Polyhydroxyalkanoate biosynthesis and simplified polymer recovery by a novel moderately halophilic bacterium isolated from hypersaline microbial mats.

AIMS: Halophilic micro-organisms have received much interest because of their potential biotechnological applications, among which is the capability of some strains to synthesize polyhydroxyalkanoates (PHA). Halomonas sp. SK5, which was isolated from hypersaline microbial mats, accumulated intracellular granules of poly(3-hydroxybutyrate) [P(3HB)] in modified accumulation medium supplemented with 10% (w/v) salinity and 3% (w/v) glucose. METHODS AND RESULTS: A cell density of approximately 3.0 g l(-1) was attained in this culture which yielded 48 wt% P(3HB). The bacterial strain was also capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] when cofed with relevant precursors. Feeding with sodium valerate (0.7 mol l(-1) carbon) at various time intervals within 36 h resulted in 3HV molar fractions ranging from 6 up to 54 mol%. Oil palm trunk sap (OPTS) and seawater as the carbon source and culture medium respectively facilitated a significant accumulation of P(3HB). Simplified downstream processing based on osmotic lysis in the presence of alkali/detergent for both dry and wet biomass resulted in approximately 90-100% recovery of polymers with purity as high as 90%. Weight-average molecular weight (M(w) ) of the polymers recovered was in the range of 1-2 × 10(6) . CONCLUSIONS: Halomonas sp. SK5 was able to synthesize P(3HB) homopolymer as well as P(3HB-co-3HV) copolymer from various carbon sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time a comprehensive study of both production and downstream processing is reported for Halomonas spp.

Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2.

During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T cells is induced upon activation and that anti-TSA-1 mAb inhibits the T cell receptor (TCR) signaling pathway in activated T cells. In the present study, we have analyzed a role of TSA-1 in thymic selection events, especially in TCR-mediated apoptosis. In in vitro experiments, anti-TSA-1 blocked anti-CD3-induced cell death of T cell hybridomas. When anti-TSA-1 was injected into newborn mice in vivo together with anti-CD3 epsilon or anti-TCR-beta, TCR/CD3-mediated apoptosis of thymocytes was almost completely blocked. The blockade of apoptosis was defined by the inhibition of, first, the decrease in total number of thymocytes; second, the decrease in percentages of CD4+ CD8+ thymocytes; and third, the induction of DNA fragmentation. However, anti-TSA-1 did not block either steroid- or radiation-induced apoptosis, indicating that a signal via TSA-1 does not inhibit a common pathway of thymocyte apoptosis. Since TCR-mediated apoptosis is pivotal in thymic ontogeny, these results suggest that TSA-1/Sca-2 is an important cell surface molecule regulating the fate of a developing T cell.

Image processing analysis of oral cancer, oral potentially malignant disorders, and other oral diseases using optical instruments.

Oral cancer screening is important for early detection and early treatment, which help improve survival rates. Biopsy is invasive and painful, while fluorescence visualization using optical instruments is non-invasive, convenient, and provides results in real time, and examinations can be repeated. The purpose of this study was to determine the usefulness of optical instruments in oral screening. A total of 314 patients who were examined using optical instruments at Tokyo Dental College between 2014 and 2018 were enrolled in this study. Fluorescence visualization images were analyzed using subjective and objective evaluations. Subjective evaluation for detecting oral cancer offered 98.0% sensitivity and 43.2% specificity. Regarding the objective evaluations for detecting oral cancer, sensitivity and specificity were 61.9% and 62.7% for mean luminance, 90.3% and 55.7% for luminance ratio, 56.5% and 67.7% for standard deviation of luminance, and 72.5% and 85.4% for coefficient of variation of luminance. Fluorescence visualization with subjective and objective evaluation using optical instruments is useful for oral cancer screening.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase.

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.

T-T cell interaction in the in vitro induction of delayed-type hypersensitivity (DTH) responses: demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of DTH responses.

C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate virus-reactive helper T cell activity. 850R X-irradiated spleen cells from vaccinia virus-primed or unprimed mice as helper cells were stimulated in vitro with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP) in the presence of normal C3H/HeN spleen cells (responding cells). After 5 days of culture, effector cells were tested for anti-TNP delayed-type hypersensitivity (DTH) responses by adoptive transfer into footpads of syngeneic C3H/HeN recipient mice together with TNP-self. The results demonstrate that spleen cells from virus-primed mice failed to enhance anti-TNP DTH responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, spleen cells from vaccinia virus-primed mice, but not from unprimed mice, could augment anti-TNP DTH responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed mice was shown to be antigen-specific, and mediated by Lyt-1+2-T cells. DTH effector cells enhanced by helper cells were also antigen-specific and Lyt-1+2-T cells. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augmented induction of anti-tumor DTH responses by stimulation with virus-infected syngeneic fibrosarcoma tumor cells. Thus, these results provide evidence for the role of antigen-specific helper T cells in augmenting the development of DTH responses to cell surface antigens including tumor antigens.

The activation of L3T4+ helper T cells assisting the generation of anti-tumor Lyt-2+ cytotoxic T lymphocytes: requirement of Ia-positive antigen-presenting cells for processing and presentation of tumor antigens.

The present study investigates the mechanisms of the recognition of tumor antigens by L3T4+ helper T cells responsible for the generation of Lyt-2+ cytotoxic T lymphocytes (CTL) against a major histocompatibility complex (MHC) Class II (Ia) antigen-negative syngeneic X5563 plasmacytoma. Treatment of X5563-immunized spleen cells with anti-L3T4 antibody plus complement (C) diminished the generation of Lyt-2+ anti-X5563 CTL. Since the contribution of L3T4+ cells was completely replaced by the addition of exogenous lymphokines, it was demonstrated that L3T4+ cells functioned as helper T cells assisting the generation of anti-X5563 CTL responses. Elimination of Ia-positive accessory cells (AC) from X5563-immunized spleen cells resulted in the abrogation of CTL generation, whereas the addition of exogenous lymphokines to AC-depleted X5563 immunized spleen cells restored the CTL response. The addition of anti-self Ia antibody to the culture also eliminated CTL responses. These observations demonstrated the requirement of Ia-positive AC for and the involvement of self Ia antigens in the activation of helper T cells. Moreover, use of tumor cells pretreated with paraformaldehyde to cultures of X5563-immunized spleen cells or adding back of AC pretreated with chloroquine to cultures of AC-depleted immune spleen cells failed to generate CTL responses. Finally, the addition of exogenous lymphokines to the above cultures resulted in appreciable restoration of CTL responses. Taken collectively, these results indicate that L3T4+ helper T cells are activated with tumor antigens processed and presented by Ia-positive AC.

Suppression of allograft responses by combining donor alloantigen-specific intravenous presensitization with suboptimal doses of FK506.

C57BL/6 (B6) mice were injected i.v. with class I H-2-disparate B10.QBR spleen cells (10(7)/mouse). This regimen, termed "donor alloantigen-specific i.v. presensitization" (DSP), induced almost complete elimination of anti-B10.QBR mixed lymphocyte reaction/IL-2 production but did not affect the generation of CTL responses. Repeated (5 or 11 times) administration in vivo (over 7 or 18 days) of FK506 at suboptimal doses (0.75-1.0 mg/kg/day) failed to eliminate the capacities to exhibit MLR/IL-2 production and to generate CTL responses. Prolongation of skin graft survival was not induced by either of a single DSP or FK treatment (0.75-1.0 mg/kg/day, 11 times during 18 days) alone, but by the combination of these. Such combined treatment also resulted in almost complete reduction of CTL responses before (5 rounds of FK injection) or after (11 rounds of FK injection) recipients were engrafted with B10.QBR skin grafts. Under conditions in which lymphoid cells from mice receiving both treatments failed to generate CTL responses, the addition of recombinant IL-2 to cultures restored the CTL generation, suggesting that CTL precursors themselves are not attenuated by the combined treatment. Both prolongation of graft survival and suppression of CTL responses were obtained when the administration of FK506 was started before but not after DSP. Prolongation of graft survival could also be obtained in class I and II MHC-disparate combinations when the combined treatment was performed in a particular protocol. These results indicate that (1) DSP alone fails to prolong graft survival in class I and class I and II MHC-disparate combinations; (2) such failure is ascribed to the induction of CTL responses by CTL precursors and CTL helpers, both of which are DSP-resistant; (3) the administration of suboptimal doses of FK506 is not sufficient for the suppression of CTL responses, but is effective selectively for suppressing DSP-resistant CTL helpers; and (4) the combination of DSP with FK506 treatment in an appropriate protocol can thus prolong graft survival through the suppression of CTL-involved as well as CTL-independent graft rejection pathways.

A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation.

Lck, a Src family protein tyrosine kinase (PTKs), is post-translationally modified by palmitoylation, a process thought to regulate the biological function, membrane affinity and glycolipid-enriched microdomain (GEM) localization of this molecule. To examine the importance of palmitoylation sites Cys3 and Cys5 in Lck, one or both of these residues was mutated to serine to create mutants S3, S5, and S3,5, respectively. Immunofluorescence and confocal microscopy of COS-7 cells transfected with these constructs showed that while S5 and S3 localized to the plasma membrane, S3,5 was localized to the cytoplasm, suggesting that palmitoylation at at least one site is essential for membrane localization. Sucrose gradient based fractionation of these mutants expressed in COS-7 cells showed that while S5 localized to GEMs in similar fashion to the wild type, GEM localization of S3 was severely inhibited. Expression of these mutants in Lck-negative JCaM1 cells showed that although S5 reconstituted activation of nuclear factor NFAT as per the wild type, S3 expression failed to do so. These results suggest that Cys3 of Lck plays a more important role than Cys5 in GEM localization and T cell activation. Additionally, it was found that the degree of T cell function recovery is positively correlated with the degree of Lck expression in GEMs.

Induction of drug-metabolizing enzymes in the rat liver by 3'-methyl-4-(dimethylamino)azobenzene.

The influence of 3-methyl-4-(dimethylamino)azobenzene(3'-Me-DAB) on the drug-metabolizing system in the liver was investigated. Feeding of 3-Me-DAB for 3 weeks at 10, 20 and 600 ppm increased the content of hepatic microsomal cytochrome P-450 slightly (up to 27% raise) but significantly. The feeding effects were also demonstrated in S-9 activity (up to 91% raise) when the mutagenicity of 3-methylcholanthrene (3-MC) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was assayed in Ames system using S-9 fraction from 3'-Me-DAB-treated rat livers.

Sulfite suppresses the mutagenic property of coffee.

The mutagenicity of instant and freshly brewed coffee on Salmonella typhimurium TA100 and TA98 without S9 mix was inactivated by sodium sulfite. Sulfite ion at a dose of 200 ppm almost completely inactivated the mutagenicity of coffee made in the ordinary way (5-15 mg dry weight/ml). Sodium bisulfite and potassium metabisulfite had similar effects. On the contrary, L-ascorbic acid enhanced the mutagenicity of coffee. Sodium sulfite also inactivated the phage-inducing activity of coffee in inductest III. Sodium sulfite completely suppressed the mutagenicities of 1,2-dicarbonyls, namely diacetyl and glyoxal. Diacetyl is present in coffee, beer, butter and other foods and drinks. Because sodium sulfite, sodium bisulfite and potassium metabisulfite are widely used as food additives, they should be useful in reducing the levels of mutagens in foods.

Roasting coffee beans produces compounds that induce prophage lambda in E. coli and are mutagenic in E. coli and S. typhimurium.

Freshly brewed blended coffee, instant coffee and instant caffeine-free coffee induced prophage lambda in lysogenic E. coli K12, strain GY5027. Because coffee prepared from green beans by the same extraction method as used for freshly brewed blended coffee had no prophage-inducing activity, this activity may be attributed to compounds produced in the roasting process. Roasting also produced compounds that were mutagenic in S. typhimurium TA100 and E. coli WP2 uvrA/pKM101.

A permease exhibiting a dual role for lysine and biotin uptake in Saccharomyces cerevisiae.

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.

[Tumor-specific immunotherapy: active immunotherapy by augmenting the induction of tumor-specific effector T cells through a T-T cell interaction mechanism].

Recent progress in tumor-specific immunotherapy was reviewed. Methods involved include a) utilization of tumor-specific monoclonal antibodies in conjugation with various anti-cancer agents, b) adoptive transfer of anti-tumor effector T cell clones elaborated in vitro in the presence of interleukin-2 and c) active tumor-specific immunotherapy by augmenting the generation of tumor specific effector T cells. This paper focused especially on the amplified induction of tumor-specific immunity by T-T cell interaction between helper T cells and anti-tumor effector T cells. Data were provided indicating successful tumor-specific immunotherapy in autochthonous as well as syngeneic tumor models and such results were discussed in the light of the future clinical application of the tumor-specific active immunotherapy.

[The augmentation of tumor-specific immunity by T-T cell interaction].

The present study establishes a tumor-specific immunotherapy model based on the principle of T-T cell interaction mechanism responsible for augmenting the induction of anti-tumor-specific effector T cells. In this model, high incidence of tumor regression was observed in hosts bearing transplantable or autochthonous tumors when these hosts, in which hapten-reactive helper T cells had been generated, received an injection of haptenic solution into the growing tumor mass. This indicated a role of hapten-reactive helper T cells in regressing the tumor. In consideration clinical application of this tumor-specific immunotherapy model, we have also tried to investigate the use of more appropriate haptenic compounds and have succeeded in synthesizing a noble haptenic compound cross-reactive with BCG, a haptenic muramyl dipeptide (MDP) derivative. The results demonstrated that prepriming of BCG and subsequent immunization with syngeneic tumor cells (mitomycin C-treated) modified with the haptenic MDP derivative resulted in the enhanced induction of tumor-specific immunity. Further investigation is in progress to establish a new tumor-specific immunotherapy model by utilizing this haptenic MDP derivative.