Annual two-dose tetanus toxoid vaccination induces protective humoral immunity to all age groups of rhesus macaques.

A tetanus outbreak occurred during 2014-2015 in the rhesus macaques reared in an open enclosure in our facility. As the soil of the facility was suspected to be contaminated with Clostridium tetani spores, there was a risk of further tetanus occurring among the macaques. To protect them from tetanus, a tetanus toxoid vaccination was recommended; however, the vaccinated elderly animals might not be effectively protected due to insufficient humoral immune responses. Hence, we evaluated the dynamics of antibody responses among rhesus macaques of all age groups vaccinated with two-dose tetanus toxoid at a 1-year interval during a 3-year follow-up study. The vaccination developed anti-tetanus toxin-specific antibodies in animals of all age groups, the antibody levels peaked 1 year after the second vaccination, and the peak levels decreased with age. However, the levels among elderly individuals (aged ≥13 years) were still higher than the threshold level, which was supposed to protect them from tetanus development. Although the rhesus macaques in our facility had a risk of occasional exposure to the spores due to the outbreak, no incidence of tetanus has ever occurred to date. These results indicate that the vaccination protocol is effective in protecting not only younger but also older animals from tetanus.

Hypercholesterolemia induced by spontaneous oligogenic mutations in rhesus macaques (Macaca mulatta).

BACKGROUND: A rhesus macaque with the fourth highest plasma cholesterol (CH) levels of 501 breeding macaques was identified 22 years ago. Seven offspring with gene mutations causing hypercholesterolemia were obtained. METHODS: Activity of low-density lipoprotein receptor (LDLR), plasma CH levels and mRNA expression levels of LDLR were measured after administration of 0.1% (0.27 mg/kcal) or 0.3% CH. RESULTS: Activity of p. (Cys82Tyr) of LDLR was 71% and 42% in the heterozygotes and a homozygote, respectively. The mRNA expression level of LDLR in the p. (Val241Ile) of membrane-bound transcription factor protease, site 2 (MBTPS2, S2P protein) was 0.83 times lower than normal levels. LDLR mRNA levels were increased for up to 4 weeks by administration of 0.3% CH before suddenly decreasing to 80% of the baseline levels after 6 weeks. CONCLUSION: Oligogenic mutations of p. (Cys82Tyr) in LDLR and p. (Val241Ile) in MBTPS2 (S2P) caused hypercholesterolemia exceeding cardiovascular risk levels under a 0.1% CH diet.

The effect of different body positions on the cardiothoracic ratios obtained by chest radiography in Japanese macaques (Macaca fuscata) and rhesus macaques (Macaca mulatta).

BACKGROUND: Although some studies have reported cardiac diseases in macaques, an adequate screening method for cardiac enlargement has not yet been established. This study aimed to evaluate the positioning of macaques for radiographs and establish reference intervals for the cardiothoracic ratio (CTR). MATERIALS AND METHODS: We developed a device for chest radiography in the sitting position and performed chest radiography in 50 Japanese and 48 rhesus macaques to evaluate the CTR and chest cavity size. RESULTS: In Japanese and rhesus macaques, the thorax height was significantly larger, the heart width was significantly smaller, and the mean CTR was significantly smaller in the sitting position than in the prone position. The reference intervals for CTR in the sitting position were 51.6 ± 4.6% and 52.2 ± 5.1% in Japanese and rhesus macaques, respectively. CONCLUSION: Thoracic radiographic images obtained in a sitting position resulted in a smaller CTR and a larger thorax height, which could be useful for detecting pulmonary and cardiac abnormalities.

Hematological and blood chemistry values in captive Japanese macaques (Macaca fuscata fuscata).

BACKGROUND: Biological information about captive Japanese macaques, including hematology and blood chemistry, is still lacking despite the fact that ethological and ecological data have accumulated during decades of field research. METHODS: Hematological (511 examinations of 280 Japanese macaques) and blood chemistry data (between 33 and 284 examinations from between 29 and 257 individual macaques) in clinically healthy, simian retrovirus-free Japanese macaques tested between 2009 and 2013 were reviewed. RESULTS AND CONCLUSIONS: Specific hematological and blood chemistry data for Japanese macaques without clinical signs of disease were provided in this study. Averages presented can be used as hematological parameters for Japanese macaques. Some differences between Japanese macaques and other closely related macaque species were found. Some parameters varied according to macaque age and sex, as well as regional origin. The data in this study will provide useful clinical indices for Japanese macaques in captive and similar conditions.

Clinical characteristics in two patients with partial lipodystrophy and Type A insulin resistance syndrome due to a novel heterozygous missense mutation in the insulin receptor gene.

AIMS: The present report aimed to clarify the clinical characteristics in a girl at the age of 12 and her mother with partial lipodystrophy and Type A insulin resistance syndrome. METHODS: We examined fat distribution in the patients using dual-energy X-ray absorptiometry, magnetic resonance imaging, and computed tomography. We performed genetic analysis to examine the causal gene for lipodystrophy and insulin resistance. RESULTS: Both patients had partial lipodystrophy and a novel heterozygous missense mutation (Asn1137 → Lys1137) in the insulin receptor gene. Because Asn1137 in the catalytic loop is conserved in all protein kinases, this mutation was thought to impair insulin receptor function. By whole-exome sequencing, we found the proband had neither mutations in candidate genes known to be associated with familial partial lipodystrophy nor novel likely candidate causal genes. Taken together, we thought that fat loss in these two patients might be caused by insulin receptor dysfunction. The proband had amenorrhea due to polycystic ovary syndrome. Her menstruation improved, as fat loss was restored during adolescence. This might be caused by improving insulin resistance due to increased levels of leptin and fat mass. CONCLUSIONS: This case might help to understand the mechanisms insulin receptor dysfunction that cause lipodystrophy.

Seasonal variation of energy expenditure in Japanese macaques (Macaca fuscata).

Animals living in seasonal environments must adapt to a wide variation of temperature changes which requires flexible adjustments of time budget and metabolic processes for efficient thermoregulation. The Japanese macaque (Macaca fuscata) is one of only a handful of nonhuman primate species that experience seasonal climates over a wide temperature range. We used behavior observations, accelerometer sensors and the doubly-labelled water (DLW) method to measure activity and total daily energy expenditure (TDEE) of M. fuscata housed in captivity but exposed to natural seasonal variations at day lengths ranging from 10 to 12 h and temperature ranging from 0° to 32°C. Although overall activity was significantly lower in winter compared to summer and autumn, we found no effect of temperature on day-time activity. However nocturnal inactivity and mean length of sleeping bouts significantly increased along a gradient of decreasing temperatures from summer through winter, suggesting the importance of adaptive behavioral thermoregulation in this species. Energy expenditure that was unaccounted for by Basal Metabolic Rate (BMR) and physical activity i.e. expended through diet-induced thermogenesis or thermoregulation was between 14% and 32%. This residual energy expenditure differed between summer/autumn and winter and was relatively consistent across individuals (approximately 5-8% higher in winter). The percentage of body fat and residual energy expenditure were negatively correlated, supporting that fat storage was higher when less energy was required for thermoregulation. Our results suggest that physiological mechanisms like behavioral and autonomic thermoregulation enable M. fuscata to adapt to wide fluctuations in environmental conditions which provide insights into the evolutionary adaptations of nonhuman primates in seasonal climate.

Night Monkey Hybrids Exhibit De Novo Genomic and Karyotypic Alterations: The First Such Case in Primates.

Using molecular chromosomal analyses, we discovered night monkey hybrids produced in captivity from matings between a female Aotus azarae boliviensis (2n = 50) and a male Aotus lemurinus griseimembra (2n = 53). The parents produced seven offspring in total, including one male and six females-a pattern consistent with Haldane's rule. Chromosomal studies were conducted on four of the hybrid offspring. Two of them showed relatively "simple" mixture karyotypes, including different chromosome numbers (2n = 51, 52), which were formed because of a heteromorphic autosome pair in the father (n = 26, 27). The other two hybrid monkeys exhibited de novo genomic and karyotypic alterations. Detailed analysis of the alterations revealed that one individual carried a mixture karyotype of the two parental species and an X chromosome trisomy (53,XXX). The second individual displayed trisomy of chromosome 18 (52,XX,+18) and a reciprocal translocation between autosomes 21 and 23 (52,XX,+18,t(21;23)). Interestingly, the second monkey exhibited mosaicism among blood cells (mos52,XX,+18[87]/52,XX,+18,t(21;23)[85]), but only a single karyotype (52,XX,+18) in skin fibroblast cells. The X- and 18-trisomies were derived from a doubling of the mother's chromosomes in early embryonic cell division, and the reciprocal translocation likely developed in the bone marrow of the offspring, considering that it was observed only in blood cells. Such occurrence of trisomies in hybrid individuals is a unique finding in placental mammals.

Endozoochorous seed dispersal by Japanese macaques (Macaca fuscata): Effects of temporal variation in ranging and seed characteristics on seed shadows.

Variation in seed shadows generated by frugivores is caused by daily, seasonal, and inter-annual variation in ranging, as well as inter-specific variability in gut passage times according to seed characteristics. We studied the extent to which seed weight, specific gravity, and daily (morning, afternoon, and evening) and inter-annual (2004 vs. 2005) variation in ranging affected seed shadows generated by wild Japanese macaques (Macaca fuscata) in northern Japan. The macaques ingested fleshy fruits of 11 species during the two year study period; Viburnum dilatatum (Caprifoliaceae: heavier seeds with higher specific gravity) and Rosa multiflora (Rosaceae: lighter seeds with lower specific gravity) were eaten frequently in both years. The travel distances of macaques after feeding on V. dilatatum and R. multiflora fruits were estimated by combining feeding locations and ranging patterns measured in the field with gut passage times of model seeds in captive animals. Median travel distances after fruit feeding were 431 (quantile range: 277-654) and 478 m (265-646), respectively, with a maximum of 1,261 m. Neither year nor time of day affected travel distances. The gut passage time of model V. dilatatum seeds was longer than that of model R. multiflora seed, but this did not affect dispersal distances. Seed shadows for both species over 2 years showed unimodal distribution (peak: 101-500 m) and more than 90%, 20%, and 3% of ingested seeds were estimated to be dispersed >100, >500, and >1000 m, respectively, the longest known distances among macaque species. R. multiflora seeds tended to be dispersed further in 2004 than 2005, but V. dilatatum seeds were not, implying that inter-annual variations in ranging pattern due to the distribution and abundance of nut fruiting could affect dispersal distance.

Cilostazol inhibits interleukin-1-induced ADAM17 expression through cAMP independent signaling in vascular smooth muscle cells.

Increased A disintegrin and metalloprotease 17 (ADAM17) expression in vascular smooth muscle cells (VSMC) is implicated in the development of cardiovascular diseases including atherosclerosis and hypertension. Although cilostazol, type III phosphodiesterase (PDE III) inhibitor, has recently been found to inhibit VSMC proliferation, the mechanisms remain largely unclear. Here, we hypothesized that cilostazol regulates the ADAM17 expression in VSMC. In cultured VSMC, interleukin (IL)-1α and IL-1ß significantly increased ADAM17 expression. MEK inhibitor U0126, NF-κB inhibitor BAY-11-7085, and siRNA targeting p65/RelA significantly inhibited IL-1α or IL-ß-induced ADAM17 expression. Cilostazol significantly inhibited IL-1α or IL-1ß-induced extracellular signal-regulated kinase (ERK) phosphorylation and ADAM17 expression. Unexpectedly, cilostamide, dibutryl cAMP, and forskolin did not affect IL-1-induced ADAM17 expression. Our results clearly demonstrated that IL-1 induces ADAM17 expression through ERK/NF-κB activation in VSMCs. Moreover, the inhibitory effects of cilostazol on IL-1-induced ADAM17 expression may be independent of the cAMP signaling pathway in VSMC. These novel findings may provide important clues to understanding the expression mechanisms of ADAM17 and the inhibitory mechanisms of cilostazol in VSMC proliferation.

Proteomic profiling of autophagosome cargo in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.

Selective autophagy regulates insertional mutagenesis by the Ty1 retrotransposon in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic components and is ubiquitously found in eukaryotic cells. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia superfamily, is the most abundant retrotransposon in the yeast Saccharomyces cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like particles (VLPs) localized in the cytoplasm. Here we show that selective autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to autophagosomes by an interaction with Atg19. We propose that selective autophagy safeguards genome integrity against excessive insertional mutagenesis caused during nutrient starvation by transposable elements in eukaryotic cells.

Selective transport of alpha-mannosidase by autophagic pathways: identification of a novel receptor, Atg34p.

In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.

Fetal sex determination of macaque monkeys by a nested PCR using maternal plasma.

Non-invasive fetal sex determination is required for biomedical studies, in which some sexual difference would be expected in fetal events, in order to make a choice of male or female fetus. To detect male fetal DNA of the sex-determining region Y gene (SRY) in maternal macaque plasma, nested real-time PCR using the SYBR Green system was developed. In all cases of pregnant macaques with male fetuses, a nested PCR product of SRY was amplified from the mother's plasma, while no amplicon was detected in any case of pregnancy with a female fetus. Interestingly, fetal SRY DNA appeared to be cleared rapidly from the maternal blood after parturition. The current method is sensitive and can be performed with a regular PCR machine.

Large congenital melanocytic nevi presenting with lissencephaly with an absent corpus callosum.

A neonatal case of provisional neurocutaneous melanosis presenting with lissencephaly is reported. Several congenital nevi were observed on the trunk and extremities of the infant, including a giant congenital hairy nevus over the skull. Brain magnetic resonance imaging revealed a marked ventricular dilatation with pachygyria and an absent corpus callosum; however, an injection of gadolinium did not demonstrate any enhanced lesions. Histopathological investigations by a brain biopsy showed a disorganized and anomalous embryonic cerebral architecture, suggesting lissencephaly. The detailed mechanism of this combined pathology is difficult to explain; however, a developmental disturbance was suggested to be present in both the neural crest cells and the neuroepithelial cells, resulting in the development of neurocutaneous melanosis accompanied with lissencephaly.

cAMP-response element binding protein (CREB) regulates cyclosporine-A-mediated down-regulation of cathepsin B and L synthesis.

Cyclosporin A (CsA) is an immunosuppressant with severe side effects including gingival overgrowth. We have previously reported that CsA impairs the activity of the lysosomal enzymes cathepsin B and L in human gingival fibroblasts (HGFs). Here, we have examined the effects of CsA on the DNA-binding activity of the cyclic AMP response element-binding protein (CREB) and cell viability, and the effects of CREB on cathepsin B and L synthesis and activity in HGFs. We have confirmed that CsA down-regulates cathepsin B and L synthesis. Further, CsA has no effect on cell viability and dramatically impairs CREB-DNA binding activity. Importantly, the synthesis of cathepsin B and L is down-regulated, and their activity is also significantly impaired in HGFs transfected with plasmid expressing dominant-negative CREB. These results suggest that CREB is essential for the CsA-mediated down-regulation of cathepsin B and L synthesis in HGFs.

The management of puerpera by visiting midwives one month after delivery.

We studied the importance of the management of puerpera by visiting midwives. We investigated their need for midwives in baby care and for themselves. The contents of their requirements were baby care counsel, breast massage and breast-feeding support. Mothers in a one-month period after delivery wanted breast-feeding support more increasingly than in one week, which means that out of the hospital, they couldn't know whether their babies were satisfied with their breast-feeding or not. We should assist them continuously to one month.

Characterization of obesity in Japanese monkeys (Macaca fuscata) in a pedigreed colony.

BACKGROUND: Japanese monkey, Macaca fuscata, is recognized as the monkey species inhabiting the northernmost area in the world, and thus likely to possess unique fat-depositing mechanisms to resist cold weather in winter. We report that obese females are present in the Wakasa group of Japanese monkey reared in an open enclosure of the Primate Research Institute, Kyoto University. METHODS AND RESULTS: Eight of 12 females were categorized as obese, showing percentage body fat of over 22%. The levels of serum leptin (mean +/- SD, 4.9 +/- 2.3 ng/ml) measured in these obese monkeys were significantly higher than those of non-obese peers of the same group (n = 4; 1.2 +/- 0.5 ng/ml) and another Japanese monkey group (Takahama, n = 14; 0.8 +/- 0.25 ng/ml); however, serum levels of adiponectin, insulin, glucose, hemoglobin A1c, and fructosamine did not differ between obese and non-obese monkeys. Few serum lipid parameters such as triglyceride and cholesterol showed lower levels in obese monkeys than their non-obese peers. CONCLUSIONS: These results show that these obese monkeys in the Wakasa group have not developed obesity-related diseases/disorders such as diabetes. In the Wakasa group, the frequency of obese individuals was high in some maternal lineages, suggesting that genetic factors responsible for obesity may have been inherited in these lineages.

Peters' anomaly with bilateral perisylvian polymicrogyria and abdominal calcification.

We report a neonatal case of Peters' anomaly with bilateral perisylvian polymicrogyria and abdominal calcification. The male infant was born after a normal labor. Bilateral central corneal opacities with iridocorneal strands indicated Peters' anomaly. The X-ray and abdominal computed tomography demonstrated multiple calcifications beneath the diaphragma around the liver and the spleen. TORCH serology was negative. Intracranial calcification was not detected. Brain magnetic resonance imaging demonstrated bilateral perisylvian polymicrogyria. Abdominal calcification was suspected to be related to vascular disruption. Bilateral perisylvian polymicrogyria has been thought to result from ischemic events such as intrauterine hypotension or vascular occlusions. Based on these considerations, we conclude that a vascular disruption sequence may an important pathogenetic mechanism of Peters' anomaly.