellular response to mating pheromone MF alpha. These results suggest that the PBS2 and RAS2 genes affect a common pathway that may communicate with the pheromone response pathway. In addition, disruption of PBS2 renders cells sensitive to high osmolarity: exposure to 0.9 M-NaCl causes growth arrest, appearance of bizarre morphological forms, and eventual death. A mutation suppressing pbs2 deletion has been found. That mutation restores full polymyxin B resistance but only partially corrects the osmotic sensitivity defect. These observations indicate that PBS2 is involved in diverse physiological pathways in yeast.

Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases.

Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on chromosome X, is not allelic to the previously described PBS1 gene (where PBS signifies polymyxin B sensitivity). Although pbs1 mutations confer resistance to high levels of polymyxin B, double mutants, pbs1 pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs1 activity. Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBS1 and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells.

Polymyxin B nonapeptide inhibits mating in Saccharomyces cerevisiae.

Polymyxin B nonapeptide enhanced susceptibility of yeast cells to various hydrophobic antibiotics and to mating pheromones. At much lower concentrations, the nonapeptide severely inhibited mating. The inhibition was caused by interference with sexual agglutination.

Effects of polymyxin B sulfate and polymyxin B nonapeptide on growth and permeability of the yeast Saccharomyces cerevisiae.

Polymyxin B, a toxic, membrane-affecting antibiotic, can be rendered harmless to yeast cells by enzymatic removal of its fatty acyl moiety. The remaining cyclic peptide portion, polymyxin B nonapeptide, has no significant effect on growth and viability but it drastically reduces mating efficiency. In addition, the cyclic peptide enhances sensitivity of cells to several drugs, presumably by increasing membrane permeability. Mutants resistant to polymyxin B are simultaneously less responsive to the combination of the nonapeptide and the drugs. This indicates that the peptide portion of polymyxin B is the moiety responsible for the permeability changes. The resistance is inherited as a simple recessive trait. The mutation has been mapped to chromosome XV of Saccharomyces cerevisiae.

Purification and characterization of an extracellular protease from Flavobacterium arborescens.

An extracellular protease from Flavobacterium arborescens has been purified to an apparent homogeneity and characterized. The enzyme is most active at pH 8-10.5, requires no metal cofactor, and is inhibited by diisopropyl fluorophosphate. The protease is nonspecific, is active at temperatures up to 60 degrees C, and is completely free of nucleases. The ease of purification and freedom from nucleolytic contaminants make the protease a useful deproteinizing agent in DNA and RNA manipulations.

A simplified method for measuring activity of beta-D-fructofuranoside fructohydrolase (invertase).

A coupled enzymatic method for the assay of invertase is described. In this method, the fructose produced from sucrose by invertase action is converted to D-sorbitol by sorbitol dehydrogenase. The reaction is monitored by the decrease in A340 mm due to the consumption of NADH. The technique is simple, sensitive, and accurate, and compares well with alternative methods which rely on determination of glucose formed in the invertase reaction.

Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum.

We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.

Cysteine biosynthesis in a fungus, Histoplasma capsulatum.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.