Development‐dependent expression of calreticulin in the brain and other tissues of the Japanese monkey,Macaca fuscata

Background Calreticulin (Crt) is a molecular chaperone localized to endoplasmic reticulum lumen. The protein is involved in the correct folding of glycoproteins and its cellular level changes depending on various physiologic conditions. Methods To clarify the Crt level in various tissues of the Japanese monkey during postnatal development, an enzyme‐linked immunosorbent assay system that we have established was applied. Results and Conclusions Calreticulin is distributed ubiquitously in various tissues. Its level in the heart, lung, liver, spleen, and kidney was high in newborns and decreased in juveniles and adults. In various cerebral areas, Crt was present in the gray matter but scarcely found in the white matter. The Crt levels in the cerebral areas were low in newborns and increase in juveniles and adults. These distribution and developmental changes in Crt might be correlated with the quality control of glycoproteins that are synthesized in respective tissues.     

Molecular cloning and gene expression of endoplasmic reticulum stress proteins in Japanese monkey, Macaca fuscata

BACKGROUND: Immunoglobulin heavy-chain binding protein (BiP), calreticulin (Crt), and protein disulfide isomerase (PDI), are major resident endoplasmic reticulum (ER) stress proteins which are involved in diverse roles relating to successful folding, assembly, intracellular localization, and degradation of other proteins. METHODS: In this study, we molecular cloned cDNAs for BiP, Crt, and PDI from Japanese monkey (Macaca fuscata), and analyzed tissue-specific expression of respective genes. RESULTS AND CONCLUSIONS: The lengths of protein-coding regions of these cDNAs for BiP, Crt and PDI are 1965, 1254, and 1533 bp, respectively. Each protein has a signal peptide and a KDEL motif in N- and C-terminal parts respectively, showing its intracellular localization to be the lumen of the ER. These stress proteins are highly conserved, showing that their similarities among mammals are more than 90% in the level of amino acid. The expression of the genes for stress proteins differed among the monkey tissues examined. BiP and PDI gene expression was predominant in secretory tissues such as liver and kidney, and brain tissues. But Crt gene expressed rather ubiquitously in a variety of tissues.     

Detection of elevated antibody against calreticulin by ELISA in aged cynomolgus monkey plasma

Calreticulin (Crt) is a molecular chaperone ubiquitously present in the endoplasmic reticulum. In non-human primates, age-related occurrence of anti-Crt antibody has not been reported. We developed an ELISA assay for an anti-Crt antibody and determined the age-related increase in the levels of anti-Crt antibody in three groups of cynomolgus monkeys: juvenile (1.5 yr), young adults (5-10 yr) and aged adults (20-34 yr). Mean ± SD auto-antibody levels at 450 nm in juvenile, young adults and aged groups were 0.23 ± 0.18, 0.30 ± 0.28, and 0.55 ± 0.33, respectively. Statistically significant differences were noted in the autoantibody levels to Crt among the aged group and juvenile or young adults. This is the first report to demonstrate the expression of anti-Crt autoantibody in aged monkeys and indicates that cynomologous monkeys may serve as an appropriate nonhuman primate model for studies of age-related alteration of immune function in elderly humans. Though preliminary, this finding merits further investigation to determine the relationship between immunosenescence and expression of antibodies to Crt.     

Distribution of three arbovirus antibodies among monkeys (Macaca fascicularis) in the Philippines

Serum samples from 54 monkeys were collected from healthy individuals in a monkey farm in Luzon island, Philippines, in 1999, and examined by IgM-capture ELISA and indirect IgG ELISA for the presence of dengue (DEN), Japanese encephalitis (JE) and chikungunya (CHIK) viruses. The positive rates for IgM ELISA were 3.7, 35.2 and 14.8% against DEN, JE and CHIK, respectively. Higher positive rates were obtained when indirect IgG ELISA was used: 100% against flaviviruses (JE or DEN) and 59.3% against CHIK virus. The results indicate a high prevalence of flavivirus infections such as JE and DEN, and a lesser prevalence of CHIK virus infections, among monkeys in the Philippines. These findings suggest possible sylvatic transmission cycles of these viruses.     

Differences among isolates of simian hemorrhagic fever (SHF) virus

Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (less than or equal to 50) in persistently infected patas monkeys (P-248, P-741). LVR lytically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.     

Rhesus monkey (Macaca mulatta) model of Helicobacter pylori: noninvasive detection and derivation of specific-pathogen-free monkeys.

BACKGROUND AND PURPOSE: Development of the rhesus monkey model of Helicobacter pylori has been hampered by problems with serodetection and by the difficulty of identifying specific-pathogen (Helicobacter)-free animals. Our purpose was to determine whether detection could be improved and to determine if pathogen-free monkeys could be derived by nursery rearing. METHODS: An enzyme-linked immunoabsorbent assay (ELISA) and a [14C]urea breath test were compared to endoscopy to determine H. pylori infection status in rhesus macaques; 18 animals were hand raised in the nursery to determine whether pathogen-free animals could be selected. RESULTS: Helicobacter pylori infection was common in colony-raised young rhesus monkeys and was nearly universal by adulthood. Serodetection, using antigen from rhesus-derived H. pylori strains, was 95% sensitive and 94% specific. The [14C]urea breath test was 96% sensitive and 88% specific for detection of chronic Helicobacter infection in rhesus monkeys. Segregation of newborn animals within the first 24 h of life was a reliable method to obtain pathogen-free rhesus monkeys. CONCLUSION: Isolation of specific-pathogen-free animals, together with better detection methods, may improve the value of the rhesus monkey model for the study of H. pylori pathogenesis, immune response, and vaccine development.     

Species preference by infant macaques with controlled social experience

In Experiment 1, infant Japanese monkeys and rhesus monkeys were artificially reared in pairs with conspecific or heterospecific monkeys. Preferences of these monkeys for a variety of pictures of Japanese monkeys and rhesus monkeys were repeatedly tested during the first 1 or 2 years of life. The duration of lever-pressing responses to see those pictures was a measure of the preference. All monkeys, Japanese or rhesus, preferred pictures of rhesus monkeys to pictures of Japanese monkeys, without regard to their social experiences. Experiment 2, with an adult Japanese monkey as the subject, and Experiment 3, with different pictures as stimuli, suggested that this preference was not a consequence of any bias in the pictures used. In Experiment 4, a Japanese monkey reared by a rhesus foster mother and rhesus monkeys reared by Japanese monkey mothers received the same preference test. The Japanese monkey infant preferred to see pictures of rhesus monkeys. However, rhesus infants did not show clear species preferences. These results suggest that infants of both Japanese and rhesus monkeys have a native tendency to prefer to see physical characteristics of rhesus monkeys over Japanese monkeys.     

High-performance liquid chromatographic analysis of the sulfation of 4-hydroxypropranolol enantiomers by monkey liver cytosol

We developed a new high-performance liquid chromatographic method using an ODS column and a chiral column for the assay of racemic 4-OH-PL sulfate and enantiomeric 4-OH-PL sulfates, respectively. The method was successfully applied to measure phenolsulfotransferase (PST) activities for 4-OH-PL in cytosolic fractions from livers of Japanese monkeys (Macaca fuscata) and for comparison with its activity of cytosolic fractions from rat, rabbit, dog, and human livers and Hep G2 cells. The activity was ranked as Hep G2 cells > monkeys = humans = dogs = rats > rabbits. To evaluate the Japanese monkey as a nonhuman animal model in drug metabolism studies, we further characterized sulfation of 4-OH-PL as a further metabolic pathway in monkey livers to compare that with human livers. Inhibition studies in which cytosolic fractions were preincubated at 43 degrees C or 2,6-dichloro-4-nitrophenol (DCNP) used as a PST inhibitor indicated that two kinds of PSTs, thermolabile, low-Km and DCNP-resistant PST and thermostable, high-Km and DCNP-sensitive PST were involved in 4-OH-PL sulfation by monkey liver cytosol, which is very similar to the reported profile of 4-OH-PL sulfation by human liver cytosol. Sulfation kinetics in a low concentration range of 4-OH-PL enantiomers demonstrated that apparent Km values were similar between human and monkey liver cytosolic fractions, but the Vmax values were different, so that intrinsic clearance values (Vmax/Km, Clint) were higher in monkeys than in humans. Furthermore, enantiomer selectivity of [R(+)-4-OH-PL > S(-)-4-OH-PL] was observed in the Vmax and CLint values of monkey liver cytosol. These results indicate that the profile of sulfation of 4-OH-PL by liver cytosolic fractions is similar in humans and Japanese monkeys.     

The serodiagnosis of Campylobacter infection in infant cynomolgus monkeys (Macaca fascicularis) 2 to 18 weeks old by enzyme-linked immunosorbent assay

We established the enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Campylobacter and applied it in defining the period of the primary infection of Campylobacter in infant cynomolgus monkeys (Macaca fascicularis). The antibody to Campylobacter spp. could be detected with only 0.25 mul of serum by using commercially available antigens and anti-cynomolgus monkey IgG antibody conjugated with alkaline phosphatase. The inhibition experiments using extracts of C. jejuni, C. fetus and Yersinia enterocolitica demonstrated that the established ELISA system could detect species-specific anti-C. jejuni and anti-C. fetus antibodies. The levels of antibodies to both C. jejuni and C. fetus were high in 2 weeks old infant cynomolgus monkeys, rapidly decreasing until 6 to 14 weeks of age. This result indicates that the antibodies detected in 2 week old infants were IgG antibodies of maternal origin transferred through placenta. The C. jejuni was isolated from infants when the level of maternal antibody became the lowest. Infant cynomolgus monkeys obviously developed IgG antibodies to C. jejuni within 4 weeks after infection. On the other hand, no antibody response to C. jejuni was found in two infants from which it could not be isolated throughout the observation period. As regards C. fetus infection, infants showed a poor antibody response although it was more frequently isolated than C. jejuni. In conclusion, the ELISA system established in the present study is useful for the serological diagnosis of C. jejuni infection during infancy in the cynomolgus monkey.     

Annual changes in hair length of the Japanese monkey (Macaca fuscata fuscata)

The hair length of Japanese monkeys was investigated for a period of one year and the molting phenomenon was clarified. Nine monkeys were employed in the study. The molting of the Japanese monkey was found to be of a seasonal type and occurred once during the year. The molting continued for one to four months in each monkey. The hair of the Japanese monkeys was wholly replaced during the period from April to August. The hair length was thus short in summer, and long in winter. Hair replacement in pregnant females began after parturition and was generally later than that in other individuals. During molting, both new and old hairs could be observed simultaneously in the same region of the body. The hair replacement ended around summer when the hair became the shortest. The new hairs continued to grow after molting and became the longest towards autumn or winter. Thus, the summer coat and the winter coat were essentially the same in the Japanese monkey. Such annual changes in the hair of the Japanese monkey were considered to be suitable for the climate of Japan.     

Detection of a unique genotype of monkey B virus (Cercopithecine herpesvirus 1) indigenous to native Japanese macaques (Macaca fuscata)

The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting the co-evolution of BV and macaques.     

Intra-troop spacing mechanism of the wild Japanese monkeys of the Koshima troop

The average frequencies of communicative behavior, social behavior, and social encounters (inter-individual proximity within three meters) per hour for a monkey were obtained in their natural habitat by tracing several adult males and females of a Japanese monkey troop living in the Koshima islet. The spatial distribution patterns and the density of troop members within the expanse of the troop at any moment were investigated by tracing several adult femals. Frequency distributions of the monkeys found within five and 10 meters were compared with a Poisson distribution. The frequencies of social encounters and of social interactions of Japanese monkeys were distinctly low, except between mothers and their offspring. The density of monkeys within the expanse of the troop at any moment was very low. Both aggressive behavior and inter-individual proximity (within three meters) were distinctly low when monkeys were foraging for natural food. An avoiding mechanism among troop members plays an important role in maintaining the social structure of these Japanese monkeys. This mechanism works in two ways: each individual does not approach others too closely; the density of monkeys within the expanse of the troop is low at all times.     

High-performance liquid chromatographic determination of the calcium channel blocker nimodipine in monkey plasma

A new high-performance liquid chromatographic (HPLC) assay was developed for the determination of nimodipine in monkey plasma. An ethyl acetate extraction procedure was employed with a reversed-phase HPLC separation for the analysis. Absolute recovery of nimodipine from plasma was over 95% with a lower limit of quantitation of 10 ng/ml. This method was applied to a preliminary pharmacokinetic study in which 0.25 mg/kg nimodipine was administered intravenously to three monkeys. Protein binding and stability of nimodipine in monkey plasma were also examined. The pharmacokinetic parameters of nimodipine in monkeys were similar to those obtained in humans and indicate that monkeys are an appropriate animal model for further pharmacokinetic investigations.     

Morphological studies ofMacaca fuscata: VI. Somatometry

Measurements of various parts of the head and body and weighing the body were carried out on about 170 adult Japanese monkeys (Macaca fuscata) and the results are noted with separate statistics for respective local groups. Intraspecific comparisons in the Japanese monkey and interspecific comparisons in macaques are discussed from the somatometrical point of view. Among macaques, the Japanese monkey has a comparatively large body, a very short tail, relatively wide biacromial and biiliac breadths, and markedly la ge intermembral and intercrural indices. The Japanese monkey itself shows various local variations. The most conspicuous difference is to be found between the so-called Yaku monkey living on Yaku islet (Yakushima), south of Kyushu, and the monkeys living in other parts of Japan, and, therefore, it is understandable that the Yaku monkey has been distinguished as a subspecies (M. f. yakui) of the Japanese monkey. The Yaku monkey has a somewhat small body, a relatively large head, wide hips, and slender hands and feet.     

Phylogenetic relationships among Japanese, rhesus, Formosan, and crab- eating monkeys, inferred from restriction-enzyme analysis of mitochondrial DNAs [published erratum appears in Mol Biol Evol 1988 Sep;5(5):601]

Mitochondrial DNA (mtDNA) polymorphisms in four species of macaques, i.e., Japanese monkey (Macaca fuscata), rhesus monkey (M. mulatta), Formosan monkey (M. cyclopis), and crab-eating monkey (M. fascicularis), were analyzed to study phylogenetic relationships. When 17 restriction enzymes of 6-bp recognition were used, 42-49 sites were observed in the samples. The estimated number of nucleotide substitutions per site among Japanese, rhesus, and Formosan monkeys ranges from 0.0318 to 0.0396, and that between the crab-eating monkey and the other monkeys from 0.0577 to 0.0653. These findings suggest that the crab-eating monkey diverged from the other three approximately 1.5-3.0 Myr before the present (Mybp) and that the Japanese, rhesus, and Formosan monkeys diverged approximately 0.9-1.8 Mybp, although the branching order cannot be determined conclusively.      

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.     

An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction

An appropriate loading control is critical for Western blot analysis. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction. This study was undertaken to look for an appropriate loading control for western blot analysis of ischemic myocardium. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. The heart tissue samples from different areas and time points after surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. However, the protein level of GAPDH was stable in the Rhesus monkey model. We concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.     

Measurement of serum IgE antibodies against Japanese cedar pollen (Cryptomeria japonica) in Japanese monkeys (Macaca fuscata) with pollinosis.

IgE antibodies against allergens of Japanese cedar (Cryptomeria japonica, CJ) pollen in the serum of seven Japanese monkeys (Macaca fuscata) with pollinosis were measured by fluorometric indirect enzyme-linked immunosorbent assay (ELISA). All of the monkeys were found to have specific IgE to the crude pollen antigen. The specific IgE levels were well correlated with those determined by the Pharmacia CAP system. IgE antibodies were then assayed with two kinds of purified allergens (Cry j I and Cry j II) by the ELISA. We found that five monkeys had specific IgE to both allergens, although the other two had IgE only to Cry j I or Cry j II; there is different immune responsiveness to the two major allergens in the monkeys.     

Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

A cytogenetic study on congenital limb malformations in the Japanese monkey (Macaca fuscata)

A cytogenetic investigation was performed on 88 Japanese monkeys (Macaca fuscata) with abnormal limbs from 11 free-ranging provisioned troops including nine individuals with abnormalities indistinguishable as to whether they were congenital or injurious. All of the monkeys with abnormal limbs including the nine questionable individuals had the same karyotypes as those of normal individuals. The chromosome number was 42, consisting of 20 bi-arm autosome pairs and a submetacentric X-chromosome and Y-chromosome. The ninth chromosome pair, which was the only chromosome pair with remarkable secondary constriction, displayed length polymorphism of the centromeric C-band and secondary constriction in both deformed and normal monkeys. These kinds of variants have also been commonly found in other monkey species, which have almost the same karyotype as the Japanese monkey and have not been reported to show frequent occurrence of limb malformation. We concluded therefore that chromosomal abnormalities could be excluded from the main causal factors for limb malformations of the Japanese monkey.