Protein-specific Monoclonal Antibodies Research Articles

Pre-operative chemotherapy for squamous cell carcinoma of the oesophagus: do histological assessment and p53 overexpression predict chemo-responsiveness?

Pre-operative chemotherapy is increasingly used in the treatment of oesophageal carcinoma. However, no features have been identified which can reliably predict a positive response to chemotherapy. The aim of this study was to examine whether histological features and p53 overexpression could predict such response. Prechemotherapy endoscopic biopsies from 55 patients, who subsequently completed two courses of chemotherapy followed by surgical resection, were studied. Patients were classified into responders and non-responders according to clinical and pathological findings. Pathological features of the endoscopic biopsies examined included adequacy of the tumour tissue, histological grade, degree of keratinisation, histologic patterns, mitotic rates and nuclear pleomorphism. Biopsy specimens were also tested for p53 overexpression using p53 protein specific mouse monoclonal antibody DO-7 on paraffin sections. Histologic features and p53 expression were correlated to chemoresponsiveness. 76% (42 of 55) of patients had sufficient biopsy tissue for assessment. Response to chemotherapy was evident in 64% (n = 27) of patients. None of the non-responders had tumours with high-grade nuclear pleomorphism compared with 37% (10 of 27) of responders (P = 0.01). All patients with high-grade nuclear pleomorphism responded to chemotherapy. No significant differences were found between the responders and non-responders with respect to tumour differentiation (P = 0.7), degree of keratinisation (P = 0.3) and mitotic rates (P = 0.8). Overall, p53 overexpression was noted in 67% (28 of 42) of patients. This was more prevalent in non-responders (12/15) compared to responders (16/27), but this was not statistically significant (P = 0.08). The degree of p53 overexpression had no significant relationship with responsiveness to chemotherapy. High-grade nuclear pleomorphism, identified on pretreatment biopsy specimens, correlated with response to chemotherapy, whereas p53 overexpression did not correlate with response. Improved tissue sampling and further investigations should be done so that the assessment of prechemotherapeutic endoscopic biopsies can have significant impact on clinical decision making.

Heparan sulfate proteoglycan expression in human lung-cancer cells.

Heparan sulfate (HS) functions as a co-factor in several signal-transduction systems that affect cellular growth, differentiation, adhesion and motility. HS, therefore, may also play a role in the malignant transformation of cells, tumor growth, cell invasiveness and the formation of tumor metastases. To explore this hypothesis, we analyzed the expression of HS and heparan sulfate proteoglycan (HSPG) in histological sections of human lung-cancer tissues and assayed for the presence of HSPGs in extracts of human lung-cancer cell lines, using a panel of native HS-, delta-HS- and HSPG (syndecan, glypican, CD44 and perlecan) core protein-specific monoclonal antibodies. Compared to normal epithelia, non-small-cell lung carcinomas, particularly poorly differentiated tumors, often expressed reduced amounts of the major cell surface-associated HSPGs (most consistently of syndecan-1). CD44 or CD44-variant proteins, in contrast, were found on all tumor cells, irrespective of their differentiation. Perlecan, a matrix-associated HSPG found in the basement membrane of normal bronchial epithelium, was consistently undetectable in invasive bronchogenic carcinomas. Staining reactions for native HS were consistently reduced in squamous-cell lung carcinomas, in the cells in contact with the stroma and in the less differentiated areas of these tumors. Reactions for delta-HS, however, were not reduced, suggesting a structural change in the HS of these tumor cells. Poorly differentiated adenocarcinomas, in contrast, yielded strong HS and delta-HS reactions. Marked differences in HSPG expression also were observed among various non-small-cell lung carcinoma cell lines. Our results suggest that poorly differentiated lung tumors have markedly altered patterns of HSPG expression, which may contribute to their invasive phenotype.

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants.

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.

Peptide mimics of a conformationally constrained protective epitopes of respiratory syncytial virus fusion protein

Aims: To identify peptides that mimic (mimotopes) conformational and protective epitopes of RSV fusion protein and to assess their efficacy as immunogens and potential vaccines. Material and methods: An 8-mer solid-phase (TG resin) library was screened with a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads, reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysed by amino acid replacement using the SPOTs method. Results: Mabs were not able to bind to the free and soluble peptides, nor did these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to produce de-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproduce this conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versions of the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance. Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibited the binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80–640); and (4) reduce significantly the viral load in the lungs of mice challenged with RSV ( P<0.01). Conclusions: This report demonstrates for the first time that: (1) a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with these mimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novel concept for the development of a vaccine against RSV.

Abundant expression of viral nucleoprotein mRNA and restricted translation of the corresponding viral protein in inclusion body polioencephalitis of canine distemper

Brain and other tissues of three dogs aged 4-21 months with inclusion body polioencephalitis caused by canine distemper virus (CDV) were examined for CDV nucleoprotein (N) antigen and mRNA distribution. Two animals (nos 3 and 1) had suddenly shown central nervous system (CNS) signs 4 days and 5 months, respectively, after vaccination with a modified live CDV vaccine; animal no. 2 had shown similar signs 4 weeks after vaccination with an unknown product. Lesions in the CNS, which were restricted to the grey matter, occurred most frequently in the diencephalon, mesencephalon, medulla oblongata and, in one animal, in the cerebral cortex. Changes were characterized by mild to moderate perivascular lymphohistiocytic cuffs, loss of neurons, neuronal necrosis, glial nodules, and oedema. Intranuclear and cytoplasmic inclusion bodies, especially prominent in neurons, were observed. By in-situ hybridization, CDV N mRNA expression was confirmed with a non-radioactively labelled N-specific mRNA probe. The corresponding RNA translation product was detected immunohistochemically with a proteinspecific monoclonal antibody. Viral antigen and mRNA were observed in the same cell types and brain compartments. However, the number of cells expressing N mRNA exceeded the number of cells containing viral antigen greatly in two animals and slightly in one. Some areas with abundant viral mRNA expression were almost completely devoid of viral antigen. mRNA and the corresponding translation product were demonstrated in neurons and less frequently in astrocytes, but not in perivascular inflammatory cells. It would appear that distemper inclusion-body polioencephalitis may be due to a non-productive CDV infection of neurons, characterized by abundant expression of CDV N mRNA and reduced translation of the corresponding viral protein. These findings suggest that in distemper the pathogenesis of grey-matter lesions differs substantially from that of white-matter lesions, which constitute the most common manifestation of distemper encephalitis.

Expression of p53 and its relationship with human papillomavirus in penile carcinomas

We studied p53 over-expression in a series of 42 primary penile carcinomas (seven verrucous carcinomas, 14 well-differentiated, 15 moderately-differentiated and six poorly-differentiated squamous cell carcinomas) from Chinese patients using the p53 protein specific mouse monoclonal antibody DO-7 on paraffin sections. p53 protein was detected in 40% (17 cases) of the tumours. The p53 staining was not observed in the penile warts (n = 6) and verrucous carcinomas (n = 7). Positive p53 staining was identified only in the less differentiated tumour cells in the periphery of the tumour cell nests in all the cases. The non-invasive dysplastic epithelium next to the tumours could also be positive for p53 protein (three out of 10 cases in which the dysplastic epithelium adjacent to the tumour was adequately sampled). Furthermore, 100% of the human papillomavirus (HPV)-positive cases showed positive p53 staining. It is concluded that p53 over-expression is present in penile squamous cell carcinomas and adjacent non-invasive tumour cells. An inverse correlation between HPV and p53 gene mutation is not observed in penile cancers.

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.

Preparation of a murine cell line which stably expresses human T lymphotropic virus type I (HTLV-I) env genome products

We prepared the murine myeloma cell line NS-1, which stably expressed the human T lymphotropic virus type I (HTLV-I) env gene. The plasmid BCMGEnv was constructed from the episomal vector BCMGSNeo, which was primarily derived from bovine papilloma virus. Transfected env expression was detected by Northern blotting, as well as by flow cytometry using envelope protein-specific monoclonal antibodies (mAb). Expression was detectable for at least seven months. The env transfectants induced syncytium formation which is characteristic of HTLV-I-infected cells, in the human uterine cervical cancer line, HeLa, and the rat cell line, XC. The requirement of envelope proteins for syncytium formation was confirmed by an inhibition assay with envelope protein-specific mAb. Therefore, env transfectants are not only stable, but also have its specific biological function. This system may be useful to analyze the initial steps of viral attachment to the cell surface and to search for the HTLV-I receptor.

Expression of the nucleoprotein of rabies virus in Escherichia coli and mapping of antigenic sites.

Investigations were performed to delineate the antigenic sites I and IV of rabies virus nucleoprotein (N), the former of which is well conserved among the rabies and rabies-related viruses. The N cDNA of the RC-HL strain was inserted into an expression vector pET3a, with which the E. coli BL21(DE3) was transformed. Upon induction with isopropyl-1-thio-beta-D-galactoside, the transformants produced a protein with a size (56 k-Da) almost identical to that of the authentic N protein. The protein also reacted with a panel of our N protein-specific monoclonal antibodies (N-MAbs) including the antibodies against the antigenic sites I and IV. By using the cDNA, various deletion mutants were generated and expressed in E. coli to examine the reactivity of mutant proteins with N-MAbs by Western blot analysis. Deletion of the C-terminal 67 amino acid residues did not abolish their reactivity with any of the N-MAbs specific for the sites I and IV. When 91 residues or more were deleted from the C-terminus, however, the protein lost the reactivity, indicating that the antigenic sites I and IV are mapped to a small region which is comprised of at most 24 amino acid residues from positions 360 to 383. Comparison of the 24-amino acid sequence with the corresponding region of N protein of several other Lyssavirus strains suggests that the antigenic site I is mapped to positions 360 to 369.

Expression of p53 in oesophageal squamous cell carcinoma in Hong Kong Chinese

We studied p53 overexpression in a series of 99 primary oesophageal squamous cell carcinomas (28 well-differentiated, 42 moderately-differentiated and 29 poorly-differentiated squamous cell carcinomas) from Chinese patients using the p53 protein specific mouse monoclonal antibody DO-7 on paraffin sections. The p53 protein was detected in 30% (30 cases) of the tumours. A significantly higher positive rate was noted in the poorly-differentiated tumours (11% for the well-differentiated, 31% for the moderately-differentiated and 48% for the poorly-differentiated tumours). In addition, strong positive p53 staining was identified only in the less differentiated tumour cells in the periphery of the tumour cell nests in all the cases and the expression was weaker in the better differentiated foci. The central keratinizing areas and the immediately adjacent tumour cells were always negative for p53. The adjacent normal oesophageal mucosa was all negative for p53 protein but the non-invasive dysplastic epithelium next to the tumours could also be strongly positive for p53 protein (four out of 14 cases in which the dysplastic epithelium adjacent to the tumour was adequately sampled). In two out of these four cases, the dysplastic epithelium showed staining for p53; even the adjacent invasive tumour was negative for p53. It is concluded that there is a strong relationship between p53 overexpression and tumour cell differentiation in oesophageal squamous carcinoma and overexpression of p53 can occur in non-invasive tumour cells.

Rotavirus serotypes and electropherotypes in Finland from 1986 to 1990.

Four epidemic seasons of rotaviruses were studied in Helsinki during 1986-1990. This is the first Scandinavian study, where both electropherotypes and serotypes are determined. Out of 5316 fecal specimens 769 (14.5%) rotavirus positive samples were detected by electron microscopy. Of these, 645 isolates (83.9%) gave a clear RNA pattern in gel electrophoresis and they clustered into 87 electropherotypes. An illustrative number of isolates representing each electropherotype (= E-type) was serotyped using VP7 protein-specific monoclonal antibodies for serotypes G1-G4 and without exceptions, within one E-type only a single serotype specificity was found. After establishment of the serotype of each E-type, the distribution of serotypes was scored as 61.2%, 2.0%, 0.5% and 29.8% for G1-G4, respectively; 6.5% remained untypable. Two seasons had one predominant E-type (Season 1, 1986-87, and Season 3, 1988-89, 84.2% and 80.6% of rotavirus positive samples, respectively). Both were followed by a season with no predominant E-type, but several minor E-types. Altogether, 5 short E-types (13/645 samples) with serotype G2 specificity were found, most of them occurring in Season 2. Only 2 E-types (3 samples) belonged to serotype G3. Group C rotavirus was found in 8 specimens. In this study a shift in serotypes, from G1 to G4, was observed in Finland in 1988/89; a similar shift was reported in many European countries at that time.

High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice.

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.

A protein-specific monoclonal antibody to rat liver beta 1--&gt;4 galactosyltransferase and its application to immunohistochemistry.

We have produced a new protein-specific monoclonal antibody (MAb) to rat liver beta 1-->4 galactosyltransferase. This MAb, GTL2, was selected as the most reactive IgG to a periodate-treated antigen. Antigen and protein specificities of GTL2 were verified by immunoblotting of a non-glycosylated recombinant protein of human galactosyltransferase and enzymatically deglycosylated rat galactosyltransferase. Using GTL2, an immunohistochemical study was done in rat liver, epididymis, and salivary glands. Intense staining was observed in Golgi areas of epididymal duct epithelial cells, and submandibular and sublingual acinar cells. Hepatocytes showed weaker staining. Immunoelectron microscopic observation revealed that the staining was exclusively localized in trans-Golgi membranes of these cells.

Mouse hepatitis virus infection utilizes more than one receptor and requires an additional cellular factor.

Mouse hepatitis virus (MHV) causes hepatitis, encephalomyelitis, respiratory and gastrointestinal ailments. Our laboratory has been particularly interested in the mechanism of neuropathogenesis of MHV. Different MHV strains have different capacities to cause central nervous system (CNS) infection. Among the neurotropic strains, there is also a large degree of variation in their ability to cause encephalitis or demyelination. Various virus variants have been obtained from the neurotropic MHV strains which show altered neuropathogenicity; for example, many of the variants which escape neutralization by the spike (S) protein-specific monoclonal antibodies differ from the parental viruses in not causing encephalitis, but still retain the ability to cause demyelination (Fleming et al., 1986; Dalziel et al., 1986). These variants have either point mutations or deletions in the viral spike protein (Gallagher et al., 1990; Wang et al., 1992). Since the S protein presumably interacts with the viral receptor on the surface of target cells, it is reasonable to assume that the difference in the viral neurotropism is caused by the variations in their ability to interact with the receptors. Therefore, it is conceivable that the MHV receptors in different cell types, e.g. neurons, astrocytes or oligodendrocytes, in CNS might be different.KeywordsViral ReceptorMouse Hepatitis VirusHoward Hughes Medical InstituteMouse Hepatitis Virus StrainMouse Hepatitis Virus InfectionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Evaluation of Urothelial Tamm-Horsfall Protein and Serum Antibody as a Potential Diagnostic Marker for Interstitial Cystitis

Demonstration of adherence of Tamm-Horsfall protein to bladder epithelium has been suggested as a potential diagnostic test for interstitial cystitis. Bladder specimens from 18 interstitial cystitis patients were evaluated by indirect immunoperoxidase techniques using a Tamm-Horsfall protein specific monoclonal antibody to determine the diagnostic value of the staining results. The study population consisted of 7 severely diseased patients who required cystectomy with urinary diversion and 11 other patients meeting National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria for interstitial cystitis. We were unable to detect intraepithelial or surface-bound Tamm-Horsfall protein in any of the biopsy tissues. Human kidney tissue, similarly fixed and processed, consistently demonstrated Tamm-Horsfall protein staining of the kidney tubules. The monoclonal antibody also reacted on Western blots against urinary Tamm-Horsfall protein. Although antibody (α-Tamm-Horsfall protein) reactivity was measured by enzyme-linked immunosorbent assay in sera from interstitial cystitis patients, the titers did not differ statistically from those measured in sera from those without interstitial cystitis. Together, these results make it unlikely that immunohistochemical detection of Tamm-Horsfall protein will have diagnostic value in interstitial cystitis. Whether Tamm-Horsfall protein has a role in the pathogenic processes involved in this disease is not yet known. These findings do not eliminate the possibility that some interstitial cystitis patients will have abnormalities associated with the biochemical and physiological functions of Tamm-Horsfall protein.

In vitro induction of interferon-like activity from rainbow trout leucocytes stimulated by Egtved virus

Interferon (IFN)-like activity was induced in vitro from blood and kidney leucocytes by infectious or inactivated Egtved virus (VHSV) serotype 1. The IFN activity in the cell culture medium was determined by spectrophotometric assessment of its ability to protect RTG-2 cells against infectious pancreatic necrosis virus infection (virus strain VR299). Characteristics of this protective activity were consistent with those described for IFN α/β of mammals. IFN synthesis was induced by either infectious or inactivated virus. The yield of IFN depended on cell origin (blood or kidney) and donor, while temperature elevation enhanced the kinetics of this synthesis. Induction was abolished by treatment of glutaraldehyde-fixed VHSV-infected EPC cells with a neutralising anti-G protein specific monoclonal antibody (MAb), whereas MAbs specific or other viral proteins had no effect. This suggests that the induction of IFN synthesis in trout could be independent, in part, from Egtved virus multiplication and thus could be triggered by contact between target fish cells and one of the virus surface components. Such a mechanism, acting immediately after the virus entry into the fish, might be involved in the virulence. Passive transfer of IFN-like activity to trout alevins resulted in protection against water borne challenge with Egtved virus. The present results encourage further investigations on the role played by IFN in the pathogenesis of viral haemorrhagic septicaemia.

Localization and ontogeny of growth hormone receptor gene expression in the central nervous system

There is literature evidence that both growth hormone (GH) and its mediator, insulin-like growth factor 1 (IGF-1), are able to act upon neuronal and glial cells in the brain. We report here the location of the GH receptor in the brain of the rat and rabbit. Receptor distribution was determined by immunohistochemistry with GH receptor/binding protein (BP) specific monoclonal antibodies and by in situ hybridization with a [ 35S]riboprobe. GH receptor/BP immunoreactivity in the rat was most prominent in the neonate and declined with postnatal age. Receptor immunoreactivity was generalised with variation in immunoreactivity in regional areas. In the rat, strongest immunoreactivity was seen in layers 2, 3, 5 and especially layer 6 of the cerebral cortex, in neurones of the thalamus and hypothalamus, in Purkinje cells of the cerebellum, in neurones of the trapezoid body of the brainstem, and in retinal ganglion cells. Glial cells, notably astrocytes were also strongly reactive, along with ependyma of the choroid plexus, ventricular lining and pia mater. In the neonatal rabbit, strongest immunoreactivity was evident in layers 2 and 3 of the cerebral cortex, in pyramidal cells of the hippocampus, and in neurones of the inferior and superior colliculi, brain stem reticular formation, dorsal thalamus and hypothalamus. A similar distribution of GH receptor mRNA was seen by in situ hybridization. The ontogeny of GH receptor/BP mRNA in whole rat brain was quantified by solution hybridization-RNAse protection assay. Contrary to its ontogeny in the liver ( Endocrinology, 113 (1983) 1325–1329) receptor mRNA decreased with postnatal age. This is the first description of divergent tissue specific ontogeny for the GH receptor/BP, and raises the possibility of a role for GH in influencing neuronal maturation and glial cell formation. Since there is evidence for GH synthesis within the brain, and receptor distribution generally correlates with IGF-1 expression, our findings raise the possibility of an endogenous GH-IGF-1 axis involved in brain growth and maturation.

Subcellular Localization of the 54-kDa Antigen of Toxoplasma gondii

A 54-kDa protein antigen of Toxoplasma gondii recently was cloned and expressed in Escherichia coli and shown to display immunoprotective properties. To determine the subcellular localization of this antigen, a fusion protein containing the 330 carboxy-terminal residues of the sequence coded by cDNA clone Tg34 was expressed in E. coli, purified, and used to raise antibodies in mice. Western blot analysis confirmed that the resulting antibody reacted with the 54-kDa antigen of T. gondii. Immunofluorescence and immunoelectron-microscopy showed that the antibody reacted with an antigen localized in the rhoptries, 1 of the organelles of the apical complex of the zoites involved in host cell invasion. Western blot studies using a recombinant fusion protein containing the full amino acid sequence encoded by the cDNA clone Tg34 and rhoptry protein-specific monoclonal antibodies (mAbs) previously described showed a reactivity of the recombinant antigen with 2 mAbs (T4 2F8 and T5 2D1) specific for the ROP2 protein, and with a mAb (T3 4A7) directed against an epitope shared by ROP2 and ROP4, but not with a mAb (T2 2H3) specific for the ROP4 protein. We thus conclude that the 54-kDa T. gondii antigen encoded by cDNA clone Tg34 is the previously described rhoptry protein ROP2.

Purification and characterization of the DNA-binding activity of the Epstein-Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) BMRF1 gene product, the EBV DNA polymerase accessory protein, under the control of the polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced two phosphoproteins of 52 and 50 kDa and one unphosphorylated protein of 48 kDa, recognized by anti-BMRF1 protein-specific monoclonal antibody. The major protein bands were 50 and 48 kDa. The expressed BMRF1 gene products were purified to near homogeneity from the nuclear extract of the recombinant baculovirus-infected insect cells by double-stranded DNA-cellulose column chromatography followed by heparin-agarose column chromatography. The purified BMRF1 gene products exhibited higher binding affinity for double-stranded DNA than for single-stranded DNA without ATP hydrolysis. The protein-DNA interaction did not necessarily require a primer terminus. The present system will open the way for the biochemical characterization of the EBV DNA polymerase accessory protein.

Monoclonal Antibody Epitope Mapping of Plasmodium falciparum Rhoptry Proteins

Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA2 from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 1l0-kDa protein. Furthermore, PLA2 activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA2 is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [3H] palmitate and [3H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [35S] methionine-labeled proteins were labeled with [3H]palmitate in ring-infected erythrocyte membranes. However, these proteins were not immunoprecipitated by a rhoptry protein-specific monoclonal antibody, 1B9. Similar label incorporation was not obtained with [3H]myristate. In Triton X-l14 solubility studies, the HMWC proteins partitioned into the aqueous phase, suggesting that they are not integral membrane proteins. In addition, the proteins were extracted by 100 mM Na2CO3, pH 11.5, and immunoprecipitated by rhoptry-specific antibody. These results suggest that the HMWC proteins may exist in a soluble and membrane bound form. The latter may participate in membrane expansion and the formation of the parasitophorous vacuole during merozoite invasion.