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Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only threeC. japonicaallergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum ofC. japonicapollen allergens demonstrates that many allergens remain to be identified.The aim of this study was to characterize a novel allergen with a high frequency of IgE binding.The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA ofC. japonicapollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition.The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients withC. japonicapollinosis and/or latex allergy to purified CJP-4.We identified CJP-4 as a novel and fourthC. japonicachitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile betweenC. japonicachitinase and latex C-serum indicated thatC. japonicachitinase should be an important pan-allergen inC. japonicapollen. [ABSTRACT FROM AUTHOR]

Background T-cell epitopes on Der 1 and Der 2 groups, the major mite allergens, have been intensively analysed, while those on the other important allergens remain to be elucidated. We have cloned four cDNAs coding for important mite allergens on the basis of frequency and capacity of IgE binding. Stimulatory action of glutathione S-transferase-fused Mag1 on lymphocytes from mite-allergic patients was relatively high among them. Objective To identify T-cell epitopes on Mag1, we studied the stimulating activity of truncated Mag1 proteins and synthetic peptides on proliferative response of lymphocytes from mite antigen-immunized mice and mite-sensitive patients. Methods Truncated Mag1 proteins were expressed as a fusion protein with β-galactosidase in Escherichia coli pop2136 carrying a variety of deleted Mag1 inserts. Murine T-cell epitope regions were estimated by the truncated antigen-induced lymphocytes proliferation assay. Overlapping peptides covering the whole sequence of the presumed T-cell epitope regions were synthesized to identify the epitope core sequences using murine and human Mag1-specific T-cell lines. Results Amino acid range 56-70 on Mag1 molecule showed remarkable stimulatory action on murine T cells, while amino acid ranges 51-65 and 86-100 had potent stimulatory activity on human T cells. Conclusions These results suggest that Mag1 is a valuable antigen suitable for studies on T-cell responses and T-cell epitopes in mice and humans. [ABSTRACT FROM AUTHOR]

The activity and the histochemical localization of γ-GTP in the liver of chronic alcoholics were investigated. Mean serum γ-GTP activity in alcoholics was 542.5 ± 337.9 milliunits/ml, and that of patients with nonalcoholic liver disease was 34.3 ± 22.6 milliunits/ml. Hepatic γ -GTP activity in alcoholics was significantly increased compared to j that in control patients (15.62 ± 9.29 versus 4.04 ± 2.67 units/g of liver; p < 0.001). A significant correlation was observed between hepatic and serum γ -GTP activity. Light microscopically, a marked γ -GTP activity was found in the bile canaliculi and a diffuse activity in the cytoplasm in alcoholic livers. By contrast, in the livers of nonalcoholic patients, only slight activity was observed in the bile canaliculi. The electron micrographs showed the enzyme was localized in the microvilli of both the bile canalicular and plasma membranes and the endoplasmic reticulum near the mitochondria in alcoholics. But a very low activity was demonstrated in the plasma membranes in the livers of nonalcoholic patients. [ABSTRACT FROM AUTHOR]

Murine peritoneal macrophages pretreated with interferon (IFN)-α and then stimulated by opsonized zymosan produced two to three times more LTB4 than untreated macrophages. However, PGE2 production was not changed by IFN-α. Meanwhile, IFN-γ did not affect the production of LTB4 and PGE2. From the results it is considered that IFN-α can modulate inflammation or host defence through the production of LTB4. [ABSTRACT FROM AUTHOR]

Summary Background Japanese cedar (Cryptomeria japonica ) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. Objective The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. Methods C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). Results The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. Conclusion Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica . [ABSTRACT FROM AUTHOR]

Nanos is a highly conserved RNA-binding protein in higher eukaryotes and acts as a key regulator protein involved in translational control utilizing the 3' untranslated region of mRNA. The C-terminal domain of Nanos has two conserved and novel CCHC-type zinc-finger motifs that are responsible for the function of Nanos. To clarify the structural basis of the function of Nanos, the C-terminal domain (residues 59-159) of zebrafish Nanos was overexpressed, purified and crystallized. The crystal belonged to space group P6(3), with unit-cell parameters a = b = 100.9, c = 71.5 A, gamma = 120 degrees. Structure determination by the MAD/SAD method is now in progress.

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.

Herpes simplex virus-1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV-1, 2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV-1 DNA or VZV DNA was detected in PBMCs of 50 age- and sex-matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV- 1 or VZV may be associated with the development of some cases of Meniere's disease.

We have recently reported that thiadiazole (TDA) derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. These compounds belong to the family of nonnucleoside reverse transcriptase inhibitors (NNRTIs). In an attempt to develop more effective and pharmacologically favorable compounds, novel TDA derivatives have been synthesized and examined for their anti-HIV-1 activity in vitro. Among them, RD4-2217 was found to be the most potent inhibitor of HIV-1 replication. It inhibited replication of the HTLV-IIIB strain in MT-4 cells at a concentration of 6 nM. RD4-2217 was also inhibitory to clinical isolates and zidovudine-resistant mutants of HIV-1. The combination of RD4-2217 with zidovudine or the protease inhibitor A-75925 synergistically inhibited HIV-1 replication. Studies on the emergence of drug-resistant mutants revealed that, although much higher concentrations (1-10 microM) were required, RD4-2217 completely suppressed the breakthrough of HIV-1 in the supernatants during long-term culturing of infected cells. Furthermore, RD4-2217 at low concentrations (10 or 100 nM), in combination with zidovudine, also completely inhibited viral breakthrough. In addition, RD4-2217 had lower lipophilicity and improved protein binding as compared to its congener RD4-2024 and loviride. These results suggest that RD4-2217, one of the TDA derivatives, is worth pursuing as a candidate drug for the treatment of HIV-1 infections.

We developed a rapid and simple method for the screening of antiviral agents against herpes simplex virus (HSV) in a model of gastrointestinal herpetic infection in vitro. This method was based on inhibition of HSV-induced cytopathogenicity in gastric adenocarcinoma MKN-28 cells, as monitored by an MTT colorimetric assay. From the various compounds that were evaluated for their activity against HSV-1 and HSV-2, brivudine (BVDU) emerged as the most effective. When the 50% effective concentration (EC50) values of the antiherpes agents in MKN-28 cells were compared with those in human embryo lung MRC-5 cells, all compounds, except for BVDU, showed higher EC50 values in MKN-28 cells. For BVDU the EC50 values in MKN-28 cells were 0.8 (HSV-1) and 0.036 (HSV-2) times the EC50 values in MRC-5 cells. Thus BVDU was 27.5 times more active against HSV-2 in MKN-28 cells than in MRC-5 cells. The MKN-28 gastric cancer cells may be useful for the rapid screening of anti-HSV agents and, in particular, those that may be useful in therapy of gastrointestinal HSV infections in gastrointestinal herpetic infection.

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 microliters of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) emerge during treatment with various reverse transcriptase (RT) inhibitors in vitro and in vivo. However, the virological nature and pathogenic importance of these mutants have not been fully elucidated. In this study, we have examined HIV-1 mutants resistant to 3'-azido-3'-deoxythymidine (AZT) and nonnucleoside RT inhibitors (NNRTIs) for their infectivity, RT activity, and replication in MT-4 cells. Although the infectivity of AZT- and NNRTI-resistant mutants was similar, the RT activity of AZT-resistant mutants was much higher than that of NNRTI-resistant mutants and their wild types. Furthermore, the RT activity of NNRTI-resistant mutants was significantly lower than that of the wild types. In contrast, the replication of NNRTI-resistant mutants was found to be greater than that of AZT-resistant mutants and the wild types. When HIV-1 proviral DNA (cDNA) synthesis was examined by PCR in MT-4 cells infected with the wild type, AZT-resistant mutant, or NNRTI-resistant mutant, the PCR signal of the NNRTI-resistant mutant was found to be much higher than those of the wild type and AZT-resistant mutant. These results suggest that the drug-resistant mutants differ from their corresponding wild types not only in drug sensitivity but also in other virological properties.

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 micrograms/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype-specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 micrograms MoAbs per 10(6) cells in 24 hr. It is practicable to use these cell lines for large-scale production of anti-P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.

Infection of human peripheral blood monocytes by varicella-zoster virus (VZV) was investigated. When freshly isolated monocytes of young adult volunteers were infected with cell-free VZV and examined by indirect immunofluorescence, specific antigens appeared at 8 hr and the number of antigen-positive cells reached the maximum between 24 and 48 hr postinfection. The proportion of antigen-positive cells to total cells was similar to that of the permissive control (HeLa cells), while very few infectious centers (IC) of monocytes were formed in comparison with the infected control cells. Monocytes isolated from infants and old persons formed a larger number of IC than those of young adults. Electron microscopic study of VZV-infected monocytes from three young adult volunteers demonstrated that the proportion of VZV particle-positive cells to total cells was similar to that of antigen-positive cells, and most of the particles seen in the nuclei were low in density and lacked a central core. These results suggest that the growth of VZV in human adult monocytes is incomplete. This restriction of VZV growth by monocytes may play an important role in defense against VZV infection.

Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella-zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type-1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone-fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.