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PubMed1985

Evaluation of chemicals used for drinking water disinfection for production of chromosomal damage and sperm-head abnormalities in mice.

Chemical oxidants are commonly added during water treatment for disinfection purposes. These chemicals have not been tested previously for their ability to induce genetic damage in vivo. Chlorine (hypochlorite and hypochlorous acid), monochloramine, chlorine dioxide, sodium chlorite, and sodium chlorate were evaluated for induction of chromosomal aberrations and micronuclei in bone marrow of CD-1 mice, and for induction of sperm-head abnormalities in B6C3F1 mice. Oral administration of chlorine at pH 8.5 (where hypochlorite predominates) at dose levels equivalent to approximately 4 and 8 mg/kg/day induced significant increases in the level of sperm-head abnormalities. There was no evidence of other effects with any of the disinfectants. Halogenated acetonitriles, which have previously been shown to form in the stomach following oral dosing of sodium hypochlorite to rats, were also tested in the sperm-head abnormality assay but gave no indication of an effect.

Animals↗
PubMed2002

Glycosaminoglycan mimetics (RGTA) modulate adult skeletal muscle satellite cell proliferation in vitro.

Muscle regeneration occurs through the activation of satellite cells, which are stimulated to proliferate and to fuse into myofibers that will reconstitute the damaged muscle. We have previously reported that a family of new compounds called "regenerating agents" (RGTAs), which are polymers engineered to mimic heparan sulfates, stimulate in vivo tissue repair. One of these agents, RG1192, a dextran derivative substituted by CarboxyMethyl, Benzylamide, and Sulfate (noted CMBS, RGTA type), was shown to improve greatly the regeneration of rat skeletal muscle after severe crushing, denervation, and acute ischemia. In vitro, these compounds mimic the protecting and stabilizing properties of heparin or heparan sulfates toward heparin-binding growth factors (HBGFs). We hypothesized that RGTA could act by increasing the bioavailability of some HBGF involved in myoblast growth and thus asked whether RGTA would alter the ability of satellite cells to proliferate. Its effect was tested on primary cultures of rat satellite cells. The RG1192 stimulated the proliferation of satellite cells in vitro in a dose-dependent manner. It appeared to be as efficient as natural glycosaminoglycans (GAGs; heparan sulfate, dermatan sulfate, or keratan sulfate) in stimulating satellite cell proliferation but was about 100 times more efficient than heparin. RG1192 stimulated satellite cell proliferation by increasing the potency of fibroblast growth factor 2 and scatter factor-hepatocyte growth factor. It also partially restored myoblast proliferation of satellite cells with chlorate-induced hyposulfation. Taken together, our results explain to some extent the improving effect of RGTA with a CMBS structure, such as the RG1192, on muscle regeneration in vivo by providing support for the hypothesis that RGTA may act by increasing the potency of some HBGFs during the proliferation phase of the regenerating muscle.

Animals↗
PubMed1993

Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95).

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate-dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner. Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion. Treatment with chlorate, an inhibitor of ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JAR-RL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate-containing proteoglycans participated in cell-cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding.

Cell Adhesion↗
PubMed2004

Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer.

BACKGROUND: Cationic polymers and liposomes are used to wrap DNA into complexes that promote its cellular uptake. The mechanisms of the uptake and the intracellular fate of these complexes are obscure, as are reasons for an unpredictable and sometimes poor efficiency of the transgene expression. Polyanionic glycosaminoglycans (GAGs) on the cell surface interact with the cationic DNA complexes and influence transfection. METHODS: The quantities of heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA) on the cell surface of mutated Chinese hamster ovary (CHO) cells and manipulated (chlorate, xyloside, chondroitinase ABC or Streptomyces hyaluronidase) smooth muscle cells were correlated with the uptake of four different DNA complexes, and the expression of the transgene. RESULTS: Two CHO mutants, without cell-surface HS and CS, showed a 1.5-6-fold increase in cellular association of DNA, and 3-25-fold increase of transgene expression, as compared with the wild type. A CHO mutant with a 5.7-fold increase of cell-surface CS, but devoid of HS, showed enhanced DNA association, but 20-40% reduction in its expression. The removal of HS, CS, or HA from the cell surface of smooth muscle cells had a minor or insignificant effect on the cell association and transfection of the carriers and only polyethyleneimine showed increased association and expression of the transgene. CONCLUSIONS: The uptake of DNA complexes varies depending on carrier, cell type and amounts of cell-surface HS, CS, and HA, whereas all GAGs inhibit the transgene expression. This implies that cell-surface GAGs probably direct complexes into intracellular compartments that do not support transcription.

Animals↗
PubMed2005

Heparin-binding epidermal growth factor-like growth factor stimulates cell proliferation in cerebral cortical cultures through phosphatidylinositol 3'-kinase and mitogen-activated protein kinase.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.

Amino Acid Sequence↗
PubMed1991

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Acetylcholinesterase↗
PubMed2001

Asymmetric autocatalysis and the origin of chiral homogeneity in organic compounds.

The discovery and development of asymmetric autocatalysis, in which the structures of the chiral catalyst and the chiral product are the same, are described. Chiral 5-pyrimidyl, 3-quinolyl, and 5-carbamoyl-3-pyridyl alkanols act as highly enantioselective asymmetric autocatalysts in the enantioselective addition of diisopropylzinc to the corresponding aldehydes, such as pyrimidine-5-carbaldehyde. 2-Alkynyl-5-pyrimidyl alkanol with an enantiomeric excess (ee) of >99.5% automultiplies practically perfectly as an asymmetric autocatalyst in a yield of >99% and >99.5% ee. Asymmetric autocatalysis with an amplification of ee has thus been realized. Consecutive asymmetric autocatalysis starting with chiral 2-alkynylpyrimidyl alkanol of only 0.6% ee amplifies its ee significantly, and yields itself as the product with >99.5% ee. The reaction of pyrimidine-5-carbaldehyde and diisopropylzinc in the presence of chiral initiators with low ee's, such as secondary alcohol, amine, carboxylic acid, mono-substituted [2.2]paracyclophane, and chiral primary alcohols due to deuterium substitution, regulates the absolute configuration of the resulting pyrimidyl alkanols, and the ee of the resulting pyrimidyl alkanol is much higher than that of the chiral initiator. Leucine and [6]helicene with very low ee's, which are known to be induced by circularly polarized light (CPL), also serve as chiral initiators to produce pyrimidyl alkanol with higher ee's. Overall, the process represents the first correlation between the chirality of CPL and an organic compound with very high ee. Chiral inorganic crystals, such as quartz and sodium chlorate, act as chiral inducers in the asymmetric autocatalysis of pyrimidyl alkanol. The process correlates for the first time ever the chirality of inorganic crystals with an organic compound with very high ee.

Aldehydes↗
PubMed1996

Heparin-binding EGF-like growth factor is an autocrine growth factor for rat gastric epithelial cells.

We examined the biological action and expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric mucosal cell line, RGM1. HB-EGF stimulated DNA synthesis of RGM1 cells in a dose-dependent manner. Mitogenic effect of HB-EGF was as potent as that of other known mitogens for gastric epithelial cells, such as hepatocyte growth factor (HGF) and transforming growth factor (TGF)-alpha. Northern blot analysis showed that RGM1 cells as well as rat gastric mucosal tissue expressed a 2.5-kilobase transcript of HB-EGF. Not only HB-EGF and TGF-alpha but also HGF caused a rapid induction of HB-EGF mRNA in the cells. Treatment with heparitinase which destroys heparan sulfate proteoglycan (HSPG) or with chlorate which inhibits sulfation of HSPG diminished [3H]thymidine incorporation of RGM1 cells in serum-free medium. In addition, a synthetic peptide corresponding to the heparin-binding domain of HB-EGF inhibits the DNA synthesis of RGM1 cells in serum-free medium in a dose-dependent manner. These results suggest that HB-EGF is an autocrine and paracrine growth factor for gastric epithelial cells and may play significant roles in mucosal repair of the stomach in cooperation with other growth factors.

Amino Acid Sequence↗
PubMed1997

Sulfated sialyl Lewis X, the putative L-selectin ligand, detected on endothelial cells of high endothelial venules by a distinct set of anti-sialyl Lewis X antibodies.

Endothelial cells of high endothelial venules (HEV) in human peripheral lymph nodes expressed a distinct type of sialyl Lewis X antigen, which was detected preferentially with a set of anti-sialyl Lewis X antibodies, 2F3, 2H5 and HECA-452 in immunohistochemistry, while another set of anti-sialyl Lewis X antibodies, FH-6 and CSLEX-1, failed to detect it. The adhesion of cells expressing L-selectin to HEV was inhibited by members of the former set of antibodies in Stamper-Woodruff assays performed on frozen sections of human peripheral lymph nodes. Transfection of a cultured endothelial cell line with a human alpha1-->3 fucosyltransferase, Fuc-T VII, resulted in the expression of a distinct type of sialyl Lewis X antigen having the reactivity similar to that of HEV; i.e., the antigen appearing on the transfectant clone was detectable only with the set of 2F3, 2H5 and HECA-452, but not with the set of FH-6 and CSLEX-1. Treatment of transfectant cells with sodium chlorate, a metabolic inhibitor of sulfation, resulted in reactivity to the members of the latter set of antibodies, suggesting that sulfation of sialyl Lewis X moiety was the cause of the discrepancy in the reactivity of the anti-sialyl Lewis X antibodies. When tested against various authentic sulfated sialyl Lewis X determinants, 6-sulfo sialyl Lewis X and 6,6'-bis-sulfo sialyl Lewis X were found to be reactive to the antibodies, 2F3, 2H5 and HECA-452, but not with antibodies FH-6 and CSLEX-1, suggesting that the distinct type of sialyl Lewis X determinant on the HEV endothelial cells and Fuc-T VII-transfected endothelial cell clone are mainly 6-sulfo and/or 6,6'-bis-sulfo sialyl Lewis X determinants.

Animals↗
PubMed2001

Syndecan-4 as antithrombin receptor of human neutrophils.

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Mechanisms of antithrombin's effects on neutrophils were, therefore, studied by testing function and expression of heparan sulfate proteoglycans in RT-PCR or flow cytometry and cell migration assays, respectively. In vitro effects of antithrombin on human neutrophil migration in modified Boyden chambers were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in neutrophils was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assay, respectively. In the presence of pentasaccharide, antithrombin lost its activity on the cells. Data suggest that antithrombin regulates neutrophil migration via effects of its heparin-binding site on cell surface syndecan-4.

Antibodies↗
PubMed1994

Inhibition of protein and lipid sulfation in oligodendrocytes blocks biological responses to FGF-2 and retards cytoarchitectural maturation, but not developmental lineage progression.

We have examined the interfaces between protein and lipid sulfation, the response of progenitor cells to specific growth factors, and developmental lineage progression. The experimental system used is the oligodendrocyte (OL), the cell responsible for myelination of axons in the central nervous system. The ability to regulate the growth and differentiation of oligodendrocytes in primary cultures growing in defined medium offers a particularly attractive model for these interrelated areas of cell and developmental biology. Sulfation was inhibited in this system by growing cells in sodium chlorate, a competitive inhibitor of sulfation. Four principal conclusions are drawn from the data. First, heparan sulfate proteoglycan is a necessary cofactor for the environmental regulation by FGF-2 of nontransformed oligodendrocyte progenitors, including both the mitogenic response and the inhibition of terminal differentiation at a specific stage of the lineage. Second, the differentiation of oligodendrocyte progenitors from an early to a mature stage can occur in the absence of sulfation. Third, however, the formation of cellular processes and myelin-like sheets is retarded, leading to the prediction that sulfated molecules are critically involved in aspects of oligodendrocyte differentiation directly relating to myelination per se. Fourth, the developmental proligodendroblast antigen, POA, is a sulfated molecule. The results provide clear evidence for the importance of sulfation for the responses of OL progenitors to FGF-2 and for OL cytoarchitectural maturation, while demonstrating an ability of OL progenitors to undergo lineage progression in its absence.

Animals↗
PubMed1998

Hepatocyte extracellular matrix modulates expression of growth factors and growth factor receptors in human colon cancer cells.

We investigated the role of hepatocyte extracellular matrix (ECM) on the growth of human colon cancer cell lines. We cultured four cell lines with different liver-colonizing potential on ECM derived from primary rat hepatocyte cultures. We investigated the effect of ECM on cell proliferation, clonal growth, and expression of growth factors and growth factor receptors. The highly metastatic cells showed better clonal growth and produced larger colonies on ECM. The proliferation of all colon cancer cell lines was enhanced on hepatocyte ECM, yet inhibited on fibroblast ECM. Screening of autocrine growth factors and receptors showed that the cells expressed growth factors and receptors of the EGF family: EGF receptor, erb-B2, amphiregulin, and cripto. The expression of cripto mRNA, but not of amphiregulin, was induced in KM12SM cells grown on ECM. All colon cancer cell lines grown on ECM showed increased expression of erb-B2. The effect of ECM on erb-B2 expression was mediated by the heparin chains of heparin proteoglycan. ECM from hepatocytes grown in the presence of nitrophenyl-beta-D-xylopyrannoside or sodium chlorate, which prevent formation of heparin proteoglycan, as well as ECM treated with heparinase, had no effect on erb-B2 expression. Our studies suggest a role for liver ECM as a determinant of colon cancer metastasis. Liver ECM acts, in part, via induction of members of the EGF family of growth factors and their receptors.

Animals↗
PubMed1998

Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells.

There is increasing evidence that the proteoglycan heparin plays a critical role in the regulation of the activity of FGF-2 by either interacting with its receptor or modifying its stability and functioning. In this study precursor cells were isolated from the rat E14 ventral mesencephalon and cultured as free floating spheres in FGF-2 alone or in combination with heparin or other related proteoglycans, including chondroitin sulfate, keratin sulfate, dermatan sulfate, or hyaluronic acid. Our results show the mitogenic effects of FGF-2 could be potentiated by heparin but not the other four proteoglycans. Sodium chlorate, which blocks the cells ability to sulfate its proteoglycans, was shown to reduce the mitogenic effects of FGF-2 alone to below that of control levels, suggesting that endogenous sulfated molecules are required for the FGF-2 effects on mesencephalic precursors. Cells expanded for 7 days with either FGF-2 or FGF-2 + heparin were plated onto a substrate and allowed to differentiate for a further 7 days in the absence of growth factors. Approximately 6% of the precursors developed into neurons whether grown with or without heparin and none were positive for TH, a marker for dopamine neurons. However, there was a significant decrease in the number of astrocytes developing from cultures grown in FGF-2 + heparin when compared to FGF-2 alone. Interestingly we could not find an EGF responsive cell in the mesencephalon at this embryonic age in the absence or presence of heparin. However, there was a synergistic effect of combining EGF + FGF-2, which could be potentiated by heparin. We conclude that heparin, but not other closely related proteoglycans, is vital for the growth of FGF-2-responsive mesencephalic neural precursors.

Animals↗
PubMed1999

Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum.

We used Stagonospora (Septoria) nodorum to explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the resident NIA1 gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expected NIA1 gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1. 1 kb of the coding region and the complete 5' region of this gene which is adjacent to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.

Blotting, Southern↗
PubMed1999

Deletion of the trichodiene synthase gene of Fusarium venenatum: two systems for repeated gene deletions.

The trichodiene synthase (tri5) gene of Fusarium venenatum was cloned from a genomic library. Vectors were created in which the tri5 coding sequence was replaced with the Neurospora crassa nitrate reductase (nit3) gene and with the Aspergillus nidulans acetamidase (amdS) gene flanked by direct repeats. The first vector was utilized to transform a nitrate reductase (niaD) mutant of F. venenatum to prototrophy, and the second vector was utilized to confer acetamide utilization to the wild-type strain. Several of the transformants lost the capacity to produce the trichothecene diacetoxyscirpenol and were shown by hybridization analysis to have gene replacements at the tri5 locus. The nit3 gene was removed by retransformation with a tri5 deletion fragment and selection on chlorate. The amdS gene was shown to excise spontaneously via the flanking direct repeats when spores were plated onto fluoroacetamide.

Amidohydrolases↗
PubMed1998

NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro.

In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. In the presence of nitrate or nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His6-tagged form (His6-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma70-dependent tac promoter for the native sigmaN-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His6-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 microM) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified regions required for both negative regulation (the terminator) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. Finally, electrophoresis mobility shift analysis revealed that His6-NasR bound specifically to nasF leader RNA. This binding was independent of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction with nasF leader RNA.

5' Untranslated Regions↗
PubMed2002

Comparison of multiplex PCR, PCR-ELISA and fluorogenic 5' nuclease PCR assays for detection of plasmid-bearing virulent Yersinia enterocolitica in swine feces.

Swine are implicated as the principal animal reservoir for plasmid-bearing Yersinia enterocolitica (YEP(+)) strains that are pathogenic to humans. To evaluate the utility of the PCR for detection of YEP(+) strains in naturally-contaminated pig feces, samples were first enriched in Irgasan ticarcillin potassium chlorate broth for 48 h at 25 degrees C and then tested by multiplex PCR, PCR-ELISA, and fluorogenic 5' nuclease PCR assays. Three different primer sets for amplification of the ail gene sequences were used in these three assays. Three out of 50 (6%) samples were positive for YEP(+) strains using the multiplex PCR targeting the chromosomal ail (170 bp) and plasmid virF (591 bp) genes. Two of the 3 samples positive by the multiplex PCR were also positive by the PCR-ELISA method using primers targeting the ail gene (425 bp). In contrast, the fluorogenic 5' nuclease PCR assay failed to detect an ail gene sequence (118 bp) in any of the 50 samples. These results indicate that the multiplex PCR was the most reliable and sensitive assay for detecting YEP(+) strains in feces among the three assays evaluated.

Animals↗
PubMed2002

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

Animals↗