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We investigated the effect of α-cyclodextrin (α-CD) on the bacterial populations of gut microbiota, production of organic acids, and short-chain fatty acids (SCFAs), and lipid metabolism in obese mice induced by feeding a high-fat diet (HFD). Male C57BL/6J mice were assigned to three diet groups: normal diet (ND) (5% [w/w] fat), HFD (35% [w/w] fat), and HFD (35% [w/w] fat) + 5.5% (w/w) α-CD for 16 weeks. Increases in body and epididymal adipose tissue weights were observed in the HFD group compared with the ND group, which were attenuated in the HFD+α-CD group. The supplementation of α-CD increased the total number of bacteria, Bacteroides, Bifidobacterium, and Lactobacillus that were decreased in gut microbiota of mice by feeding the HFD. Importantly, α-CD administration increased the concentrations of lactic acid and SCFAs, such as acetic, propionic, and butyric acids, and decreased glucose concentrations in cecal contents. Furthermore, supplementation of α-CD upregulated the gene expression of peroxisome proliferator-activated receptor (PPAR)γ involved in adipocyte differentiation and PPARα involved in energy expenditure and downregulated that of sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase involved in fatty acid and triglyceride synthesis in adipose tissue. This study revealed that the alteration in gut microbiota and increased production of lactic acid and SCFAs by supplementation of α-CD have beneficial antiobesity effects via modulating the expression of genes related to lipid metabolism, indicating a prebiotic property of α-CD. © 2018 BioFactors, 2018.

Macaca fascicularis and Macaca mulatta are two of the most commonly used laboratory macaques, yet their genetic differences at a genome-wide level remain unclear. We analysed the multilocus DNA sequence data of 54 autosomal loci obtained from M. fascicularis samples from three different geographic origins and M. mulatta samples of Burmese origin. M. fascicularis shows high nucleotide diversity, four to five times higher than humans, and a strong geographic population structure between Indonesian-Malaysian and Philippine macaques. The pattern of divergence and polymorphism between M. fascicularis and M. mulatta shows a footprint of genetic exchange not only within their current hybrid zone but also across a wider range for more than 1 million years. However, genetic admixture may not be a random event in the genome. Whereas randomly selected genic and intergenic regions have the same evolutionary dynamics between the species, some cytochrome oxidase P450 (CYP) genes (major chemical metabolizing genes and potential target genes for local adaptation) have a significantly larger species divergence than other genes. By surveying CYP3A5 gene sequences of more than a hundred macaques, we identified three nonsynonymous single nucleotide polymorphisms that were highly differentiated between the macaques. The mosaic pattern of species divergence in the genomes may be a consequence of genetic differentiation under ecological adaptation and may be a salient feature in the genomes of nascent species under parapatry.

Impaired axonal transport may promote pathogenesis in neurodegenerative disorders, such as Alzheimer's disease (AD). We previously showed that tau, amyloid precursor protein (APP), and intracellular amyloid β-protein (Aβ) accumulate in the nerve-ending fraction of aged monkey brains, perhaps because of impaired axonal transport. In the present study, we assessed age-related changes of axonal transport motor proteins in aged monkey brains. Western blotting showed that kinesin, dynein, and dynactin (DYN) localizations dramatically changed with aging, and dynein level in nerve-ending fractions increased significantly. Coimmunoprecipitation analyses showed that DYN–dynein intermediate chain (DIC) interactions decreased, suggesting that age-related attenuation of this interaction may cause the impairment of dynein function. Moreover, RNAi-induced down-regulation of DIC in human neuroblastoma cells caused endogenous tau and APP to accumulate, and their subcellular localizations were also affected. Our findings suggest that aging attenuates DYN–DIC interaction, representing one of the risk factors for age-related impaired dynein function and even for accumulation of disease proteins. © 2007 Wiley-Liss, Inc.

A novel system for substrate-specific activation of carboxylic acids leading to the formation of carboxamides has been developed in our laboratory. A combination of a water-soluble dehydrocondensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), and an inverse phase-transfer catalyst (IPTC) (hydroxypropyl)cyclodextrin (HP-β-CD), in a water/ether biphasic solvent system was found to be most effective. A lipophilic carboxylic acid with a strong affinity for the cavity of HP-β-CD can be selectively transferred to the aqueous phase and predominantly reacts with DMT-MM, dissolving in the aqueous phase. The substrate specificity was similar to that observed with a complex artificial enzyme based on CD. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

We previously reported that peripheral lymphocytes from about 12% of cynomolgus monkeys lacked reactivity with anti-rhesus monkey CD3 monoclonal antibody (FN18). The nucleotide sequence analysis of the genes encoding CD3 component proteins revealed that a single amino acid substitutions found in the CD3epsilon chain determined the phenotype. In this study, we attempted to develop a method based on the restriction fragment length polymorphism (RFLP) and apply it for determination of the genotypes of individual monkeys. Comparison of the phenotype determined by fluorescence-activated cell sorter analysis with the genotype determined by RFLP analysis revealed that the FN18 -positive trait was dominant over the FN18-negative trait. It was also revealed that allele frequency was significantly different among macaques depending on the geographical region where their ancestors were derived from.

The age-related increase of peripheral CD4+ CD8+ double-positive (DP) T cells in cynomolgus monkeys has been reported previously. Because the percentage of DP T cells in cynomolgus monkeys increases abruptly in parallel with the thymic involution occurring at around 11 years of age, it was suggested that thymic involution was associated with this increase. Therefore, a longitudinal study was carried out over 5 years to clarify the exact time when DP T lymphocytes start to increase in relation to the thymic involution. Twelve cynomolgus monkeys at 6 years of age were classified into three groups, based on their percentage of DP T cells, as follows: DP-High (5% DP T cells); DP-Middle (1-5% DP T cells); and DP-Low (1% DP T cells). In the DP-High group, the percentage of DP T cells showed an abrupt increase, of10%, in monkeys at 7 years of age, and the prevalence of this subset correlated with a distinctive increase in the percentage of memory T cells (CD4+ CD29(high), CD8+ CD28-), indicating an association with the maturation of immune function, including thymic involution. To assess the thymic function, the coding joint of T-cell receptor excision circles (cjTREC) levels in sorted T cells were analysed by polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA). The cjTREC in the T cells of the DP-High group (4362 +/- 3139 copies/105 T cells) was significantly lower than that (22 722 +/- 4928 copies/105 T cells) of the DP-Low group. Moreover, the mean copy number of cjTREC in naive T cells was also significantly different between the DP-High and the DP-Low group (0.457 +/- 0.181 and 1.141 +/- 0.107, respectively). These findings suggest that thymic involution has an influence on the age-related increase of DP T cells in cynomolgus monkeys.

The FN18 monoclonal antibody (mAb), directed to CD3 molecules, did not react with the lymphocytes of some cynomolgus monkeys (Macaca fascicularis), because of the polymorphism of the CD3epsilon chain. The epitope recognized by the FN18 mAb was successfully expressed on COS7 cells upon transfection of plasmid DNA coding for the CD3epsilon derived from T cells of a FN18 positive cynomolgus monkey. By construction and expression of plasmid DNA encoding the mutant CD3epsilon, the amino acid residue at position 67 was demonstrated to be involved in the formation of an epitope recognizable by the FN18 mAb.

A vest and tethering system was applied to Japanese macaques (Macaca fuscata) to collect blood samples under less stressful conditions. The level of plasma cortisol was assayed to assess the amount of stress. The pattern of plasma cortisol fluctuation during 24 hours was consistent under non-stress conditions. The plasma cortisol level was increased significantly by encounter and venipuncture. The increase was especially noteworthy when the experimental macaques encountered a large adult male, reflecting the strong stress on macaques. As venipuncture is a common medical procedure, and the encounter between macaques who have never met before occurs frequently, this vest and tethering system is useful for the assessment of the amount of stress in the daily care of macaques in a laboratory.

Summary Telomere length is a good biomarker to study the cellular senescence as well as aging of an organism, because it regulates the replicative capacity of vertebrate somatic cells. To demonstrate age-related telomere length dynamics in the peripheral blood mononuclear cells (PBMC) of the cynomolgus monkey, we introduced a novel method of measuring telomere length by fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH). A highly significant correlation was observed between the intensity of telomere-specific fluorescence by Flow FISH and telomere length by Southern blot analysis (R = 0·923, n = 22). The intensity of telomere fluorescence in PBMC significantly decreased with age in 55 monkeys aged from 0 to 34 years and this decrease corresponded to the loss of 62·7 base pairs per year (R = − 0·52, P

Background The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning. Methods Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34+ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells. Results Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes. Conclusions Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

Autoimmune hemolytic anemia (AIHA) is a disorder associated with the destruction of red blood cells (RBCs) by autoantibodies. We report a rare case of AIHA in an infant rhesus macaque (Macaca mulatta) which received a continuous administration of four drugs, a dopamine agonist. dopamine receptor inhibitor, and two gamma-aminobutyric acid receptor inhibitors into the brain during the course of neurophysiological experiments. The main clinical findings were severe anemia and splenomegaly. Hematological and serological examinations revealed the appearance of peripheral erythroblasts and autoantibodies against RBCs. Medical treatments, including washed RBC transfusion and corticosteroids, transiently improved the animal's anemia, but euthanasia was decided on 331 days after the start of the experiment. The pathological findings revealed severe anemia, splenomegaly, and extramedullary hematopoiesis in the liver and kidneys. These findings and the clinical course suggest that this anemia was a warm-antibody type of AIHA induced by the administration of the drugs for the neurophysiological experiment.

Objective To define which regions of the type II collagen (CII) molecule result in anticollagen antibody production and the subsequent development of autoantibodies in a collagen-induced arthritis (CIA) nonhuman primate model. Methods Male and female cynomolgus monkeys (2-6 of each sex per group) were immunized with either chicken (Ch), human, or monkey (Mk) CII, or with cyanogen bromide (CB)-generated peptide fragments of ChCII emulsified in Freund's complete adjuvant. Monkeys were observed for the development of arthritis, and sera were collected and analyzed for anticollagen antibody specificity by enzyme-linked immunosorbent assay. Results Overt arthritis developed in all groups of monkeys immunized with intact CII and with all major CB peptide fragments of ChCII except CB8. Onset and severity of arthritis correlated best with serum anti-MkCII antibody levels. The levels of IgG autoantibody to MkCII were a result of the cross-reactivity rate of anti-heterologous CII antibodies with MkCII, which was based on the genetic background of individual monkeys rather than on sex differences. Conclusion CII from several species and disparate regions of the CII molecule were able to induce autoantibody-mediated arthritis in outbred cynomolgus monkeys. The strong anti-MkCII response suggests that epitope spreading or induction of broad-based CII cross-reactivity occurred in these animals. Autoantibody levels to MkCII were higher in CIA-susceptible monkeys than in resistant monkeys, despite comparable antibody levels in response to the various immunizations of CII. These results closely parallel the type of anticollagen responses found in sera from rheumatoid arthritis patients. Perhaps this can be accounted for by similar major histocompatibility complex heterogenicity associated with an outbred population, or maybe this is a primate-specific pattern of reactivity to CII.

We have previously reported a cynomolgus monkey (Macaca fascicularis) pedigree with early onset macular degeneration that develops drusen at 2 yr after birth. In this study, the molecular composition of drusen in monkeys affected with late onset and early onset macular degeneration was both characterized. Involvement of anti-retinalautoimmunity in the deposition of drusen and the pathogenesis of the disease was also evaluated. Funduscopic and histological examinations were performed on 278 adult monkeys (mean age=16.94 yr) for late onset macular degeneration. The molecular composition of drusen was analyzed by immunohistochemistry and/or direct proteome analysis using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Anti-retinal autoantibodies in sera were screened in 20 affected and 10 age-matched control monkeys by Western blot techniques. Immunogenic molecules were identified by 2D electrophoresis and LC-MS/MS. Relative antibody titer against each antigen was determined by ELISA in sera from 42 affected (late onset) and 41 normal monkeys. Yellowish-white spots in the macular region were observed in 90 (32%) of the late onset monkeys that were examined. Histological examination demonstrated that drusen or degenerative retinal pigment epithelium (RPE) cells were associated with the pigmentary abnormalities. Drusen in both late and early onset monkeys showed immunoreactivities for apolipoprotein E, amyloid P component, complement component C5, the terminal C5b-9 complement complex, vitronectin, and membrane cofactor protein. LC-MS/MS analyses identified 60 proteins as constituents of drusen, including a number of common components in drusen of human age-related macular degeneration (AMD), such as annexins, crystallins, immunoglobulins, and complement components. Half of the affected monkeys had single or multiple autoantibodies against 38, 40, 50, and 60 kDa retinal proteins. The reacting antigens of 38 and 40 kDa were identified as annexin II and mu-crystallin, respectively. Relative antibody titer against annexin II in affected monkeys was significantly higher than control animals (P

The dehydration of aldoximes with 2-methylene-1,3-dioxepane (MDO) proceeds smoothly in the presence of a catalytic amount of Lewis acid such as scandium(III) triflate to give corresponding nitriles in moderate to high yields under mild conditions.

4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was quantitatively synthesized by the coupling of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine in THF, and fully characterized. Condensation of car☐ylic acids and amines by DMTMM proceeded effectively in THF to give the corresponding amides in good yield.

Selective formation of carboxamides in an alcohol or water by an exceptionally convenient one-step procedure in which a condensing agent is simply added to a mixture of acids and amines has been achieved successfully by using a new condensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). Activation of carboxylic acids by DMT-MM in the presence of amines and subsequent aminolysis of the resulting acyloxytriazine in alcoholic solvents occurred selectively and led to the formation of carboxamides in excellent yields. The rate of aminolysis of the acyloxytriazine intermediate can be estimated to be about 2×104 times greater than that of methanolysis. The amide/ester selectivity observed using DMT-MM was much larger than that obtained with DCC or EDC. Condensation of polar substrates, such as amino acid esters and their hydrochlorides, glucosamine hydrochloride, sodium acetate and dicarboxylic acids, proceeded successfully in MeOH, water or aqueous MeOH in good yields. The present reaction is technically quite simple and easy to achieve. It proceeds by simple mixing of acids, amines and DMT-MM without any additives, and the MeOH is readily removable by a rotary evaporator after completion of the reaction.

Vitamin D-binding protein (DBP) of crab-eating macaques (Macaca fascicularis) was examined by means of three electrophoretic methods. DBP phenotypes were observed to be one or two bands in each method. All of DBP molecular variants could be detected by the simultaneous typing with these three methods. Family analysis suggested that DBP variants followed the mode of autosomal codominant inheritance. A total of 17 phenotypes governed by at least 11 alleles were observed in the populations of Malaysia, Indonesia, and the Philippines. The genetic variability was high in Malaysian and Indonesian populations but low in the Philippine population.

The mode of inheritance of four different red cell antigens (Ecy, Fcy, Gcy, and Hcy), detected by alloimmune reagents prepared by us for the cynomolgus monkey, was studied. The results of the segregation analysis suggest that each antigen is inherited in an autosomal dominant manner. Both the random sample analysis and family study revealed that two of these red cell antigens (Ecy and Fcy) probably represent the products of two independent genes while the expression of the other two antigens (Gcy and Hcy is governed by two autosomal codominant alleles. Thus, three simian-type blood group systems could be demonstrated by the four newly developed alloimmune reagents. We use the following nomenclature for these blood groups: the E and F blood groups, each with two phenotypes, antigen positive (E and F) and antigen negative (e and f), and the GH blood group having three phenotypes (G, H, and GH).