Fibrosin, a novel fibrogenic cytokine, modulates expression of myofibroblasts.

Granulation tissue fibroblasts, or myofibroblasts are characterized by the presence of alpha smooth muscle actin fibers (alpha SMA). These specialized cells are involved in wound contraction and in retractile phenomena observed during fibrotic disease. Myofibroblasts have also been shown to play a role in embryonic development. Growth factors such as Transforming growth factor beta TGFbeta and Nerve growth factor (NGF) can modulate the differentiation of myofibroblasts. In this report, we show that in vitro application of fibrosin, a novel fibrogenic cytokine, stimulates expression of alpha SMA-producing cells at least four-fold above that observed in control cultures. In addition, administration of fibrosin in a wound healing model in mice stimulates increased numbers of myofibroblasts 7 days after injury, when compared with untreated, or, control, wounded mice. These results suggest that fibrosin plays an important role in up regulating the appearance of myofibroblasts during wound healing, and possibly in fibrotic diseases. It may, therefore, be important in the process of scarring.

Fibrosin: a novel lymphokine in wound healing.

Several growth factors are actively synthesized during wound repair and function to stimulate different cell types involved in the process of healing. Fibrosin is a novel fibrogenic lymphokine that stimulates several biological activities that relate to in vivo scarring. To investigate the role of fibrosin, we used "punch biopsy" and linear wounding procedures in a murine model of wound healing. Histological examination showed that recombinant fibrosin stimulated epithelialization of wounds and accelerated healing of both punch biopsy and linear wounds. Fibrosin enhanced healing of linear wounds by reducing the time for healing by approximately 30-40%. From our data we estimated the healing time of control wounds to be 22-24 days; wounds treated with fibrosin appeared to heal in 14-16 days. Our observations suggest that fibrosin enhances wound healing and may be involved in accelerating epithelialization, collagen matrix formation, and also remodeling of the extracellular matrix in vivo. Thus fibrosin may function during different phases of wound healing and act as a potent inducer of scar formation and wound healing. This finding may have direct clinical applications.

sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis.

sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv(+)-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.

Glycotyping of prostate specific antigen.

Measurement of serum levels of the prostate specific antigen (PSA) is now widely used for the diagnosis of prostate cancer and benign prostate hyperplasia. This serum marker is of value since it is derived only from the tissue of interest, but increased levels of PSA in serum do not allow a completely clear cut diagnosis of benign versus malignant changes. Since PSA is a glycoprotein with one asparagine linked oligosaccharide, and since malignant transformation often leads to an increased branching of such oligosaccharides, we initially studied the asparagine linked structures on PSA made by a cell line derived from malignant metastatic prostate tissue. We observed that unlike normal PSA, which bears only biantennary oligosaccharides, PSA from the metastatic cell line has a mixture of biantennary and triantennary oligosaccharides. Further experiments will reveal carbohydrate differences derived from the PSA from sera or, prostate tissue of normal versus prostate cancer patients, and of the utility of such carbo-hydrate differences as a possible diagnostic marker for prostate cancer.

Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins.

The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption. CaMNN9 has significant homology with S. cerevisiae MNN9, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in Candida. CaMNN9 resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of CaMNN9 leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with beta-1,3-glucanase. Like all members of the S. cerevisiae MNN9 gene family the Camnn9Delta strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the Camnn9Delta strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of MNN9 gene family members CaVAN1 and CaANP1 in the Camnn9Delta strain showed that transcription of those genes is not affected in the absence of CaMNN9 transcription. Our results suggest that, while the role MNN9 plays in glycosylation in both Candida and Saccharomyces is conserved, loss of MNN9 function in C. albicans leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target.

Fibrosin: A novel lymphokine in alcohol-induced fibrosis.

In this study, we examined the role of fibrogenic cytokines in alcohol-induced fibrosis. In particular, we examined the production of a novel fibrogenic cytokine, fibrosin, among others, by fibroblasts in response to ethanol in vitro; we also studied the production of fibrosin in an animal model of alcohol-induced liver injury. This model system utilizes the intragastric feeding rat model in which rats are fed different dietary fats and ethanol or dextrose. Our study showed that physiologic concentrations of ethanol directly induced proliferation of fibroblasts in vitro and also stimulated the production of cytokines. In particular, fibrosin, the novel fibrogenic cytokine, was produced. Other cytokines such as TGFbeta, IL-6, and TNFalpha were also induced. Also, exposure of fibroblasts to interleukin-1beta, interleukin-6, and tumor necrosis factor alpha induced production of fibrosin. In the fish oil-ethanol-fed rats which showed fibrotic lesions in the liver, fibrosin mRNA as well as protein was expressed. Fibrosin was not detected in control rats not exhibiting fibrosis. These studies show that ethanol can directly stimulate fibroblast proliferation and production of fibrogenic cytokines. It is likely that fibrosin, which may be derived from inflammatory cells, contributes to alcohol-induced hepatic fibrosis in vivo.

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Cloning and analysis of the cDNA for human fibrosin, a novel fibrogenic lymphokine.

Several diseases are complicated by tissue fibrosis, an outcome of chronic inflammation. Investigations have shown that soluble mediators produced by inflammatory cells may be a molecular link between chronic inflammatory cells and scarring. Using the murine model of schistosomiasis for studying chronic inflammation, a novel fibrogenic lymphokine, fibrosin, was isolated and characterized. Subsequently, we cloned the cDNA for murine fibrosin from a cDNA library derived from a mitogen-stimulated lymphocyte cell line, CDC25. In the current study, we cloned human fibrosin from cDNA libraries derived from human placenta and human peripheral blood lymphocytes. The isolated cDNA has an open reading frame spanning 531 nucleotides. Human fibrosin has considerable homology with murine fibrosin at the nucleotide as well as the amino acid level. And, like the murine fibrosin, it has no significant homology with nucleotide sequences encoding other proteins archived in the GenBank database. A 36-amino acid synthetic peptide constructed from the deduced amino acid sequence of human fibrosin is biologically active at subnanomolar concentrations. The availability of recombinant human fibrosin may allow us to better understand the involvement of this new lymphokine in certain chronic inflammatory as well as other diseases.

Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus.

The lumens of the endoplasmic reticulum and Golgi apparatus are the subcellular sites where glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins, proteoglycans, and lipids occur. Nucleotide sugars, nucleotide sulfate, and ATP are substrates for these reactions. ATP is also used as an energy source in the lumen of the endoplasmic reticulum during protein folding and degradation. The above nucleotide derivatives and ATP must first be translocated across the membrane of the endoplasmic reticulum and/or Golgi apparatus before they can serve as substrates in the above lumenal reactions. Translocation of the above solutes is mediated for highly specific transporters, which are antiporters with the corresponding nucleoside monophosphates as shown by biochemical and genetic approaches. Mutants in mammals, yeast, and protozoa showed that a defect in a specific translocator activity results in selective impairments of the above posttranslational modifications, including loss of virulence of pathogenic protozoa. Several of these transporters have been purified and cloned. Experiments with yeast and mammalian cells demonstrate that these transporters play a regulatory role in the above reactions. Future studies will address the structure of the above proteins, how they are targeted to different organelles, their potential as drug targets, their role during development, and the possible occurrence of specific diseases.

Elevated expression of chitinase 1 and chitin synthesis in myosin II-deficient Saccharomyces cerevisiae.

To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed. Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells. The increase in soluble activity was correlated with an increase in enzyme levels. Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold. These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells.

Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis.

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.

Chitinases are a multi-gene family in Aedes, Anopheles and Drosophila.

Degenerate primers were used to amplify by the polymerase chain reaction (PCR) DNA fragments from the chitinase genes of five insect species: Aedes aegypti, Anopheles freeborni, Anopheles gambiae, Anopheles stephensi and Drosophila melanogaster. As many as four different products were found for each species; each deduced protein sequence having greatest homology to chitinase sequences from other species of insects and the crustacean, Penaeus japonicus. The four PCR products of A. aegypti hybridize to two loci, with three of the products derived from either three tightly linked genes or a single gene with three catalytic domains. Southern blot hybridizations of the PCR products from the species of Anopheles suggest a similar arrangement.

Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene.

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Cloning and expression of chitinases of Entamoebae.

Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cysts formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.

Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae.

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis.

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungus Ustilago maydis was amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product of Umchs5 has highest similarity with class IV chitin synthases encoded by the CHS3 genes from Saccharomyces cerevisiae and Candida albicans, chs-4 from Neurospora crassa, and chsE from Aspergillus nidulans. Umchs5 null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase, Umchs6. It is suggested that multigenic control of chitin synthesis in U. maydis operates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.