The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.     

Mouse hepatitis virus receptor activities of an MHVR/mph chimera and MHVR mutants lacking N-linked glycosylation of the N-terminal domain

Mouse hepatitis virus binds to the N-terminal domain of its receptor, MHVR, a murine biliary glycoprotein with four immunoglobulin-like domains (G.S. Dveksler, M. N. Pensiero, C. W. Dieffenbach, C. B. Cardellichio, A.A. Basile, P.E. Elia, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 90:1716-1720, 1993). A recombinant protein with only the anchored N-terminal domain was not a functional receptor, but a recombinant protein with the N-terminal domain of MHVR linked to the second and third immunoglobulin-like domains and anchor from the mouse poliovirus receptor homolog, mph, was a functional receptor for mouse hepatitis virus. The native four-domain MHVR has 16 potential N-linked glycosylation sites, including three on the N-terminal domain. Recombinant proteins lacking each one of these three sites or all three of them were functional receptors. Thus, glycosylation of the N-terminal domain is not required, but a glycoprotein longer than the N-terminal domain is required for virus receptor activity.     

Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.     

Mutational analysis of the hepatitis C virus RNA helicase

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.     

Development of a monoclonal antibody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor

We measured dorsal hippocampal activity accompanying sighs and apnea using reflectance imaging and electrophysiologic measures in freely behaving cats. Reflected 660-nm light from a 1-mm2 area of CA1 was captured during sighs and apnea at 25 Hz through a coherent image conduit coupled to a charge coupled device camera. Sighs and apnea frequently coincided with state transitions. Thus, state transitions without apnea or sighs were separately assessed to control for state-related activity changes. All dorsal hippocampal sites showed discrete regions of activation and inactivation during transient respiratory events. Imaged hippocampal activity increased 1-3 s before the enhanced inspiratory effort associated with sighs, and before resumption of breathing after apnea. State transitions lacking sighs and apnea did not elicit analogous optical activity patterns. The suprasylvian cortex, a control for site, showed no significant overall reflectance changes during phasic respiratory events, and no discrete regions of activation or inactivation. Spectral estimates of hippocampal electroencephalographic activity from 0-12 Hz showed significantly increased power at 3-4 Hz rhythmical slow activity before sighs and apnea, and increased 5-6 Hz rhythmical slow activity power during apnea, before resumption of breathing. Imaged activity and broadband hippocampal electroencephalogram power decreased during sighs. We propose that increased hippocampal activity before sigh onset and apnea termination indicates a role for the hippocampus in initiating inspiratory effort during transient respiratory events.     

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor

1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.     

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.     

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13-36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50-65% in the CA3 stratum oriens 110-140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110-120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1-3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.     

A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.     

Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.     

Purification and proteic characterization of a Cuban strain of the hepatitis A virus

The testicular toxicities of gallium arsenide (GaAs), indium arsenide (InAs) and arsenic trioxide (As2O3) were examined by repetitive intratracheal instillation using hamsters. GaAs (7.7 mg/kg) and As2O3 (1.3 mg/kg) were instilled twice a week a total of 16 times and InAs (7.7 mg/kg) was instilled a total of 14 times. GaAs caused testicular spermatid retention and epididymal sperm reduction, though the degrees were less severe than those in rats shown in our previous experiment. InAs and As2O3 did not show any testicular toxicities. Serum arsenic concentration in GaAs-treated hamsters was less than half of that in As2O3-treated hamsters in which no testicular toxicities were found. Serum molar concentration of gallium was 32-times higher than that of arsenic in GaAs-treated hamsters. Therefore gallium may play a main role in the testicular toxicity of GaAs in hamsters.     

Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in a murine model

Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.     

A novel monoclonal antibody permitting recognition of NKT cells in various mouse strains

OBJECTIVES: The purpose of this study was to determine whether restrictive left ventricular (LV) filling patterns are associated with diastolic ventricular interaction in patients with chronic heart failure. BACKGROUND: We recently demonstrated a diastolic ventricular interaction in approximately 50% of a series of patients with chronic heart failure, as evidenced by paradoxic increases in LV end-diastolic volume despite reductions in right ventricular end-diastolic volume during volume unloading achieved by lower body negative pressure (LBNP). We reasoned that such an interaction would impede LV filling in mid and late diastole, but would be minimal in early diastole, resulting in a restrictive LV filling pattern. METHODS: Transmitral flow was assessed using pulsed wave Doppler echocardiography in 30 patients with chronic heart failure and an LV ejection fraction < or = 35%. Peak early (E) and atrial (A) filling velocities and E wave deceleration time were measured. Left ventricular end-diastolic volume was measured using radionuclide ventriculography before and during -30-mm Hg LBNP. RESULTS: Nine of the 11 patients with and 2 of the 16 patients without restrictive LV filling patterns (E/A > 2 or E/A 1 to 2 and E wave deceleration time < or = 140 ms) increased LV end-diastolic volume during LBNP (p = 0.001). The change in LV end-diastolic volume during LBNP was correlated with the baseline A wave velocity (r = -0.52, p = 0.005) and E/A ratio (r = 0.50, p = 0.01). CONCLUSIONS: Restrictive LV filling patterns are associated with diastolic ventricular interaction in patients with chronic heart failure. Volume unloading in the setting of diastolic ventricular interaction allows for increased LV filling. Identifying patients with chronic heart failure and restrictive filling patterns may therefore indicate a group likely to benefit from additional vasodilator therapy.     

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.     

Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor

Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,beta2 receptor-ligand interactions in the normal development of peripheral lymphoid organs and in triggering cytolytic activity and NF-kappaB activation in certain cell types, our finding implies that the HCV core protein may aggravate these biological functions of LT-betaR, resulting in pathogenesis in HCV-infected cells.     

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.     

A randomized, double blind, placebo controlled multicenter trial of murine anti-CD4 monoclonal antibody therapy in rheumatoid arthritis

To assess the feasibility and advantages of functional motion-mode (M-mode) sonography in pediatric patients with various non-cardiac M-mode applications, 50 patients aged 6 days to 14.5 years with megaureter, diaphragmatic palsy and spinal cord malformation were studied. In addition to initial conventional brightness-mode gray-scale ultrasound. M-mode sonography was performed for evaluation of motion and possible impairment. The findings were compared with other imaging results. The impact of the sonographic results on clinical management was evaluated retrospectively. Impaired motion was demonstrated by conventional sonography in all cases. Only M-mode sonography enabled adequate documentation for comparison with follow-up examinations, but allowed only semiquantitative assessment. Thus even gradual improvement or deterioration can be followed in an unbiased way by using a better-documented investigation for comparison, though an objective 'ranking', especially of diaphragmatic movement, could not be achieved. M-mode sonography enables a non-invasive and non-ionizing evaluation and documentation of motion impairment in pediatric patients. It improves documentation of motion and enables a better comparison with previous findings. It is especially useful for follow-up in evolving disease.     

Mutational analysis of the endothelin-B receptor gene in Japanese Hirschsprung's disease

BACKGROUND/PURPOSE: Hirschsprung's disease (HD, HSCR) is one of the most common diseases in the field of pediatric surgery. It is well known that the aganglionic bowel is primarily a causative factor of dismotility of distal narrow segment. Recent studies have shown that mutations in endothelin-B receptor (EDNRB), endothelin-3, RET, glial cell line-derived neurotrophic factor (GDNF) genes are responsible for the occurrence of congenital aganglionosis. Here, the authors describe two new mutations of the EDNRB gene in Japanese patients with HD. RESULTS: One patient had a heterozygous point mutation at the splice donor site of intron 3, leading to premature termination of translation of EDNRB mRNA. Another patient has a heterozygous missense mutation (N1041) in exon 1, but the same mutation was found in two of 50 normal individuals, so the mutation may be a noncausative polymorphism of the EDNRB gene. CONCLUSION: These results provide further evidence that a spectrum of different mutations within the EDNRB gene are responsible for HD.     

Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.