Polymorphism, Heteroplasmy, Mitochondrial Fusion and Diabetes

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPAls that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the influence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.     

Chemoenzymatically synthesized glycoconjugate polymers

Glycoconjugate polymers with poly(vinyl alcohol) (PVA) backbone were synthesized via a chemoenzymatic method. The sugar alcohols of maltose and lactose were submitted to transesterification in the presence of lipases. The esterification was achieved with high selectivity and yield, and the resulting maltitol and lactitol 6-vinyl sebacates were polymerized by a conventional radical initiator with hydrogen peroxide and ascorbic acid. The glycoconjugate polymers carrying alpha-glucose and beta-galactose as recognition signals showed the biological activity such as lectin recognition abilities and hepatocyte adhension. The biodegradability of these polymers was modest but higher than PVA.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

Synthesis of Methylenecyclobutyl- and Cyclobutenyl Adenine,Potent Antiviral Carbocyclic Analogues of Oxetanocin A

Abstract

9-Cyclobutyladenines bearing both methylene and hydroxymethyl groups, 3 and 4, were prepared by dehydration of carbocyclic oxetanocin A (1a). Introduction of a double bond into cyclobutane ring was achieved by allylic oxidation of N ⁶-benzoyl-9-[3-methylenecyclobutyl]adenine (12), which after several steps, afforded 9-[3-(hydroxy-methyl)-2-cyclobutenyl)adenine (5).     

Identification of the mouse killer immunoglobulin-like receptor-like (<Emphasis Type="Italic">Kirl</Emphasis>) gene family mapping to Chromosome X

Natural killer (NK) inhibitory receptors, which recognize major histocompatability complex (MHC) proteins in humans, are known as killer Ig-like receptors (KIRs) and are encoded by a multi-gene immunoglobulin (Ig) superfamily. In a screen for genes differentially expressed in the mouse thymus, we discovered the first close rodent homologue of the NK receptor KIR family, which we named KIR- Like (Kirl). KIRL1 shares 40% amino acid identity with primate KIR family members, with the majority of the homology contained within the Ig-like ectodomains. KIRL1 is more similar to the KIRs than to any other known member of the Ig domain-containing leukocyte receptor superfamily. This highly significant homology suggests that the KIR family did not arise independently in primates, as has been previously suggested, but rather evolved from a primordial gene already present in the common rodent/primate ancestor. KIRL1 lacks the cytoplasmic protein motifs that mediate inhibition in KIRs (immunoregulatory tyrosine inhibiting motif, ITIM); KIRL1 also lacks the transmembrane activation signature (a conserved K residue involved in association with the immunoregulatory tyrosine activating motif-containing DAP12 molecule) found in some KIRs. Nevertheless, we hypothesize that Kirl1 is functional, for the following reasons: (1) Kirl1 mRNA is expressed at high levels in immature thymocytes; (2) Kirl1 is regulated during thymocyte development; (3) KIRL1 protein is detected in thymus. We also show that the mouse genome contains a closely related, transcribed gene, which we name Kirl2. Kirl2 encodes a KIR-like molecule with three Ig-like domains and also lacks tyrosine-based immunoregulatory motifs in its cytoplasmic region.     

Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins

A molecular test for Alzheimer's disease could lead to better treatment and therapies. We found 18 signaling proteins in blood plasma that can be used to classify blinded samples from Alzheimer's and control subjects with close to 90% accuracy and to identify patients who had mild cognitive impairment that progressed to Alzheimer's disease 2-6 years later. Biological analysis of the 18 proteins points to systemic dysregulation of hematopoiesis, immune responses, apoptosis and neuronal support in presymptomatic Alzheimer's disease.     

Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4(+) T cell lines generated in vitro were adoptively transferred into Rag-2(-/-) mice with primed B cells derived from Dsg3-immunized Dsg3(-/-) mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Ralpha in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.