Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

Rapid Purification and Crystallization of Thermostable Malate Dehydrogenase Isolated from a Thermophilic Bacterium,Thermus flavus AT 62

A structural study of the water-soluble dextran made by Leuconostoc mesenteroides strain C (NRRL B-1298) was conducted by enzymic degradation and subsequent ¹³C-NMR analysis of the native dextran and its limit dextrins. The α-l,2-debranching enzyme removed almost all of the branched D-glucose residues, and gave a limit dextrin having a much longer sequence of the internal chain length (degree of linearity: n = 24.5 compared with the value of n = 3.3 for the native dextran). The degree of hydrolysis with debranching enzyme corresponded to the content of α-1,2-linkages determined by chemical methods, which suggested that most of the α-l,2-linkages in the dextran B-1298 constituted branch points of a single D-glucose residue. A synergistic increase of susceptibility of the dextran B-1299 was observed by simultaneous use of debranching enzyme and endodex-tranase. ¹³C-NMR spectral analysis indicated the similarity of structure of dextran B-1298 to that of B-1396, rather than that of B-1299. Occurrence of α-l,3-linkages in the limit dextrin was supported by a newly visualized chemical shift at 83.7 ppm.     

A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.     

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein—approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.     

HIV/AIDS in Asia: The shape of epidemics and their molecular epidemiology

The Asia-Pacific region is a home to 60% of the population in the world and to approximately one quarter of people with HIV/AIDS. Close to a million of people has been infected and a half million people died of AIDS annually in Asia, becoming the second largest epicenter of global AIDS epidemic. Molecular epidemiology has been useful tool to track a course of HIV spread. In-depth knowledge from the studies on molecular epidemiology elucidates the dynamics of HIV spread and the interrelationship of epidemics in the different regions in Asia. Foundation items: Grant support from Ministry of Health, Labour and Welfare and Ministry of Education, Science and Technology in Japan; Japanese Foundation for AIDS Prevention.     

The process of cardiorespiratory autoresuscitation in intact newborn rats

To examine the process of spontaneous autoresuscitation and the recovery of the hypoxic ventilatory response (HVR) after prolonged anoxia, we monitored respiratory frequency (f, by body plethysmography) and heart rate (HR, by ECG) in intact newborn rats (n = 12, day 2-4) before, during, and after 100% N2 exposure. The rat before anoxia showed signs of HVR: f changes at acute hypoxia (10% O2) and hyperoxia (100% O2). During anoxia, the spontaneous respiratory movement "gasping" appeared for 21 min (mean). At O2 restoration (with 100% O2), gasping stopped and no respiratory flow was detected for 1 min. One rat failed to autoresuscitate and had heart arrhythmia during the transient apnea, but 11 rats recovered respiration after the HR acceleration. Despite the successful autoresuscitation, the rats did not show HVR at 10 min into the recovery period and the recovery of HVR required more than 30 min. The results indicate that O2 inhalation is useful to trigger autoresuscitation even when the rat has already been in a state of profound hypoxic depression, but the rat becomes transiently insensitive to HVR after autoresuscitation. We estimate that reform of the respiratory control system in newborn rats is not yet firmly established to track HVR early in the recovery phase after prolonged anoxia.