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Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor beta chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24(+) fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV(+) gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I-restricted, antigen-specific CD8(+) CTLs are specifically expanded within EBV(+) gastric carcinoma tissue.

A novel interleukin-2 (IL-2)-dependent cell line, HANK1, was established from a patient with CD56+ NK/T-cell lymphoma arising in the retroperitoneum. Morphologically, HANK1 is a pleomorphic large cell line with irregular nuclei, which contains azurophilic granules in the cytoplasm. Immunophenotypic analysis showed that HANK1 expressed CD2, CD3epsilon, CD56, TIA-1, granzyme B, and HLA-DR, but no other T-lineage markers. These features were the same as seen in the original tumour, and are highly characteristic of nasal and 'nasal-type' NK/T-cell lymphoma as described in the proposed W.H.O. classification. Genotypically, this cell line also demonstrated the germline configuration of the T-cell receptor beta, gamma and the immunoglobulin heavy chain genes and clonal integration of the Epstein-Barr virus (EBV) together with antigen expression with a type II latency pattern (LMP-1+ and EBNA2-). Furthermore, Southern blot analysis using the EBV termini as probes confirmed its derivation from the original lymphoma, and revealed that it contained multiple copies of the EBV genome. Dose-dependent growth on IL-2 was observed in an in vitro study with a doubling time of 3 d at maximal stimulation. These data indicate that HANK1 seemed to preserve the biological characteristics of the original tumour and therefore may serve as a good model for the further analysis of unusual 'nasal-type' NK/T-cell lymphoma.

We have identified the herpes simplex virus type 2 (HSV-2) UL55 gene product using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL55 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 23 kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the UL55 protein within and at the periphery of the nucleus as discrete granules at late times post-infection, and nuclear fractionation studies showed that the protein was associated with the nuclear matrix of infected cells. Moreover, these discrete regions containing the UL55 protein were found to be adjacent to compartments, designated assemblons, containing the capsid protein ICP35. However, the UL55 protein was not detected in purified virions. These results suggest that the UL55 protein of HSV-2 may play an accessory role in virion assembly or maturation.

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G1, S, and G2/M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G2/M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G2/M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

One supposes that herpes simplex virus US3 gene product possessing serine/threonine protein kinase activity is a cytoplasmic enzyme. To determine its subcellular localization during viral replication we prepared an antiserum to a synthetic oligopeptide corresponding to the N-terminal region of the US3 protein of HSV type 2 strain 186. The US3 protein first appeared in the cytoplasm of infected cell at 4 h postinfection but strong fluorescence was detected in the nuclei at 8 h postinfection. At 12 h postinfection fluorescence was mainly detected in the cytoplasm, again. Further, the US3 protein expressed alone was widely distributed throughout the cell, indicating that the US3 protein by itself can be localized in the nuclei even in the absence of any other viral proteins. These observations suggest that the HSV-2 US3 protein kinase may function not only in the cytoplasm but also in the nuclei.

We have identified the herpes simplex virus type 1 (HSV-1) US10 gene product using rabbit polyclonal antisera raised against a recombinant 6xHis-US10 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with 34 and 36 kDa proteins in HSV-1 KOS-infected cells as shown by Western blotting and immunoprecipitation experiments. The 36 kDa protein was immunoprecipitated with the US10 antiserum from 32P-labelled lysates of Vero cells infected with HSV-1 KOS, demonstrating that the US10 protein was phosphorylated. Indirect immunofluorescence studies localized the US10 protein mainly to nuclei as large discrete particles at later times post-infection (p.i.), and nuclear fractionation studies revealed that the protein was tightly associated with the nuclear matrix. Moreover, analysis of isolated intracellular capsids showed that both phosphorylated and unphosphorylated forms of the US10 product were also associated with the capsid/tegument. These results indicate that the US10 gene of HSV-1 encodes a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix.

The Epstein-Barr virus (EBV) DNA polymerase (Pol) holoenzyme is an essential enzyme required for ori-Lyt dependent EBV DNA replication. Using singly primed M13ssDNA circles as template, the EBV DNA Pol holoenzyme synthesized DNA chains greater than the unit length of M13 ssDNA in addition to full length products even at a low ratio of polymerase molecule per templates. The long replication products consisted of circular double-stranded DNA with single-stranded tails that were sensitive to mung bean nuclease. Reconstitution of the EBV Pol holoenzyme by preincubation of BALF5 Pol catalytic subunit and BMRF1 Pol accessory subunit in vitro resulted in reproduction of the strand displacement DNA synthesis. Thus, the EBV DNA Pol holoenzyme by itself is able to produce strand displacement coupled to the polymerization process in a highly processive way in the absence of any other protein.

The proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His.Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed in Escherichia coli and also recognized 56- to 57-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite) using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was not detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK II) and also resistant to CK I-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region.

Epstein-Barr virus(EBV) is a human herpesvirus with a linear double stranded DNA, 172 kbp in length. The EBV has both a latent state and a lytic replicative cycle. Latency replication is dependent on cellular DNA replication machinery except for a single EBV-encoded protein, EBNA1. The EBV genome is maintained as a circular plasmid molecule and ori P, the latent phase replication origin of EBV, mediates this type of replication. The immediate-early BZLF1 protein disrupts viral latency through transactivation of early EBV genes. After induction of the lytic phase of viral replication, the EBV genome is amplified 100- to 1000-fold. The replication product is a head to tail concatemer which is presumed to be synthesized via a rolling circle mechanism initiated from the lytic phase replication origin, ori Lyt. EBV encodes seven viral genes that are essential for ori Lyt-dependent DNA replication. A number of features of EBV DNA replication make it a unique model system for the study of eukaryotic DNA replication. Here, the molecular mechanism of the lytic phase of the EBV DNA replication is discussed.

By a pulse-chase approach combined with immunoprecipitation, we showed that the newly synthesized precursors of glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) were associated with calnexin, a membrane-bound chaperone in the endoplasmic reticulum. Kinetics of association and dissociation was quite different among the precursors. For the precursors of gC and gD of HSV-1, the maximal association was observed immediately after the synthesis and they dissociated rapidly with half-times of 25 min for gC and 30 min for gD, respectively. In contrast, the precursor of gB showed prolonged association with calnexin. Their maximum association was observed 30 min after the synthesis, and thereafter, they dissociated slowly with a half-time of 70 min. The results suggest that, while glycoproteins that have a rapid processing rate, such as gC and gD of HSV-1, rapidly associate with calnexin and their association is quite short, those that take much time to be processed to a mature form, such as gB of HSV-1, have prolonged association kinetics with calnexin. Complete inhibition of the binding of these glycoprotein precursors to calnexin by tunicamycin or castanospermine indicates the importance of partially trimmed N-linked oligosaccharides for their association.

Epstein-Barr virus (EBV) BALF2 gene product is one of the essential components in the lytic phase of the EBV DNA replication. The BALF2 protein was purified to near homogeneity from the nuclear extract of B95-8 cells with virus productive cycle induced by chemical agents. SDS-polyacrylamide gel electrophoresis showed the presence of a single polypeptide with a molecular weight of 130 K, which was identified as BALF2 protein by Western immunoblot analysis. On Superose 6 HR 10/30 gel filtration the BALF2 protein eluted at a position corresponding to an apparent molecular mass of approximately 128 K, indicating that the BALF2 protein behaves as a monomer in solution. The purified BALF2 protein bound to single-stranded DNA preferentially over double-stranded DNA or single-stranded RNA. Replication of singly primed M13 single-stranded DNA by the EBV DNA polymerase complex in the absence of the BALF2 protein exhibited a highly processive mode of replication and generated full length products in addition to some bands of pausing sites. Although the addition of the BALF2 protein did not affect the replication rate, the average chain length of the replication products was slightly increased with eliminating bands of pausing sites. Similar effects were observed with the reconstituted polymerase complex composed of the BALF5 and BMRF1 Pol subunits. On the other hand, in the absence of the BALF2 protein, the BALF5 Pol catalytic subunit alone extended the primer slightly and paused at specific sites on M13 ssDNA template where stable secondary structure is predicted. However, addition of the BALF2 protein, in contrast to the case of herpes simplex virus ICP8 which does not affect the overall distribution of length of the replication products synthesized by the HSV Pol catalytic subunit (Gottlieb et al., 1990, J. Virol. 64, 5976-5987), stimulated DNA synthesis and yielded a distribution of replication products with long lengths in addition to full length products. Although the BALF2 protein behaved as if it converts a low processive enzyme of the EBV Pol catalytic subunit to a highly processive form like the BMRF1 Pol accessory subunit, challenger DNA experiments revealed that the EBV Pol catalytic subunit is transferred to challenger DNA even in the presence of the BALF2 protein. It is therefore likely that the EBV BALF2 protein functions to melt out the regions of secondary structure on the single-stranded DNA template, thereby reducing and eliminating pausing of the EBV DNA polymerase at specific sites. These properties indicate that the EBV BALF2 protein acts as a single-stranded DNA-binding protein during lytic phase of EBV DNA replication.

Human cytomegalovirus glycoprotein B (gB) is synthesized as a 105-kDa nonglycosylated polypeptide and cotranslationally modified by addition of N-linked oligosaccharides to a 160-kDa precursor in the endoplasmic reticulum (ER). It is then transported to the Golgi complex, where it is endoproteolytically cleaved to form the disulfide-linked mature gp55-116 complex. Pulse-chase experiments demonstrate that the 160-kDa gB precursor was transiently associated with calnexin, a membrane-bound chaperone, in the ER. The association was maximal immediately after synthesis, and they dissociated with a half-time of 15 min. Complete inhibition of binding by tunicamycin or castanospermine indicates the importance of N-linked oligosaccharides for it. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that during an initial stage in the biogenesis, the 160-kDa gB precursor was first synthesized as a fully reduced form and rapidly converted to an oxidized form, with a half-time of 18 min. Both forms of the gB precursor could bind to calnexin. The kinetics of the conversion from the fully reduced to the oxidized form coincided with that of dissociation of the 160-kDa gB precursor from calnexin, suggesting that the two steps are closely related.

Herpes simplex virus type 2 (HSV-2) gene US3 encodes a serine-threonine protein kinase. We previously described the isolation of a US3-inactivated mutant which is able to replicate in Vero cells but not in murine macrophages. To learn more about the biological role of the US3 protein kinase, we have sought to identify the target proteins of the enzyme. Studies of in vitro phosphorylation with extracts of infected cells demonstrate that the US3 protein kinase is involved in phosphorylation of the UL12 alkaline nuclease in vitro, suggesting that the nuclease is a possible target of the protein kinase.

Human cytomegalovirus (HCMV) infection results in a marked reduction in the surface expression of class I major histocompatibility complex antigens on the host cells, which is thought to be one of the means for HCMV to evade the host immune system. To clarify the precise mechanism(s) of this phenomenon, we investigated the fate of the heavy chain of class I major histocompatibility complex antigens in HCMV-infected human embryonic lung fibroblasts (HEL) by pulse-chase analysis and immunocytochemical techniques. In HCMV-infected HEL, the heavy chain was synthesized at an increasing rate. However, instead of being transported to the cell surface through the Golgi apparatus, it was retained in the endoplasmic reticulum (ER) without acquisition of a complex-type N-linked oligosaccharide. In addition, it was rapidly degraded, with a half time of 20 min, and the amount of the heavy chain remaining at the end of 3 h of chase was 10% (or less) of that initially synthesized. ER degradation of host glycoproteins in HCMV-infected HEL was selective for the heavy chain, since the posttranslational processing of the transferrin receptor in these cells was not affected. The heavy chain degradation in infected cells was resistant to inhibitors of a lysosomal proteolytic pathway and to metabolic poisons. These observations suggest the presence of an energy-dependent nonlysosomal proteolytic pathway in the ER.

To evaluate the value of cytology of pure pancreatic juice (PPJ) and tumor marker determination in PPJ and serum for the diagnosis of early pancreatic cancer (EPC), PPJ was obtained endoscopically from 16 patients with EPC (< 20 mm, confined to pancreas), 16 patients with chronic pancreatitis (CP), and 20 controls. Cutoff levels of CEA, CA19-9, and POA in PPJ were set from ROC curves at 40 ng/ml, 7,500 U/ml, and 1.5 U/ml, respectively. For the differentiation of EPC from CP, the sensitivity of CEA, CA19-9, and POA was 71.4, 42.9, and 50%, respectively; specificity was 93.3, 46.7, and 80%, respectively; and diagnostic accuracy was 82.8, 44.8, and 65.5%, respectively. Determination of serum tumor markers was useless. Sensitivity of cytology was 75%, specificity was 93.8%, and diagnostic accuracy was 84.4%. Combined cytology and CEA determination in PPJ increased the diagnostic accuracy to 93.1%. The combination was useful in supporting and supplementing endoscopic retrograde cholangiopancreatography (ERCP) findings for the correct diagnosis in 11 and 4 patients, respectively, with EPC; in one patient EPC was correctly diagnosed on ERCP findings alone. One of 16 patients with CP showed false-positive results. We conclude that cytology and CEA determination in PPJ with ERCP is a useful combination for the diagnosis of pancreatic cancer even in early stages.

Herpes simplex virus type 2 (HSV-2) gene US3 has been shown to encode a serine-threonine protein kinase. In this study, we have tried to identify target proteins of the US3 protein kinase using a US3 lacZ insertion mutant of HSV-2. When permeabilized cells were labelled with [gamma-32P]ATP under the optimum conditions for the US3 enzyme, the most striking difference between wild-type HSV-2 strain 186- and mutant-infected cells was observed in the phosphorylation of proteins ranging in M(r) values from 14K to 22K. Studies of in vitro phosphorylation with purified virions and with cells infected with a US9-defective HSV-1 mutant suggested that a tegment phosphoprotein encoded by the US9 gene may be a target of HSV-2 US3 protein kinase.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.

Background: Recurrence in Stage I non-small cell lung cancer was examined with respect to vascular invasion and the immunohistochemical expression of sialyl-dimeric Lewisx (SLX) and proliferating cell nuclear antigen (PCNA)., Methods: One hundred twenty-eight patients with Stage I non-small cell lung cancer who had a curative resection were the subjects of this study. Using tumor tissues fixed in formaldehyde solution, blood vessel invasion (BVI) and lymphatic invasion stained with Victoria blue-hematoxylin and eosin and the immunohistochemical expression of SLX and PCNA were retrospectively studied with respect to postoperative recurrence., Results: By univariate analysis, BVI and SLX and PCNA expression were significantly important factors of disease-free survival (P < 0.01). The disease-free survival of the patients with both BVI and SLX expression was significantly shorter than that of the patients with BVI but negative SLX expression (P < 0.02). In 35 patients with recurrence, tumors with PCNA expression showed a significantly shorter time to recurrence compared with tumors without PCNA expression (P < 0.01)., Conclusions: BVI and SLX expression may be important determinants of recurrence. PCNA may be a determinant of time to recurrence.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to successive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 microM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.

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.

We have investigated the pathogenicity of a US3 protein kinase-deficient mutant (L1 BR1) of herpes simplex virus type 2 (HSV-2) for 4-week-old ICR mice to define the role of the viral protein kinase in virus-host interaction. When mice were intraperitoneally infected with 10(5)PFU of L1 BR1, the virus disappeared from the peritoneal cavity by 2 days postinfection and failed to induce any significant histopathological changes in the liver and spleen although viral antigens were occasionally detected in the epithelial cells of small bile ducts and small vascular wall. The parental virus (HSV-2 186) and a revertant of the mutant (L1 B-11) both caused severe hepatitis, and viral antigens were clearly detected in the hepatocytes and Kupffer cells in the focal necrotic areas. Both of the virulent viruses, unlike L1 BR1, could produce infectious progeny and cytopathic effects in freshly harvested peritoneal macrophages. The growth of L1 BR1 in peritoneal macrophages was restricted at a stage of or prior to viral DNA synthesis but after the induction of viral DNA polymerase. In addition, the production and/or the spread of mutant in mouse embryo fibroblasts (MEF) was found to be much more effectively suppressed by cocultivation of peritoneal macrophages than that of 186. An almost complete inhibition of L1 BR1-plaque formation was observed at a macrophage-to-MEF ratio of 4:1. These results suggest that the attenuation of L1 BR1 following intraperitoneal infection is primarily due to its high sensitivity to intrinsic and extrinsic inhibition of peritoneal macrophages and that the US3 protein kinase may play a role in viral DNA replication in peritoneal macrophages.

Epstein-Barr virus (EBV) DNA polymerase possesses a proofreading 3'-to-5' exonuclease activity (Tsurumi, T. (1991) Virology 182, 376-381). The 3'-to-5' exonuclease activity can be selectively inhibited by ribonucleoside 5'-monophosphates, while no inhibition of the DNA polymerase activity can be observed even when the template/primer concentrations are rate-limiting. Deoxynucleoside monophosphates except 5'dGMP have almost no effect on the exonuclease activity. Of the four ribonucleoside monophosphates, 5'GMP is the most potent (62% inhibition at 5 mM). The kinetic study shows that 5'-GMP inhibits the exonuclease activity competitively with respect to DNA template/primer. During DNA polymerization process the EBV DNA polymerase catalyzes the DNA-dependent conversion of complementary deoxynucleoside triphosphate to monophosphate form. With poly(dT).oligo(rA) as a template primer, selective inhibition of the exonuclease activity by 5'-GMP results in a decrease in the amount of free dAMP generated which is complementary to the template DNA, suggesting the functional relationship between the editing exonuclease activity and the chain elongation activity of the EBV DNA polymerase molecule.

Seventy-four patients with lung cancer, resected consecutively from April 1989 to August 1990, were divided into (1) 21 with diploid tumors having a single G0/G1 peak and a coefficient of variation (CV) of 4.9 or less, (2) 18 with peridiploid tumors having a single G0/G1 peak and a CV at 5.0 or more, and (3) 35 with aneuploid tumors having multiple G0/G1 peaks. Aneuploid tumors had higher frequencies of lymphatic invasion and metastasis to the mediastinal lymph nodes. To evaluate the relationship between ploidy tumor status and immunologic competence of the regional lymph nodes, histologic findings and the proportion of killer T-lymphocytes were examined in the dissected lymph nodes. Aneuploid tumors had significantly lower proportions of paracortical hyperplasia and killer T-lymphocytes than did diploid and peridiploid ones in the nonmetastatic lymph nodes of N0 and N1 disease. These findings suggest the possibility that a decline in the antitumor competence of these lymph nodes could cause metastasis to the nodes. The recurrence rates were 19% in diploid, 33% in peridiploid, and 54% in aneuploid tumors, and the 2-year survival rates were 87%, 78%, and 44%, respectively. Peridiploid tumors showed intermediate values between diploid and aneuploid in terms of immunologic competence, recurrence rate, and survival. They were assumed to have a different proportion of aneuploid cells than the other two.

Nine patients underwent operation for pleural dissemination of thymoma and received radiotherapy postoperatively. In 5 patients dissemination was resected along with the invasive thymoma at the initial operation. In 6 patients resection of a part of the diaphragm was needed. In 4 patients recurrent dissemination of thymoma appeared in the phrenico-costal sinus, where not only the location of dissemination but also postoperative radiotherapy had not been performed properly at the time of the previous operation. Seven are alive 2 months to 7 years 10 months after the first operation for dissemination. Two died respectively 7 years and 3 years 5 months after the first operation for dissemination.

The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.

Epstein-Barr virus (EBV) DNA polymerase mediates viral DNA replication during the lytic phase of the EB virus life cycle. In order to characterize its enzymatic activities EBV DNA polymerase was purified more than 1200-fold from chemically induced B95-8 cells. One polypeptide with molecular weight of 110,000 corresponded to the predicted EBV DNA polymerase, whereas the other polypeptides did not. A 3'-to-5' exonuclease activity was copurified with the EBV DNA polymerase through the course of the purification. Unlike HSV DNA DNA polymerase, 5'-to-3' exonuclease activity was not associated with the EBV DNA polymerase on the final step chromatography of single-stranded DNA agarose column. The associated 3'-to-5' exonuclease activity was stimulated by ammonium sulfate like the polymerase activity. It exhibited DNA-dependent nucleotide turnover activity and preferentially excised a terminal mismatched nucleotide on hybridized polynucleotides compared to the correctly paired substrate, indicating that the 3'-to-5' exonuclease may play a role in proofreading in the polymerization process.

Forty-six cases of resected lung cancer, including 20 cases at Stages I and II and 26 cases at Stage III (N2), were subdivided into two groups: a good prognosis group with a longer survival period and a poor prognosis group in which the patients died earlier of the cancer. From paraffin-embedded lymph node tissues of these patients, the authors examined DNA histogram pattern and DNA content, using flow cytometry, and histologic hyperplasia of germinal center and paracortical area; they also evaluated their correlation with the prognosis. In the good prognosis group at Stages I and II, paracortical hyperplasia (PH) of the lymph nodes was observed significantly more frequently. In the good prognosis group at Stage III, the incidence of PH, G2M phase in the DNA histograms, and DNA content were all significantly higher. DNA content was positively correlated with the grade of PH.

Surgical treatment for bullous emphysema using Gore-Tex sheet and mechanical stapler is reported. The techniques were followed in a Juetnner's report: Small cut is made at the center of a 10 cm square of a Gore-Tex sheet, through which the area of bullous lung planned for resection is tented out with a lung forceps. A stapling instrument charged with a 4.8 mm clips is placed over the sheet around the suture line, and closed over both sheet and lung parenchyma. The excess sheet is removed after resection of the bullous lung. Five cases of bullous emphysema with a history of pneumothorax were successfully treated by the above method. Air leakage disappeared on the first postoperative day at latest. We assume Gore-Tex sheet, although a non-absorbable material, is in use as well as polydioxanone ribbon for prevention of air leakage in surgical cases of bullous emphysema.

Electrolytes undergo electrostatic resistances in reaching membrane surfaces and passing through membrane pores when weakly charged dialysis membranes are used in hemodialysis treatments. Diffusive permeability to an electrolyte of weakly charged dialysis membranes depends upon the effective charge density of the membranes, which varies with ionic strength. The authors carried out dialysis experiments at a temperature of 310 K, with 32P-Na2HPO4 in aqueous NaCl and bovine serum, to obtain diffusive permeability to an inorganic phosphate ion of regenerated cellulose, and polymethylmethacrylate membranes of various fixed charge densities at varying ionic strengths ranging from 1.66 to 100 mol/m3. Diffusive permeability to an inorganic phosphate ion increased with ionic strength. Bovine serum, having an ionic strength of approximately 150 mol/m3, gave higher diffusive permeability to an inorganic phosphate ion. The internal structure of dialysis membranes, such as pore size, surface porosity, and tortuosity, also affects diffusive permeability. The electrolyte diffusion theory gives an effective charge density of -5.3 mol/m3 for the cellulosic membrane. In conclusion, ionic strength enhances diffusive permeability to an inorganic phosphate ion in both aqueous NaCl and bovine serum. Inorganic phosphate ion transport through negatively charged dialysis membranes depends upon the degree of dissociation of inorganic phosphate, the molecular size and valence of hydrated inorganic phosphate ion, the effective charge density and internal structure of the membrane, and ionic strength.

Hepatitis B virus (HBV) can be effectively removed from HBV-positive plasma by filtration with a Bemberg Microporous Membrane (BMM) with a pore size of 30 nm or less, however considerable amounts of macromolecular IgM and Factor VIII are trapped in the BMM. We report that HBV-free plasma with adequate amounts of all of the plasma proteins can be obtained by double filtration with a BMM with a port size of 50 nm. Thus it may be possible to remove HBV or other transfusion-associated viruses from plasma by BMM filtration with good recovery of all of the plasma components.

Infection of Vero cells with herpes simplex virus type 1 results in the appearance in soluble extracts of a DNA primase activity. The partially purified enzyme, Mr, approximately 100,000, is identical in resistance to alpha-amanitin, pH profile, Mg2+ dependence, salt sensitivity, and KmATP to the catalytic core of Vero cell mitochondrial RNA polymerase. Moreover, the products synthesized are those expected of an RNA polymerase rather than a DNA primase. Inasmuch as the enzyme is not present in soluble extracts of uninfected Vero cells, we presume that the specific appearance of RNA polymerase in extracts of herpesvirus-infected cells results from infection-induced disruption of the mitochondrial membrane, followed by release of the enzyme into the cytosol.

A 56 years old male with chronic pancreatitis complained of intractable abdominal pain, anorexia, emaciation and peripheral edema. Medical treatment initiated only partial improvement in the general condition and hypoproteinemia. Endoscopic retrograde cholangiopancreatography revealed multiple filling defects in the dilated main pancreatic duct. Endoscopic aspiration of pure pancreatic juice yielded numerous protein plugs. The endoscopic removal of protein plugs from the pancreatic duct resulted in remarkable improvement in symptoms, laboratory findings and ERCP findings. We consider this procedure to be an important new treatment of chronic pancreatitis.

Clinical features and validity of a duodenofiberscopic examination for the diagnosis of carcinoma of the papilla of Vater were evaluated on 13 cases preoperatively diagnosed endoscopically. Followings are the conclusions derived. 1) Average age was 59.8 years. Incidence was equal among men and women. 2) As initial symptoms, general fatigue and easy fatigability or symptoms of cholangitis should be emphasized. 3) Other important symptoms include jaundice, fever with chills and weight loss. Abdominal pain, when present, was noted as one of symptoms of cholangitis in most cases. 4) Important laboratory findings include elevated serum bilirubin and alkaline phosphatase, elevated erythrocyte sedimentation rate, positive occult blood in stool. 5) Carcinomas of the papilla of Vater were divided into 3 types (type I, II and III) according to endoscopical and pathological findings. Clinical features and laboratory findings were discussed in relation to the type of lesions. Characteristic endoscopic findings of each type of the lesions were described. Validity of aspiration cytology, pancreatocholangiography and biopsy under duodeno-fiberscopic observation was also discussed. As a result, duodenofiberscopy was considered to be the most useful method for the diagnosis of carcinoma of the papilla of Vater because it provides us with an opportunity to perform simultaneously an endoscopic observation, aspiration cytology, pancreatocholangiography and biopsy.

The synthesis of 3 polypeptides with molecular weights of 103-, 80- and 78-kdalton (K) was dramatically accelerated when the cultures of human embryonic fibroblasts were transferred from 37 to 40 degrees C. The induction of these polypeptides was not observed if actinomycin D was added to the cultures at the beginning of the rising of the temperature, indicating that this response may be mediated by increased transcription of their messenger RNA.

Immunotherapy with levamisole (LMS) and combined splenectomy were performed in the cases of stages III and IV gastric cancer of the upper and middle stomach and associated with total gastrectomy. The cell-mediated immunity and the survival rates for both groups were compared with controls. Combined splenectomy was not effective for cases of stage III gastric cancer, but was effective for those in stage IV. Comparing the cumulative survival rates of the immunotherapy group with the controls, cases of stages III and IV gastric cancer without splenectomy, the survival rate of those with immunotherapy was higher than that of the control group in the cases of the stage III, but the result was reverse in cases of stage IV gastric cancer. Changes in the cell-mediated immunity correlated with the survival rates.

We propose a new method to rid solutions of a virus by using a novel regenerated multilayered structured cellulose membrane (BMM). When the filtrate of human immunodeficiency virus (HIV) preparation was obtained through BMM it showed no infectivity. Electron microscopic observation revealed that HIV was completely caught by the multilayers of the BMM. Conveniently, BMM was seldomly found to adsorb protein molecules and also to have a high filtration rate. These characteristics may have a use in the removal of other variously sized pathogenic agents from plasma.

Some patients on hemodialysis have elevated plasma phosphate values. The chemical and physical properties of dialysis membranes may cause reduced inorganic phosphate clearance, leading to hyperphosphatemia. The zeta potential was determined by a streaming potential method using an electrolytic, aqueous solution to elucidate discrepancies in phosphate ion transport through cellulosic and polymethylmethacrylate membranes. The authors also carried out dialysis experiments at 310K to obtain solute permeability for H32PO4(2-), overall mass transfer coefficient for HPO4(2-), and pure water permeability. This study demonstrates that permeability to inorganic phosphate ion does not vary with zeta potential for cellulosic membranes, but polymethylmethacrylate membranes with highly negative zeta potentials may suppress phosphate removal from patients on hemodialysis.

We have developed a new cellulose membrane in which the active hydroxyl and carboxyl groups of the original cellulose fibers are masked with cationic synthetic polymers. The membrane was shown by in vitro and clinical tests to affect the activation of the complement system very little, and it retains the high dialysis efficiency, good tolerance to steam autoclave sterilization, and economical production cost characteristics of the original cellulose membrane. It thus appears that the new cellulose provides the optimum combination of qualities needed in a hemodialysis membrane.

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.

The experimental and clinical effects of bestatin were examined and the results obtained were as follows. Bestatin which is an immunomodulator discovered by Umezawa did not increase the bone marrow stem cells examined by the method of the spleen colony assay in the 60Co irradiated mouse. However, it prolonged the survival time slightly when it was administered 10-25mg/kg intraperitoneally. Clinically, bestatin was administered to the patients with gastrointestinal cancer. It did not influence on the PHA-induced lymphocyte blastformation rate, but it increased the peripheral lymphocyte count and PPD skin reaction in the cases of curative resection and increased the peripheral lymphocyte counts in the cases of nonresection after 1 month of the operation.

When influenza A/RI/5+ virus-infected cells were incubated in medium to which 2 micrograms of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding-like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine-treated cells by freeze-thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine-substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5+, because of the inability of M protein to associate with the plasma membrane.