The coxsackievirus and adenovirus receptor.

The coxsackievirus and adenovirus receptor (CAR) has been studied extensively since its identification and isolation in 1997. The CAR is an immunoglobulin superfamily protein with two extracellular Ig-like domains, a single membrane-spanning sequence, and a significant cytoplasmic domain. It is structurally and functionally similar to the junctional adhesion molecules. The amino terminal domain, distal from the membrane, has been structurally characterized alone, bound to the adenovirus fiber knob, and, in full-length CAR, docked in the canyon structure of the coxsackievirus capsid. Although the past decade has produced a burst of new knowledge about CAR, significant questions concerning its function in normal physiology and coxsackievirus-related pathology remain unanswered.

QTL associations for density and diameter in Pinus radiata and the potential for marker-aided selection.

A large full-sib family of radiata pine ( Pinus radiata Donn. ex D. Don) was used for quantitative trait locus (QTL) detection and independent verification. QTL detection experiments were carried out for juvenile wood density (JWD) and stem diameter at breast height (DBH) using selective genotyping. Evenly spaced RFLP and microsatellite markers were selected from an existing linkage map. QTLs were verified in an independent set of progeny from the same family. Based on map location, at least eight QTL positions for JWD and two for DBH were detected and verified. The percent variance accounted for by the markers ranged from 0.78% to 3.58%, suggesting a genomic architecture of many genes with small effect. Two unrelated "bridging" families were chosen as candidates for marker-aided selection (MAS), and six microsatellite markers showing an association with JWD or DBH were tested in these families. Of these, four markers showed a consistent association with JWD in one or both of the bridging families. Results from this study provide a basis for MAS in P. radiata.

The effect of an imprecise map on interval mapping QTLs.

The statistical analysis of quantitative trait locus (QTL) experiments relies on the use of a linkage map of the markers genotyped. Such a map is, at best, a good estimate of the true map. Resources might be diverted into developing better marker maps or improved maps become available after the analysis, raising concerns over the original analysis. It is therefore important to understand the sensitivity of QTL analysis to map inaccuracy. We have used simulation methods to investigate the consequences of an incorrect map on the results of a QTL analysis using interval mapping. Backcross data sets were generated with a particular map and then analysed with both the correct map and incorrect maps. If the incorrect maps maintained the true linkage groups (i.e. no markers were incorrectly assigned to another linkage group), the accuracy of the map had little or no impact on the ability to detect QTLs, the true significance levels of the tests or the relative placement of QTLs. When a marker was incorrectly placed on another linkage group, there was a small increase in the level of the test. After adjusting for this increase, there was a decrease in power to detect a QTL near the misplaced marker. This decrease was of a similar magnitude to that found when using a single-marker analysis compared with interval mapping. These results mean that QTL analyses can proceed without the need for very accurate marker maps, and that estimated QTL positions can be translated onto updated maps without the need for reanalysis.

Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence.

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.

Coxsackievirus and adenovirus receptor (CAR) binds immunoglobulins.

The coxsackievirus and adenovirus receptor protein (CAR) serves as the cell surface receptor for group B coxsackieviruses and most adenoviruses, but the physiological function and ligand for this protein remain to be described. An affinity column was constructed with the recombinant extracellular domain of the CAR (rECAR) to isolate potential ligands by affinity chromatography. Immunoglobulins G and M were consistently isolated from human sera passed through the column, suggesting that the CAR may be an immunoglobulin-binding protein. Further investigation revealed that the affinity-purified immunoglobulins bound to rECAR-coated immunoassay plates, and the peroxidase-labeled rECAR bound the immunoglobulins on ligand-overlay blots. The peroxidase-labeled rECAR was incorporated into immunoprecipitates formed between the affinity-purified immunoglobulins and rabbit antibodies against human immunoglobulins, but not into immunoprecipitates formed between mouse IgG and rabbit antibodies against mouse IgG. The CAR present in HeLa cell lysates also bound to the affinity-purified immunoglobulins on Immobilon membranes, showing that the association is not limited to the recombinant protein. These results demonstrate that the CAR binds IgG and IgM present in serum, and reveal a direct interaction between the coxsackievirus and adenovirus receptor and the immune system.

Receptor for the group B coxsackieviruses and adenoviruses: CAR.

Considerable progress towards the characterisation of the long-sought receptor, CAR (coxsackievirus and adenovirus receptor), shared by group B coxsackieviruses (CVB) and most adenoviruses (Ad) has been made since it was isolated and cloned in 1997. The primary sequence of CAR shows that it is a member of the immunoglobulin superfamily of proteins, containing two Ig superfamily domains: an amino-terminal V-like module and a C2-like module. The CAR cytoplasmic domain, representing nearly one-third of the protein, is separated from the C2-like module by a single membrane-spanning sequence. The structure of the CAR V-like module complexed with the Ad fibre knob has been determined using recombinant proteins, and reveals three CAR modules associated with a single knob. Although recombinant CAR expressed in mammalian cells confers permissivity to CVB infection, details of the interaction between CAR and CVB remain to be elucidated. The expression of CAR appears to be highly regulated with respect to both cell type and developmental age. In rodents, CAR is expressed at high levels just before birth, and declines thereafter. Expressed levels have been found to increase in regenerating muscle and in response to immunological mediators or inflammation, and in RD cells and umbilical vein endothelial cells in response to high cell density. These studies indicate that CAR expression is highly regulated, but the mechanisms and molecules that mediate the expression remain to be discovered. The physiological function of CAR and its natural ligand also remain to be discovered. In addition, while CAR expression generally correlates with viral tropism, the relationship between the physiological function of CAR and the pathologies of CVB and Ad infections remain to be described.

Limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) on HeLa cells exposed to trypsin.

Trypsin treatment of HeLa cells results in a limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) after which the cleaved CAR remains cell-associated and tryptic peptides remain associated through disulfide bonds. Trypsin-treated HeLa cells remain susceptible to infection with coxsackievirus B and produce progeny virus at 8 h post-infection in amounts comparable to cells with intact CAR. HeLa cells remove the proteolysed CAR within 15 h and require over 24 h to restore intact CAR to control levels. As turnover is relatively slow, physiological functions that require intact CAR protein may be compromised for more than 24 h following trypsin treatment. Moreover, since removal of proteolysed CAR proceeds at more than twice the replacement rate, trypsin treatment disrupts the receptor-per-cell steady state for at least 24 h.

Tissue factor encryption/de-encryption is not altered in the absence of the cytoplasmic domain.

Since the cytoplasmic domain of tissue factor (TF) appears to have a role in TF function beyond coagulation, experiments were conducted to determine whether the cytoplasmic domain also has a role in regulating procoagulant activity of TF present in the cell membrane. TF encryption was quantitated in human YU-SITI, U87-MG, and mouse 3T3 cells which were transfected for expression of human tissue factor or a construct lacking the cytoplasmic domain (TF(CD)). Comparison of intact cells (encrypted) with fully disrupted cells (de-encrypted) showed that TF and TF(CD) were equally encrypted with respect to function in fX activation. Moreover, cells expressing TF and TF(CD) were indistinguishable in their procoagulant responses to A23187-calcium and varied concentrations of nonionic detergents. TF in membrane vesicles spontaneously shed by U87-MG cells was largely, but incompletely, de-encrypted, and the degree of de-encryption was independent of the cytoplasmic domain. We conclude that the predominant mechanism(s) for encrypting TF procoagulant activity is independent of the cytoplasmic domain.

Phase variation of the gonococcal siderophore receptor FetA.

FetA, the recently characterized gonococcal ferric enterobactin receptor, exhibited extremely rapid phase variation between high- and low-expression levels. The frequency of phase variation was approximately 1.3% in both directions in gonococcal strain FA1090. FetA expression in the 'high phase' was significantly greater than the level of expression in the 'low phase'. Expression levels correlated with the number of cytosine residues in a string of cytosines located close to the transcriptional start site for fetA between the putative -10 and -35 consensus sequences. Antibody production against FetA commonly occurs in infected patients, and we therefore hypothesize that phase variation reflects a balance between the advantages of being able to use a ferric siderophore as an iron source and evasion of the host immune response.

Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density.

Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B.

Ferric enterobactin binding and utilization by Neisseria gonorrhoeae.

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.

Tissue factor and cell morphology variations in cell lines subcloned from U87-MG.

Tissue factor is heterogeneously distributed within and among cells in cultures of U87-MG, a glioblastoma-derived line. The heterogeneity among cells may reflect the presence of distinct populations within the U87-MG cultures. This hypothesis has been confirmed by cloning of five distinct sublines from the parent population. These subpopulations have remained distinct through 4 months of growth in culture and one cycle of cryogenic preservation and thawing. The cultures differ in growth rates, amounts of tissue factor activity expressed, tissue factor antigen measured by flow cytometry, and patterns of tissue factor distribution studied by immunofluorescence microscopy. Characterization of these sublines allowed us to recognize that the tissue factor distribution on polarized cells (e.g. spindle-shaped) differed from that on cells with less polar morphologies. Finely speckled tissue factor staining tended to be localized to polarized aspects of the cell body where actin stress fibers are commonly present, whereas larger distinct foci of tissue factor were present in regions of membrane spreading. These results show that tissue factor is distributed differently in distinct regions of plasma membrane differentiation. Furthermore, the isolation of distinct stable subpopulations by dilutional cloning of U87-MG cultures serves as a reminder that cell culture heterogeneity can complicate experiments using molecular genetic manipulation of cultured cells which require clonal isolation of genetically altered lines.

Activation of coagulation factor X in human semen.

Tissue factor (TF) is an essential cofactor for factor VII (fVII) in initiating blood coagulation. Recently, TF was shown to be present in human semen and to be associated with prostasomes that originate from prostatic secretions. In the blood coagulation cascade, the complex of TF and activated factor VII (fVIIa) can activate both factor X and factor IX, by limited proteolysis. In the present study, we investigated the ability of semen to activate factor X. We also determined that factor X was activated predominantly by TF-fVIIa and that most of the TF was present in the seminal plasma, consistent with prostasome localization. No endogenous factor X was detected in semen, but activation of added factor X occurred in the absence of added fVIIa. Subsequent experiments showed that seminal plasma contains endogenous fVII-like activity, but the addition of more fVIIa increased factor X activation. Thus, while seminal plasma contains significant amounts of TF, its potential to activate factor X is limited by fVII availability and by the absence of endogenous factor X. Evaluation of semen specimens from infertility patients revealed a 16-fold variation in TF-fVII activity. No relationship between TF and number of days of abstinence, specimen pH, sperm count, or sperm motility was evident. Additional factor X-activating potential, independent of further TF activity, was generated in seminal plasma after treatment of semen with calcium and ionophore A23187. Production of this additional activity was blocked by the addition of anti-TF antibody during the activation. Since there is no factor X endogenous to semen, the additional activity stimulated by A23187 appears to be due to an endogenous, non-factor X substrate for TF-fVII in semen. This endogenous substrate may be either factor IX or a novel new substrate for TF-fVIIa. Future experiments will test these hypotheses.

Tissue factor cytoplasmic domain peptide is multiply phosphorylated in vitro.

Human tissue factor was phosphorylated when incubated with lysates of U87-MG cells or fractions from preparative isoelectric focusing of the lysates. The cytoplasmic domain peptide, isolated following chemical cleavage at cysteine 245, focused on PhastGel IEF near pH 3.4, indicating the presence of three phosphate groups. A peptide corresponding to the carboxyl-terminal cytoplasmic domain (residues 245-263) was synthesized and shown to be a protein kinase substrate when incubated with lysates of U87-MG cells and radiolabeled ATP. As found with full-length tissue factor, the TF(245-263) peptide was phosphorylated at all three serines, but a diphosphate form was also identified. TF(245-263) was phosphorylated in the absence of calcium as well as in the presence of calphostin C, indicating that phosphorylation can be independent of protein kinase C. These results reveal that tissue factor can be multiply phosphorylated in vitro, and that the synthetic TF(245-263) cytoplasmic domain peptide serves as a model substrate.

Purification of the putative coxsackievirus B receptor from HeLa cells.

We have identified a protein expressed by human and murine cells susceptible to coxsackievirus B3 (CVB3) infection and purified it from HeLa cells. This protein of approximately 45,000 Mr is expressed by HeLa cells and mouse fetal heart fibroblasts (susceptible to infection), and not by C3H murine fibroblasts or the human RD cell line (resistant). The protein was isolated from Triton X-100- deoxycholate lysates of HeLa cells by chromatography on concanavalin A-Sepharose, Affi-gel Blue, Phenyl Sepharose, and PBE94. The CVB3-binding fraction from PBE94 was blotted from SDS-polyacrylamide gel onto PVDF membrane for amino acid sequencing. Approximately 2 pmoles of CVB3-binding protein provided assignments for 26 consecutive residues: LSITTPEEMIEKAKGETAYLPXKFTL. This sequence corresponds neither to decay accelerating factor nor to nucleolin, both of which have previously been identified as CVB3-binding proteins, but does match two entries in GenBank. These data show that we have purified a novel CVB3-binding protein, the characteristics of which suggest the CVB group receptor has been purified. Identification of 26 amino acid residues in the protein and corresponding GenBank enteries will accelerate study of CVB tropism and the diseases caused by these viruses.

Cloning and sequencing of a Haemophilus ducreyi fur homolog.

Iron regulation in a growing number of bacterial species is being attributed to the presence of a fur (ferric uptake regulation) regulatory system. In the presence of iron, Fur acts as a classical negative regulator, binding conserved sequences within the promoter of iron-repressible genes and blocking transcription. Western blot analysis utilizing Escherichia coli Fur antisera detected a band of approximately 17 kDa in soluble extracts of Haemophilus ducreyi. Additionally, Southern blot hybridization of the H. ducreyi chromosome with a meningococcal fur probe indicated that H. ducreyi might contain a fur homolog. This putative fur homolog was cloned into the E. coli vector pACYC184. This clone was capable of repressing expression of a normally Furregulated lacZ fusion in the fur-background of E. coli strain H1780. The deduced amino acid sequence shows H. ducreyi fur to be 54% identical and 73% similar to E. coli fur, containing putative DNA-binding and metal-binding domains. These data demonstrate that H. ducreyi has a functional fur system.

Manifestation of cryptic fibroblast tissue factor occurs at detergent concentrations which dissolve the plasma membrane.

Cultured fibroblasts treated with increasing concentrations of detergents expressed only encrypted levels of tissue factor activity (measured by fX activation in the presence of fVIIa), characteristic of undamaged cells, until each detergent reached a critical concentration at which the cryptic tissue factor activity was manifested. Beyond the narrow ranges of concentrations over which the detergents stimulated tissue factor activity, the detergents were inhibitory. Studies with Triton X-100 and octyl glucoside revealed that manifestation of tissue factor activity coincided with breakdown of the plasma membrane. The magnitude of the increased tissue factor activity differed among detergents, with octyl glucoside giving the largest response. The tissue factor that was active after Triton X-100 treatment remained mostly associated with the insoluble cell residue, whereas the concentration of octyl glucoside which stimulated activity released tissue factor activity into the supernatant. Radiolabeled antibody against human tissue factor was used to show that a small percentage of the total accessible tissue factor remained in the insoluble fraction after treatment with either non-ionic detergent. Chromatographic analysis of lipids extracted from cells treated with detergents and dansyl chloride showed dansyl-reactivity of phosphatidylserine on intact cells, and solubilization of membrane lipids at sublytic concentrations of detergents. These findings reveal that there is a critical level of detergent-induced membrane damage at which tissue factor activity is maximally expressed, in essentially an all-or-none manner. The results are consistent with a major role for phospholipid asymmetry in regulation of tissue factor specific activity, but require either maintenance of asymmetry during sublytic detergent perturbation of the plasma membrane or additional control mechanisms.

Fibroblasts restrict tissue factor from vesicles which form in response to low concentrations of detergent.

Studies of tissue factor activity on fibroblasts have found that manifestation of the otherwise cryptic activity is evoked by Triton X-100 or octyl glucoside at concentrations that lyse the cells. Even though sublytic concentrations of the detergents extract membrane lipids into the soluble phase, they were without effect on tissue factor activity. Those experiments led us to conclude that either the fibroblasts maintain plasma membrane lipid asymmetry even as lipids are extracted by the detergents, up to the onset of lysis, or additional mechanisms for regulation of tissue factor specific activity were operative. Using phase contrast and immunofluorescent microscopy, we now show that at least one additional regulatory mechanism is indeed operative. In response to sublytic concentrations of octyl glucoside or Triton X-100, the cells release vesicles from which tissue factor antigen is excluded. Lytic concentrations of the detergents preclude this segregation, leaving only low amounts of tissue factor antigen associated with the adherent cytoskeletons. Two-color staining reveals marked tissue factor-actin filament co-localization, which implies the potential for cytoskeletal participation in the observed tissue factor segregation. We propose that tissue factor activity is indeed regulated by the phospholipids with which it is associated and the degree to which phosphatidylserine is available on the membrane surface, but the cells possess additional mechanisms by which the association of tissue factor with potentially procoagulant membrane domains is controlled.

Human placental tissue factor: protease susceptibility of extracellular and cytoplasmic domains.

We have examined the effects of seven proteases on human placental tissue factor in Triton X-100, focusing on extracellular and cytoplasmic domains recognized by monoclonal antibodies HTF1, C28 1.1, and C28 2.1. Plasmin produced peptides recognized on Western blots by C281.1 but not HTF1. None of the other proteases destroyed the extracellular epitope without also removing the cytoplasmic epitope, and both trypsin and chymotrypsin removed the cytoplasmic epitope with little effect on the extracellular domain. Proteinase K destroyed both epitopes, as did neutrophil elastase when used at a relatively high concentration. When digests were sampled over time and reconstituted with lipids for determination of tissue factor activity, only proteinase K consistently produced a loss in tissue factor activity at four hours. After 24 hr, other enzymes also decreased the recovered activity, with the order of effectiveness elastase > trypsin > chymotrypsin.

Staurosporine blocks down-regulation of monocyte-associated tissue factor.

Inflammatory agents including bacterial endotoxin (LPS) and low concentrations of phorbol myristate acetate (PMA) stimulate human peripheral blood monocytes to transiently express tissue factor procoagulant activity. Concentrations of PMA that cause the cytosol-to-membrane translocation of protein kinase C (PKC) (10(-9)-10(-7) M) induce a rapid decrease in monocyte tissue factor activity. Staurosporine, an inhibitor of protein kinase C, enhances the stimulatory effect of low concentrations of PMA on monocyte expression of tissue factor activity and blocks suppression of tissue factor activity at high PMA concentrations. Furthermore, staurosporine prolongs LPS-induced tissue factor expression in monocytes. These results suggest that protein kinases modulate tissue factor activity in human monocytes by regulating both induction and down-regulation.