Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6

The activity and expression of superoxide dismutase (SOD) was analyzed in a copper-tolerant yeast, Cryptococcus sp. N6. Using cell extracts, two distinct bands exhibiting SOD activity appeared on native PAGE: one band, with higher mobility, appeared when the cells were grown without CuSO₄, and the other band appeared when the cells were grown with 10 mM CuSO₄. Cells grown with 3 mM CuSO₄ produced both SOD isoforms. Western blot analysis, using a monoclonal antibody against human SOD-1, showed that SOD protein was expressed in the absence of CuSO₄ and that the expression level increased when the cells were grown with 3 or 10 mM CuSO₄. The molecular weight of SOD from strain N6 was approx. 18 kDa. Treatment of the cells with the protein synthesis inhibitor, cycloheximide at 0.5 g ml^–1, did not affect cell growth in the absence of CuSO₄ but significantly inhibited growth in the presence of 10 mM CuSO₄ and inhibited expression of SOD protein. This suggests that SOD may play a role in cell growth in the presence of high concentrations of CuSO₄.     

Study of cognitive function among the Adult Health Study (AHS) population in Hiroshima and Nagasaki

Although neuropsychological dysfunction is found among A-bomb survivors exposed in utero as it is among patients who receive central nervous system radiotherapy, neuropsychological examinations have not been conducted on the survivors. Its prevalence may be increased as a result of the increased rate of strokes reported among those exposed to a high radiation dose. In this study, we examined the effects of radiation exposure on cognitive function among adult survivors in the Adult Health Study (AHS). The study subjects were men and women born prior to September 1932 who had undergone biennial examinations during the period 1992-1996 in Hiroshima or 1993-1998 in Nagasaki. We evaluated cognitive performance for 3,113 subjects with the Cognitive Abilities Screening Instrument (CASI), and we examined the relationship between cognitive performance and potentially related factors (sex, age, city where the subjects were exposed, years of education, and radiation dose). In contrast to exposure to radiotherapy, exposure to atomic bomb radiation had no apparent effect on cognitive function. Factors that did affect cognitive function were age, sex, city and years of education. Further investigation, including examination of other neurological functions, is required before a final conclusion regarding radiation-induced neurological dysfunction can be reached.     

Decreases in percentages of naïve CD4 and CD8 T cells and increases in percentages of memory CD8 T-cell subsets in the peripheral blood lymphocyte populations of A-bomb survivors

Our previous studies have revealed a clear dose-dependent decrease in the percentage of na?ve CD4 T cells that are phenotypically CD45RA+ in PBL among A-bomb survivors. However, whether there is a similar radiation effect on CD8 T cells has remained undetermined because of the unreliability of CD45 isoforms as markers of na?ve and memory subsets among the CD8 T-cell population. In the present study, we used double labeling with CD45RO and CD62L for reliable identification of na?ve and memory cell subsets in both CD4 and CD8 T-cell populations among 533 Hiroshima A-bomb survivors. Statistically significant dose-dependent decreases in the percentages of CD45RO-/CD62L+ na?ve cells were found in the CD8 T-cell population as well as in the CD4 T-cell population. Furthermore, the percentages of CD45RO+/CD62L+ and CD45RO+/CD62L- memory T cells were found to increase significantly with increasing radiation dose in the CD8 T-cell population but not in the CD4 T-cell population. These results suggest that the prior A-bomb exposure has induced long-lasting deficits in both na?ve CD4 and CD8 T- cell populations along with increased proportions of these particular subsets of the memory CD8 T-cell population.     

Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.     

In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity

Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8(+) T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.     

Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.     

Telomerase inhibiting activity in vitro from natural resources, marine algae extracts

We attempted to screen for telomerase inhibitingactivity in vitro from a total of 304 marine algae samples which were collected from various Japancoasts by nonradioisotope telomeric repeatamplification protocol (Non-RI TRAP) assay using humanleukemia MOLT-4 cells. Ten of the MeOH extracts andtwo of the PBS extracts from those algal samplesshowed telomerase inhibiting activity. In particular,the MeOH extract from a green alga, Caulerpasertularioides strongly inhibited telomerase activitywhen added to MOLT-4 cell culture at a level of 1.25%(v/v), suggesting that it may be possible to developa novel anti-cancer agent in view of its specificanti-telomeric property.     

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.     

Biochemical characterization of a thermostable β-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K _m value was 10.35 mg/ml and the k _cat value was 588.24 s^?1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.