In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.

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Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5, 000 to 30, 000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between Gram-negative and Gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.

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The duplicative mutation of an Ala-Val-Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C β-lactamase caused substrate specificity extension to oxyimino β-lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the β-lactams (M. Nukaga et al, 1995, J. Biol. Chem. 270, 5729-5735). In order to confirm the universality of this phenomenon among other class C β-lactamases, the duplicative mutation was applied to a class C β-lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae β-lactamase amino acid sequence. The counterpart sequence to the Ala-Val-Arg of the E. cloacae enzyme in C. freundii β-lactamase was identified to be Pro-Val-His. A Pro-Val-His sequence was inserted just after the native Pro-Val-His sequence at positions 208 to 210 in the C freundii β-lactamase. The resulting mutant of C. freundii β-lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino β-lactams. Nearly the same result was obtained with the insertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequence. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C β-lactamases produced by Gram-negative bacteria.

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We investigated the relationship between the binding activity to galactosylceramide (GalCer) and the arthritis induction activity of Borrelia japonica. The B. japonica strains maintained the ability to induce arthritis in inbred C3H/HeN and immunodeficient SCID mice, but the ability was lower than that of Borrelia burgdorferi sensu stricto virulent strain 297. Histopathological changes were restricted to the joints, and a marked effusion of polymorphonuclear neutrophils into the joint space was found. The binding activity of B. japonica strains to GalCer was lower than that of the virulent strain 297 but higher than that of the high-passage strain 297. The lower infectivity and virulence of B. japonica may explain its lower binding ability to GalCer.

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Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.

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Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site II. Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.

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A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerise activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors. The nonnucleoside RT inhibitors are quite specific for HIV-1 RT but not human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) RT. We have investigated the property of SIV/HIV-1 chimeric viruses in which portions of SIV_MAC RT were exchanged with the corresponding domain of HIV-1 RT; amino acids 176-190, 176-383 and 176-495 of HIV-1 RT. The chimeric virus, which was substituted amino acids 176-190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors [nevirapine, HEPT derivative (E-EBU-dM) and TIBO derivative (R82913)]. To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the K_m and V_max values, and the K_i value of HEPT derivative calculated for the DNA polymerase activity. This study reveals that amino acids 176-190 of SIV_MAC RT were important for the enzymatic activity and the SIV/HIV-1 chimeric RT, which had amino acids 176-190 of HIV-1, was sensitive to the nonnucleoside RT inhibitor.

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In order to minimize the denaturation of proteins in milk, normal cow's milk was pasteurized at 61C for 20min. The protective effects of the thus prepared skim milk (low-heat skim milk) on exogenous and endogenous infection were examined as compared with conventional skim milk which was pasteurized at 121C for 2sec. The antibody titers to Listeria monocytogenes and Escherichia coli of low-heat skim milk were almost equal to that of raw milk, while no antibody was detected in the conventional skim milk. When mice were given low-heat skim milk or conventional skim milk, the incidence of the translocation of orally inoculated Listeria monocytogenes to the spleen was lower in the low-heat skim milk group than that in the conventional skim milk group. The life span of 7Gy X-ray irradiated mice given low-heat skim milk was significantly prolonged in comparison to that of mice given conventional skim milk. However, there were no differences in the number of bacteria in the feces or IgA production by Peyer's patch cells between the two groups. These results suggest that antibodies in low-heat skim milk, which still have reactivity to exogenous or indigenous bacteria, may contribute to the protective effects against bacterial infection.

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We have previously shown that calprotectin, the most abundant cytosolic protein existing in polymorphonuclear leukocytes (PMNs), induces apoptotic cell death in various tumor cells, suggesting that calprotectin is an effector molecule against tumor cells in PMNs. To explore the cell death-inducing mechanism of the factor, we examined the involvement of target protein synthesis and generation of reactive oxygen species (ROS) in the reaction. Calprotectin induced cell death in MM46 mouse mammary carcinoma cells after a 14-16hr lag time. When the factor was removed from the medium up to about 12hr after culturing, the effect was diminished. The induction of cell death by calprotectin was markedly inhibited by the presence of the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. However, the addition of these inhibitors after 12hr of culturing was unable to inhibit the reaction. Up to 12hr of culturing, the net protein synthesis of MM46 cells was augmented by the presence of calprotectin, but thereafter was impaired. The induction of cell death was also inhibited by the antioxidative reagents N-acetyl-L-cysteine (NAC) or propyl gallate. The addition of NAC even 15hr later significantly attenuated the calprotectin effect. Flow cytometry analysis showed that calprotectin began to increase the ROS content in MM46 cells after 8-12hr of culturing, and that the increase was abrogated by the antioxidants. Thus, protein synthesis and ROS generation may be essential elements in the early or later phases of the cell deathinducing reaction of calprotectin, respectively.

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Campylobacter jejuni is a frequent cause of enteritis and sometimes it requires antimicrobial therapy. We have studied the evolution of resistance to nine antibiotics from 1990 to 1994 and investigated how frequently gyrA mutations are involved in the acquisition of quinolone resistance. The percentage of chloramphenicol-, clindamycin-, tertracycline- and amoxicillin plus clavulanic acid-resistant strains has remained practically unchanged and erythromycin and gentamicin resistance has decreased, whereas the percentage of ampicillin-, nalidixic acid- or ciprofloxacin-resistant strains has almost doubled in the follow-up period, from 56 to 76% for ampicillin- and from 47.5 to 88% for quinolone-resistant strains. This study clearly shows that a mutation in Thr-86 to Ile or Lys is a frequent mechanism associated with the acquisition of a high level of resistance to quinolones in clinical isolates of C. jejuni.

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A primer pair, PB and BSH, which amplified alts, a portion of Candida albicans-specific repetitive sequence, RPS, gave stable and reproducible fingerprint patterns of the strains by polymerase chain reaction (PCR). We applied this method to clinical isolates of C. albicans for strain discrimination. Using PCR fingerprint patterns, we could analyze the relatedness of C. albicans strains including those isolated from children with leukemia and their bedside parents. The results indicated that PCR analysis targeting an alt region gives rise to the same conclusion as the previous study obtained by SmaI RFLP analysis.

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Susceptibilities of several preparations of Staphylococcus aureus cells to various peptidoglycan hydrolases with known bond specificity were analyzed by zymography. The substrates were intact S. aureus cells, cells boiled in the presence of SDS and cells treated with trichloroacetic acid after treatment with boiling SDS solution (TCA-cells). Twofold dilution of lysostaphin (LS), lysozyme (LZ), S. aureus 51kDa glucosaminidase (GL) or S. aureus 62kDa amidase (AM) were electrophoresed, and the minimal enzyme dose showing a visible bacteriolytic band was defined as MBD (minimal bacteriolytic dose). Under the same experimental conditions, this method gave reproducible results. As the substrate for zymogram, TCA-cells were the most sensitive to LS, LZ and AM, whereas the three substrate were equally sensitive to GL. A zymographic analysis of methicillin-resistant S. aureus treated with methicillin together with previous studies suggest that this method can be used for the preliminary characterization of S. aureus cell wall peptidoglycan.

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Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5kb single-stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5kDa).

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We found and sequenced intervening sequences (IVSs) in the PCR-amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9-100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.

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