Biomedical subjects

Z Yao

Publications and source records attributed to Z Yao.

At least 217 records · Page 12Linked to original sources

PubMed1994

Human recombinant TNF-alpha on localization of 90Y-radioantibody in human tumour xenografts.

The effect of recombinant human tumour necrotic factor-alpha (TNF-alpha) on the intratumour and whole-body distributions of 90Y-labelled C110 anticarcinoembryonic antigen (CEA) monoclonal antibody (MAb) was studied using nude mice bearing two different tumours. The nude mice were injected subcutaneously with the CEA-positive LS174T colorectal cancer xenograft and the CEA-negative H-MESO-1 malignant mesothelioma xenograft. One hour before injection of radiolabelled MAb, mice were injected intravenously with human recombinant TNF-alpha (3 mg per mouse) or saline, and biodistributions of radiolabel were determined by tissue counting and whole-body autoradiography (ARG). Twenty-four hours after injection, TNF-alpha administration increased radioactivity in the LS174T tumour by 57% (17.30 +/- 1.61 vs. 9.83 +/- 1.55% ID g-1, P < 0.01), while decreasing radioactivity in blood and other normal organs. Diminished but similar effects on radioantibody biodistribution were seen at 48 and 72 hours. TNF-alpha did not affect specific MAb localization in the control H-MESO xenograft. Tumour:blood ratios were increased from 0.7 to 1.7 at 24 h with TNF-alpha administration. Pretreatment with TNF-alpha may be of value in increasing specific localization of monoclonal antibodies in tumour tissue.

Animals↗

PubMed1994

Monophosphoryl lipid A protects against endotoxic shock via inhibiting neutrophil infiltration and preventing disseminated intravascular coagulation.

The major objective of the present study was to determine the effects of a partial structure of the lipid A moiety of gram-negative lipopolysaccharide, monophosphoryl lipid A (MLA), on endotoxin-induced mortality and disseminated intravascular coagulation (DIC) in rats. A second objective was to examine the role of polymorphonuclear neutrophil invasion to visceral organs, including lung, liver, heart, and kidney in the pathogenesis of the compromised multiorgan function which occurs in endotoxic shock. Finally, a third aim was to determine if the potential protective effects of MLA might be mediated via inhibiting neutrophil invasion to various visceral organs. Male Sprague-Dawley rats (220-260 g) were fasted over night and used the following day. In control rats, endotoxin (S. abortus equi LPS, 15 mg/kg, i.v.) produced a 89% mortality at 48 hr following its administration, and gross pathological and laboratory signs of DIC at 3 hr after injection. The latter included increased serum fibrin(ogen) degradation products (FDP, 24.00 +/- 7.81 vs. 0 micrograms/ml, P < .05), prothrombin time (PT, 16.20 +/- 1.12 vs. 13.03 +/- 0.20 sec, P < .05), and activated partial thromboplastin time (APTT, 32.70 +/- 3.83 vs. 20.11 +/- 0.60 sec, P < .05), and decreased plasma fibrinogen (233.2 +/- 41.6 vs. 406.3 +/- 23.2 mg/dl, P < .05) as well as evidence of gross visceral hemorrhage. Pretreatment with MLA (5 mg/kg) for 24 hr produced a marked reduction in endotoxin-induced mortality at 48 hr (0% versus 89% in controls) and inhibited all of the manifestations of DIC produced by endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The ATP-dependent potassium channel: an endogenous cardioprotective mechanism.

The major objectives of the present study were to examine the effects of ischemic preconditioning and two endogenous substances, adenosine and acetylcholine (ACh), on myocardial infarct size to determine the role of the cardiac KATP channel in mediating these effects. Barbital-anesthetized open-chest dogs subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion were used. Preconditioning elicited by 10 min of LAD occlusion or intracoronary infusion of adenosine (400 micrograms/min) or ACh (10 micrograms/min) for 10 min, followed by 10 min of reperfusion or drug-free period before the 60-min occlusion period, markedly reduced myocardial infarct size to a similar extent compared to that in control dogs that were infused with an equivalent volume of saline into the LAD for 10 min followed by a 10-min saline-free period before the 60-min ischemic insult. The infarct size-limiting effects of these three endogenous interventions were totally abolished by the specific KATP-channel blockers glibenclamide or 5-hydroxydecanoate. These results indicate that cardiac KATP-channel activation is an important endogenous protectant against ischemia-reperfusion injury.

Acetylcholine↗

PubMed1994

Increased production of interleukin 4 in children with simple idiopathic nephrotic syndrome.

Sera and peripheral blood mononuclear cells (PBMC) were collected from 42 children with simple idiopathic nephrotic syndrome (INS) aged from 1.5 to 14 years and 28 age and sex-matched healthy children. The levels of IgE in serum and interleukin 4 (IL-4) in supernatant of PHA-activated PBMC were tested by ELISA. A significantly high incidence of allergic diseases was noted in INS children and their first-degree relatives, and there was a close association between atopy and INS. Besides, the levels of IgE and IL-4 were increased in nephrotic state (P < 0.001 both). IgE normal and IL-4 slightly lower in remission state (P < 0.2, P < 0.01 respectively). A positive correlation was noticed between IL-4 and IgE, and between IL-4 and 24-hour proteinuria (r = 0.472, r = 0.562; P < 005, P < 0.01 respectively). These showed a close association between atopy and INS. Both IL-4 and IgE, the important mediators in atopy, showed abnormal change in patients. The increased IL-4 production of T cells might account for the elevated serum IgE level. IL-4 may have a pathogenetic role in INS, but it still remains speculative.

Adolescent↗

PubMed1993

Expression of human apolipoprotein B100 in transgenic mice. Editing of human apolipoprotein B100 mRNA.

Apolipoprotein B (apoB) is a large glycoprotein that circulates in plasma as a major constituent of numerous lipoproteins. ApoB exists in two forms: apoB48 and apoB100. ApoB48 is identical in sequence to the N-terminal region of apoB100 and is generated by sequence-specific mRNA editing of the apoB100 transcript. Here, we describe the development of a line of mice expressing a human apoB transgene driven by promoter/enhancer sequences from the transthyretin gene. In these mice, immunodetectable human apoB100 is synthesized by the liver, kidney, and brain. Human apoB100 is found in low concentration (approximately 0.1 mg/dl) in the plasma of the transgenic mice and circulates in the low density lipoprotein fraction. The hepatic human apoB100 transcripts undergo mRNA editing at only slightly lower efficiency than the endogenous mouse apoB100 message. Therefore, there is no absolute species specificity to the apoB100 mRNA editing process.

Animals↗

PubMed1993

Hepatic polysomes that contain apoprotein B mRNA have unusual physical properties.

To characterize the association of the mRNA for apoprotein B (apoB) with ribosomes, rat hepatic cytoplasmic extracts were fractionated by density gradient centrifugation. On linear sucrose gradients, the sedimentation velocity of the 14.4-kilobase apoB mRNA was retarded compared to the mRNAs for other hepatic proteins, which were concentrated in fractions containing the bulk of the polysomes. This unusual distribution of apoB mRNA could not be explained by cotranslational association of nascent apoB peptides with lipids, based on experiments using either detergents to delipidate proteins or puromycin to release nascent peptides from polysomes. The results were also not the result of the editing of apoB mRNA, since the sucrose gradient distributions of both edited and nonedited forms were similar. In contrast, the distribution of a 3'-truncated apoB mRNA (apoB-42, 5.8 kilobases) expressed in rat hepatoma cells resembled that of mRNA of a typical hepatic protein. As opposed to the sedimentation velocity results, on equilibrium density gradients most hepatic apoB mRNA was found in the fraction that contained polysomes. Based on these data, the elongation rate of nascent apoB, and the calculated translational yield of apoB mRNA, we conclude that the majority of rat hepatic apoB mRNA must be part of polysomal complexes with unusual physical properties related to the presence of sequence(s) in the 3'-region of the message. These sequences may either be primary determinants of structural features or binding sites for protein factors that effect conformational changes.

Animals↗

PubMed1993

Long-term reversal of diabetes by the injection of immunoprotected islets.

The intraperitoneal injection of insulin-producing islets immunoprotected by an alginate-poly(amino acid) membrane is a potential method of reversing diabetes without the need for lifelong immunosuppression. Previous attempts to demonstrate this technology in large animals have failed, preventing application in humans. We have determined that key factors responsible for these past failures include cytokine (interleukins 1 and 6 and tumor necrosis factor) stimulation by mannuronic acid monomers from alginate capsules with weak mechanical integrity, which results in fibroblast proliferation. With this insight, we formulated mechanically stable microcapsules by using alginate high in guluronic acid content and report prolonged reversal of diabetes in the spontaneous diabetic dog model by the intraperitoneal injection of encapsulated canine islet allografts. Euglycemia, independent of any exogenous insulin requirement, was noted for up to 172 days. Graft survival, evidenced by positive C-peptide release, was noted for as long as 726 days in a recipient receiving a single injection of immunoprotected islets. Histological evidence of viable islets retrieved from the peritoneal cavity 6 months posttransplant confirmed the biocompatibility and immunoprotective nature of this capsule formulation. The finding that intraperitoneal injection of alginate-immunoprotected islets, a minimally invasive surgical procedure, is effective in prolonged (> 1 year) maintenance of glycemic control, without the need for lifelong immunosuppression, may have significant implications for the future therapy of type I diabetes in humans.

Alginates↗

PubMed1993

Studies directed toward the design of orally active renin inhibitors. 1. Some factors influencing the absorption of small peptides.

A systematic evaluation of structure-absorption relationships using a high throughput intraduodenal rat screening model has led to the delineation of a set of structural parameters that appear to govern bioavailability in a series of peptide-based renin inhibitors. Optimum structures, exemplified by 25 and 41, incorporated a single, solubilizing substituent at the C- or N-terminus combined with a lipophilic P2-site residue. Both inhibitors gave unprecedented plasma drug levels upon intraduodenal administration to monkeys, and the calculated bioavailability for 41 (14 +/- 4%) is the highest reported for any peptidic renin inhibitor.

Animals↗

PubMed1993

Studies directed toward the design of orally active renin inhibitors. 2. Development of the efficacious, bioavailable renin inhibitor (2S)-2-benzyl-3- [[(1-methylpiperazin-4-yl)sulfonyl]propionyl]-3-thiazol-4-yl-L-alanine amide of (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane (A-72517).

Employing a set of empirical guidelines for the design of well-absorbed renin inhibitors, we have followed two strategies to improve potency while maintaining bioavailability. One process involved incorporation of an extended N-terminal residue bearing a weakly basic substituent and is exemplified by compound 25. The other approach centered on the inclusion of an N-terminal sulfonamide and culminated in the discovery of inhibitor 32 (A-72517). Both 25 and 32 showed excellent bioavailability in the rat and ferret (> 25%) and, while subject to hepatic elimination in the monkey, were efficacious in this species.

Animals↗

PubMed1993

Polymorphism of the DQA1 promoter region (QAP) and DRB1, QAP, DQA1, DQB1 haplotypes in systemic lupus erythematosus. SLE Study Group members.

We have investigated the DNA polymorphism for the DQA1 promoter region (QAP) and HLA-class II DRB1, DQA1, and DQB1 genes in 178 central European patients with Systemic lupus erythematosus (SLE) using polymerase chain reaction and Dig-ddUTP labeled oligonucleotides. Increased frequencies of DRB1*02 and *03 are confirmed by DNA typing. In addition, the frequencies of DQA1*0501, *0102 and DQB1*0201, *0602 alleles are increased in the patients as compared to controls. The strongest association to SLE is found with DRB1*03 and DOB1*0201 alleles (p < 10(-7), p corr. < 10(-5) and p < 10(-6), p corr. < 10(-4), respectively). By investigating the DQA1 promoter region in the SLE patients we have detected nine different QAP variants. Increased frequencies of QAP1.2 and QAP4.1 are observed in patients as compared to controls (p < 0.05, p corr. = n.s.). Analysis of linkage disequilibria demonstrates a very strong association between QAP variants and DQA1, DRB1 alleles. Certain QAP variants are completely associated with DQA1 and DRB1 alleles, whereas others can combine with different DQA1 and DRB1 alleles. All DRB1*02-positive patients and controls carry QAP1.2, and all DRB1*03-positive patients and controls carry QAP4.1. Conversely, the QAP1.2 variant appears only in DRB1*02 haplotypes, while the QAP4.1 variant can be observed in DRB1*03, *11, and *1303 haplotypes. Based on the strong linkage disequilibria between DRB1-DQA1-DQB1 genes and between DRB1-QAP-DQA1, we have deduced the four-point haplotypes for DRB1-QAP-DQA1-DQB1 in patients and controls. Two haplotypes DRB1*02-QAP1.2-DQA1*0102-DQB1*0602 and DRB1*03-QAP4.1-DQA1*0501-DQB1*0201 are significantly increased in patients as compared to controls (p < 0.01, p corr. = n.s., RR = 1.8 and p < 10(-7), p corr. < 10(-5), RR = 3.1, respectively). The analysis of relative risks attributed to the various alleles of QAP, DQA1, and DQB1 as well as the investigation of the deduced DRB1-QAP-DQA1-DQB1 haplotypes leads to the conclusion that QAP4.1 and DQA1*0501 on the DR3 haplotypes are probably not involved in SLE susceptibility.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗

PubMed1993

HLA-DP/DR interaction in early onset pauciarticular juvenile chronic arthritis.

We investigated the polymorphic second exon of the HLA-DPB1 and HLA-DRB1 genes, using in vitro DNA amplification by polymerase chain reaction (PCR) and oligonucleotide hybridization in 136 patients with early onset pauciarticular juvenile chronic arthritis (EOPA-JCA) and 199 healthy controls. The analysis of the HLA-DRB1 system revealed that most of the DRB1 alleles are not indifferent with respect to susceptibility to EOPA-JCA. There is a hierarchy of susceptible (DRB1*08, DR5), "permissive" (DRB1*01), moderately "protective" (DR2, DRB1*04), and "protective" (DRB1*07) alleles. In contrast, no hierarchy could be shown for the HLA-DPB1 system. DPB1*0201 was found to be susceptible. The relatively frequent alleles DPB1*0402 and DPB1*0401 seem to be indifferent. The associations with DPB1*0201, DR5, and DRB1*08 are independent of each other: that is to say they, are not brought about by linkage disequilibrium. The susceptible alleles DPB1*0201 and DR5 show evidence for interaction in the pathogenesis of EOPA-JCA. Interaction seems likely between DPB1*0201 and DRB1*08, DR5 and DRB1*08, or between DR6 and DRB1*08. The strongest interaction exists between DPB1*0201 and a common DQ factor associated with both DR5 and DRB1*08. Finally, we observed a hierarchy among the various marker combinations, where the risk of developing EOPA-JCA increases with the number of associated markers present in an individual.

Alleles↗

PubMed1993

No direct correlation between HLA-DPB1 and antibodies against recombinant Ro (SS-A)/La (SS-B) proteins in systemic lupus erythematosus. SLE Study Group.

We investigated the association of HLA-DPB1 alleles with the occurrence of autoantibodies against Ro (SS-A) or La (SS-B) using recombinant 52 kD-Ro, 60 kD-Ro and La proteins in 177 German patients with systemic lupus erythematosus (SLE). A significant increase in the frequency of DPB1*0101 is observed in SLE patients compared to healthy controls (Pcorr.. < 0.004). Antibodies against 52 kD-Ro, 60 kD-Ro and La are tested by ELISA and are found with a frequency of 25.4%, 33.9% and 17.5% in the patients, respectively. An association with HLA-DPB1*0101 is observed for antibodies against La (P < 0.01) and 52 kD-Ro (P < 0.01), but not for 60 kD-Ro in the absence of La/52 kD-Ro. Since there is a strong linkage disequilibrium between DPB1*0101 and DR3 in the normal population and in SLE patients, and since there is an association between DR3 and SLE, as well as between DR3 and the occurrence of recombinant Ro/La antibodies in SLE patients, we investigated whether DPB1*0101 is associated per se or via linkage disequilibrium with DR3. DPB1*0101 in the absence of DR3 is not more common in patients than in controls and not in patients with autoantibodies to Ro and La than without autoantibodies. We conclude that there is no evidence for a direct involvement of DPB1*0101 in the production of Ro/La autoantibodies in SLE patients.

Alleles↗

PubMed1993

Radioimmunotherapy for unresectable hepatocellular carcinoma using 131I-Hepama-1 mAb: preliminary results.

Twenty-three patients with surgically verified unresectable hepatocellular carcinoma (HCC) have been treated by intrahepatic arterial administration of 131I-labeled anti-HCC monoclonal antibody (Hepama-1) combined with hepatic artery ligation. Radioimmunoimaging demonstrated that the median tumor/liver ratio was 2.1 (1.1-3.6) at day 5. A decline in alpha-fetoprotein level and shrinkage of tumor were observed in 75% (12/16) and 78% (18/23) of patients respectively. Sequential resection was done in 11 patients (48%) after treatment. The surgical specimens revealed massive necrosis of tumor, but residual cancer cells were found at the edge of the specimens. Anti-antibody was determined in 43% (10/23) of patients 2-4 weeks after the administration of 131I-Hepama-1 mAb. No marked toxic effects were noted. It is suggested that 131I-Hepama-1 mAb might be of value as one of the multimodality treatments for unresectable HCC.

Animals↗

PubMed1993

Cardioprotective effects of monophosphoryl lipid A, a novel endotoxin analogue, in the dog.

OBJECTIVE: The major objective of the present study was to determine the effects of a new endotoxin analogue, monophosphoryl lipid A (MLA), on myocardial infarct size in dogs. A second aim was to determine if potential cardioprotective effects of MLA might be mediated via an enhancement of antioxidant defence mechanisms. METHODS: Barbiturate anaesthetised dogs were subjected to 60 min left circumflex coronary artery occlusion followed by 5 h reperfusion. Either of two different doses of MLA (30 and 100 micrograms.kg-1) or an equivalent volume of vehicle were given intravenously 24 h prior to the infarct experiments. Transmural myocardial blood flow was measured at 30 min of occlusion by the radioactive microsphere technique and infarct size was determined at the end of 5 h of reperfusion by triphenyltetrazolium staining. Tissue catalase and myeloperoxidase activities were measured at 5 h of reperfusion as indices of antioxidant activity and neutrophil infiltration, respectively. RESULTS: There were no significant differences between groups in systemic haemodynamic variables, myocardial oxygen demand, ischaemic bed size, or coronary and collateral blood flow to the ischaemic region. However, administration of MLA produced a marked dose dependent reduction in myocardial infarct size: 19.8(SEM 3.7)% and 14.1(2.5)%, respectively, v 32.7(2.9)% in the vehicle control group, p < 0.05. Pretreatment with either 30 or 100 micrograms.kg-1 of MLA resulted in small increases in tissue catalase activity in the non-ischaemic region of the heart: 0.169(0.033) and 0.197(0.013) K.g-1, respectively, v 0.136(0.013) K.g-1 tissue in the control; however, the increases were not statistically significant by ANOVA. Myeloperoxidase activity in the border zone immediately adjacent to the infarct was markedly decreased in both MLA treated groups: MLA 30 micrograms.kg-1, 2.69(0.82); MLA 100 micrograms.kg-1, 2.49(0.47), v control group, 5.81(1.20) units.g-1 tissue; p < 0.05. CONCLUSIONS: These data are the first to show a marked cardioprotective effect of a lipid A derivative of endotoxin in an in vivo model of myocardial infarction. Although the mechanism responsible for the reduction in infarct size by MLA is unknown, a reduction in neutrophil migration at the site of ongoing tissue injury, the border zone, may be partially responsible.

Animals↗

PubMed1993

Effects of monophosphoryl lipid A on myocardial ischemia/reperfusion injury in dogs.

We wished to determine if the previously observed cardioprotective effects of monophosphoryl lipid A (MLA, 65 micrograms/kg intravenously, i.v.), an endotoxin derivative, were time related and mediated by an enhancement of antioxidant defense mechanisms, i.e., myocardial catalase and superoxide dismutase (SOD) activities and neutrophil infiltration as assessed by myeloperoxidase (MPO) activity. We also wished to study the effect of pretreatment with MLA on vascular endothelial and smooth muscle function in vivo and in vitro. Barbital-anesthetized dogs were subjected to 60-min left circumflex coronary artery (LCX) occlusion followed by 5-h reperfusion. Myocardial catalase, SOD, and MPO activities were measured at the end of 5-h reperfusion. Pretreatment with MLA 24 h before ischemia produced a significant reduction in myocardial infarct size as measured by triphenyltetrazolium staining (15.3 +/- 4.4 vs. 30.9 +/- 5.2% in controls, p < 0.05), but 1-h pretreatment with MLA had no protective effect. MLA pretreatment for 24 h resulted in marked reduction (p < 0.05) in MPO activity in the border zone surrounding the infarct. Although a trend indicated an increase in catalase activity in the 24-h pretreatment group, no significant changes were observed in either catalase or SOD activities among the three groups. The cardioprotection produced by MLA was independent of differences in collateral blood flow to the ischemic region assessed by radioactive microsphere technique, systemic hemodynamics, myocardial oxygen demand, and ischemic bed size. Responses of the LCX bed to intracoronary acetylcholine (ACh) or nitroglycerin (NTG) in vivo and responses of isolated femoral artery rings to the endothelium-dependent vasodilators, ACh, A23187, bradykinin, or the nonendothelium-dependent vasodilator, sodium nitroprusside (SNP) in vitro were significantly decreased in the MLA 1-h pretreatment group but not in the 24-h pretreatment group. Incubation of the femoral artery rings from the MLA 1-h pretreatment group with 3 mM L-arginine for 1 h reversed the decreased endothelium-dependent responses to ACh and A23187, but not those to bradykinin. These results indicate that (a) the MLA-induced myocardial infarct size reduction was pronounced when MLA was administered for 24 h but was not evident at 1-h pretreatment; (b) a decrease in neutrophil infiltration into the site of ongoing tissue damage might be partially responsible for the protection; (c) vascular endothelial and/or smooth muscle function were transiently decreased by MLA administration and returned to nearly normal levels 24 h after treatment; and (d) the effect of MLA on endothelium-dependent responses might be mediated by the L-arginine/nitric oxide (NO) pathway.

Animals↗

PubMed1993

DNA typing for HLA-DPB1-alleles in German patients with systemic lupus erythematosus using the polymerase chain reaction and DIG-ddUTP-labelled oligonucleotide probes. Members of SLE Study Group.

Genomic DNA of 178 German Caucasian patients with systemic lupus erythematosus are studied for HLA-DP locus by using PCR and DIG-ddUTP-labelled oligonucleotide probes. A significant increase of DPB1*0101 is observed in SLE patients compared with healthy controls (chi 2 = 15.27, p.c. < 0.004). DPB1*0501 and *0901 are also slightly increased (chi 2 = 5.85, P < 0.05, p.c. = NS; chi 2 = 5.64, P < 0.05, p.c. = NS). There is no significant difference in frequency of DP alleles between male and female patients. Since a linkage disequilibrium between HLA-B, DR and DP loci is found in our SLE patients, an analysis is performed assessing the relative importance of these HLA-markers to SLE. The results show that the increase of DPB1*0101 in SLE patients is associated with the HLA-B8, DR3 haplotype and it suggests a more important role for HLA-B8, DR3 or genes within this haplotype than for DPB1*0101 in the genetic predisposition for SLE.

Alleles↗

PubMed1993

Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system.

The unique short region of the varicella-zoster virus (VZV) genome contains two open reading frames which encode glycoproteins designated gpI and gpIV (herpes simplex virus homologs gE and gI, respectively). Like its herpesviral counterpart gE, the VZV gpI gene product functions as a cell surface receptor (V. Litwin, W. Jackson, and C. Grose, J. Virol. 66:3643-3651, 1992). To evaluate the biosynthesis of the two VZV glycoproteins and further explore their relationship to one another, the two glycoprotein genes were individually cloned into a pTM1 vector under control of the T7 promoter. Transfection of the cloned gpI or gpIV construct into HeLa cells previously infected with vaccinia recombinant virus expressing bacteriophage T7 polymerase resulted in a much higher level expression of each VZV glycoprotein than previously achieved. Synthesis of both gpI and gpIV included intermediary partially glycosylated forms and mature N- and O-linked final product. Transfections in the presence of 32Pi demonstrated that the mature forms of both gpI and gpIV were phosphorylated, while similar experiments with [35S]sulfate showed that only the mature gpI was sulfated. When gpI and gpIV were coexpressed in the same cell, the two glycoproteins were complexed to each other, as both proteins could be immunoprecipitated by antibodies against either gpI or gpIV. Coprecipitation did not occur as a result of a shared epitope, because gpI expressed alone was not precipitated by antibody to gpIV, and gpIV expressed alone was not precipitated by antibody to gpI. Pulse-chase analysis demonstrated that the gpI-gpIV association occurred early in processing; furthermore, this complex formation interfered with posttranslational modifications and thereby reduced the M(r)s of the mature forms of both gpI and gpIV. Similarly, the molecular masses of the cotransfected gene products corresponded with those of the infected cell glycoproteins, a result which suggested that authentic gpI and gpIV were ordinarily found within a complex. Thus, the adjacent open reading frames 67 and 68 code for two glycoproteins which in turn form a distinctive sulfated and phosphorylated cell surface complex with receptor properties.

Antibodies, Monoclonal↗

PubMed1993

Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.

Amino Acid Sequence↗