Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

Dietary nucleic acid and intestinal microbiota synergistically promote a shift in the Th1/Th2 balance toward Th1-skewed immunity

BACKGROUND: Intestinal microbiota are known to play an important role in the establishment of oral tolerance, thereby protecting the organism from food allergies. Dietary intake of nucleic acid (NA) is also reported to have such an anti-allergic effect; however, one unsolved question is whether or not dietary NA would act through a process of toll-like receptor 9 signaling activated by DNA containing a CpG motif, a well-known sequence leading to immunostimulatory activity. In this study, we focused on the question of whether the addition of dietary NA lacking CpG motifs would allow continued modulation of the Th1/Th2 balance. METHODS: Germ free (GF) and Bifidobacterium-infantis-monoassociated BALB/c mice were maintained on either an NA-free casein diet or on an NA-supplemented casein diet for 4 weeks. Thereafter, both the in vivo anti-casein antibody levels and in vitro splenocyte cytokine secretion pattern were evaluated. RESULTS: Feeding with a casein diet elicited a substantial increase in the serum anti-casein-specific IgG1, IgG2a, and IgE levels of GF mice fed the NA free-diet. The in vitro cytokine production profile showed that enhanced IL-4 production in the GF mice fed the NA free-diet was markedly reduced by the supplementation with dietary NA in both the GF and B.-infantis-monoassociated mice. In addition, IFN-gamma secretion increased in the B.-infantis-reconstituted mice fed the diet containing NA. CONCLUSIONS: These results suggest that dietary intake of NA devoid of CpG motifs may prevent the development of allergies via acceleration of Th1-dominant immunity.     

Peroxyl radical scavenging capability of fish sauces measured by the chemiluminescence method

Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis. A radical scavenger as an antioxidant has been sought in foods. Fish sauces are traditional Asian fermented seasonings. Using the luminol chemiluminescence method, the peroxyl radical scavenging capability of fish sauces was examined. From the IC50 values, many fish sauces have been shown to have a strong scavenging capability as well as soy sauces. A scavenging mechanism is also proposed.     

Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands

Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-beta and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-beta, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.     

Epithelial myoepithelial tumour of the tracheal gland.

A case of epithelial myoepithelial tumour originating from the tracheal gland in a 57 year old woman is described. The tumour was removed by segmental tracheal resection and end-to-end anastomosis. Histologically, the tumour comprised clear cells and presented a monophasic pattern. Immunohistochemical analysis showed that the tumour cells were positive for both S-100 protein and smooth muscle actin. suggesting that this tumour resembles a subtype of epithelial-myoepithelial carcinoma described in the 1990 WHO international classification of salivary glands. Although some reports describe a clear cell dominant epithelial myoepithelial carcinoma, in this case local invasiveness or regional lymphnode metastasis was not proved through investigation. It is therefore concluded that this was an epithelial myoepithelial tumour rather than a carcinoma.     

Isolation and characterization of a highly divergent HIV-2[GH-2]: generation of an infectious molecular clone and functional analysis of its rev-responsive element in response to primate retrovirus transactivators (Rev and Rex).

A highly divergent HIV-2 designated as HIV-2[GH-2] was obtained from an AIDS-related complex (ARC) patient in Ghana. A full-length molecular clone of this isolate was obtained and a biologically active clone was constructed. Its restriction pattern differed from that of prototype HIV-2[GH-1] in 25 of 35 restriction sites, but was strikingly similar to a previously characterized HIV-2 isolate from a Ghanaian (HIV-2ALT). The conserved integrase region (288-bp fragment) previously displayed 95% identity with that of ALT but 17-20% divergence from the HIV-2 prototype member, and a new distinct subgroup (HIV-2b) of HIV-2 consisting of GH-2 and ALT was postulated (Miura et al. 1991.) These isolates, however, were biologically distinguishable from each other by its replication capacity in a monocyte line, U937, in which GH-2 could not grow but ALT grew well. In addition, the nucleotide sequence of the LTR of this new isolate displays 21% divergence from that of prototype HIV-2[GH-1], but the core enhancer, Sp1 binding sites and TATA box were conserved. Although the 3' half of the env gene sequence which is deleted in HIV-2ALT clone showed 27% diversity from the prototype, functional differences in the rev-responsive element were not observed.     

Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes

To understand how CD8 expression is regulated during the transition process from CD4+8+ (CD4 and CD8 double positive, DP) to CD4-8+ (CD8 single positive, CD8SP) cells in the thymus, the involvement of Runx proteins in the alteration of chromatin configuration was investigated. Using the chromatin immunoprecipitation assay, we first demonstrated that Runx proteins bind to the stage-specific CD8 enhancer, as well as the CD4 silencer, in CD8SP thymocytes. Among Runx family members, Runx3 expression was initiated in DP thymocytes receiving a positive selection signal and increased in concert with differentiation to the CD8SP stage. Furthermore, reactivation of the CD8 gene, as well as CD4 silencing, was suppressed in positively selected thymocytes of Runx dominant-negative transgenic mice. These results suggest that Runx proteins, especially Runx3, are involved in lineage specification of CD8 T cells and provide important information for understanding the mechanism for the mutually exclusive expression of coreceptors in mature thymocytes.     

Various types of corticotectal neurons of cats as demonstrated by means of retrograde axonal transport of horseradish peroxidase

Summary The retrograde labeling of cortical neurons with horseradish peroxidase (HRP) was used to investigate the morphological features of neurons in various cortical areas projecting to the superior colliculus in the cat.Corticotectal cells were found to be labeled in layer V of the entire cerebral cortex. The number of labeled cells and their locations varied according to the sites of injections of HRP in the colliculus. Most of the Corticotectal cells identified in the present study were small (9–20 m in diameter, 66%) and medium (20–40 urn, 30%) pyramidal neurons and only 4% of them were large (more than 40 m). The labeled cells, 261 in total number, had somal diameters of 20.8±8.0 m (mean and SD). The range of sizes of the labeled neurons was different in different cortical areas. For example, the labeled neurons in the Clare-Bishop area had a greater proportion of large diameter cells than in other areas.The present findings are largely in agreement with the previous data of anterograde degeneration methods with respect to the topographical correlation of the Corticotectal projections. However, in some cortical areas, e.g., the sensorimotor and the first visual (area 17) cortex of the lateral surface of the hemisphere, relatively small numbers of Corticotectal neurons appear to have been labeled by retrogradely transported HRP. The sparsity of the labeled neurons in certain cortical areas may reflect the existence of Corticotectal neurons with axon collaterals supplying brain structures other than the superior colliculus.Abbreviations A.c. Aqueductus cerebri - AEct Gyrus ectosylvius anterior - AEs Sulcus ectosylvius anterior - AI Stratum album intermediale - AL Gyrus lateralis anterior - AP Stratum album profundum - AS Gyrus sylvius anterior - Cd Nucleus caudatus - F.l.m. Fasciculus longitudinalis medialis - GI Stratum griseum intermediale - GP Stratum griseum profundum - GS Stratum griseum superficiale - Ic Inferior colliculus - L Left - MEct Gyrus ectosylvius medius - MS Gyrus sylvius medius - MSup Gyrus suprasylvius medius - N.r. Nucleus ruber - O Stratum opticum - P Pontine nuclei - P.c. Pedunculus cerebri - PEct Gyrus ectosylvius posterior - P.g. Periaqueductal gray matter - PSigm Gyrus sigmoideus posterior - PSup Gyrus suprasylvius posterior - R Right - Sc Superior colliculus - S.n. Substantia nigra - Z Statum zonale - II Optic nerve - III and IV Motor nuclei of cranial nerves     

High expression of the CD30 molecule in human decidual cells.

CD30 is a receptor-type membrane protein that belongs to the nerve growth receptor superfamily. It is expressed on Hodgkin's cells and activated lymphocytes, as well as in some human malignancies including malignant lymphomas, embryonal carcinomas, and other mesenchymal tumors. However, whether it is expressed in normal tissues remains unclear. To study the expression and biological function of CD30, we first examined various human tissues by immunohistochemistry. The monoclonal antibody Ber-H2 intensely stained the membranes of decidual and endometrial cells with decidual change in frozen sections. Western blots of these tissues with Ber-H2 revealed bands of 120, 105, and 90 kd as found on CD30-expressing cells. Northern blots of these tissues using a CD30 cDNA probe detected mRNAs of the same molecular mass and variety as those in the positive control cell line HUT 102. These results indicated that CD30 is expressed in human endometrial tissue with decidual change and imply that CD30 expression in endometrial tissue is induced by hormonal control.