Chimpanzee Personality and the Arginine Vasopressin Receptor 1A Genotype.

Polymorphisms of the arginine vasopressin receptor 1a (AVPR1a) gene have been linked to various measures related to human social behavior, including sibling conflict and agreeableness. In chimpanzees, AVPR1a polymorphisms have been associated with traits important for social interactions, including sociability, joint attention, dominance, conscientiousness, and hierarchical personality dimensions named low alpha/stability, disinhibition, and negative emotionality/low dominance. We examined associations between AVPR1a and six personality domains and hierarchical personality dimensions in 129 chimpanzees (Pan troglodytes) living in Japan or in a sanctuary in Guinea. We fit three linear and three animal models. The first model included genotype, the second included sex and genotype, and the third included genotype, sex, and sex × genotype. All personality phenotypes were heritable. Chimpanzees possessing the long form of the allele were higher in conscientiousness, but only in models that did not include the other predictors; however, additional analyses suggested that this may have been a consequence of study design. In animal models that included sex and sex × genotype, chimpanzees homozygous for the short form of the allele were higher in extraversion. Taken with the findings of previous studies of chimpanzees and humans, the findings related to conscientiousness suggest that AVPR1a may be related to lower levels of impulsive aggression. The direction of the association between AVPR1a genotype and extraversion ran counter to what one would expect if AVPR1a was related to social behaviors. These results help us further understand the genetic basis of personality in chimpanzees.

A case of maxillary sarcoma in a chimpanzee (Pan troglodytes).

Oral malignancy is rare in chimpanzees. A 34-year-old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.

Haplotype analysis of the human alpha2-HS glycoprotein (fetuin) gene.

Alpha2-HS glycoprotein (AHSG), which is equivalent to fetuin in other species, is a protein found in human plasma. AHSG is polymorphic with two common alleles and many variants. To examine the intragenic haplotypes and their diversity at this locus, a contiguous genomic DNA sequence (10.3 kb) was analyzed in 20 samples (40 chromosomes), and haplotypes were determined for 309 subjects. Judging from the aligned nucleotide sequences and the conserved amino acid residues comparing human and chimpanzee AHSG, it was concluded that the type 1 allele is probably older and has evolved into four major suballeles. The type 2 allele was generated from one branch of the type 1 allele. AHSG*3 and *5 variants were each found to have a single nucleotide change in exon 7, resulting in the change of an amino acid residue from Arg299 to Cys and from Asp258 to Asn, respectively. It was noted that the AHSG*3 mutation gives rise to an additional cysteine residue, which possibly affects the conformation of the protein. The AHSG gene was found to have a low mutation rate and no apparent recombination events. Furthermore, the detected substitutions were nonhomogeneously distributed at this locus. In particular, four nonsynonymous substitutions were concentrated in the carboxyl-terminal domain.

Induction of adrenomedullin by hypoxia in cultured retinal pigment epithelial cells.

PURPOSE: To explore the effects of hypoxia on the production and secretion of adrenomedullin (ADM) and endothelin (ET)-1 in human retinal pigment epithelial (RPE) cells. METHODS: RPE cells were cultured under normoxic or hypoxic (1% O2) conditions. Expression of ADM and ET-1 was examined by Northern blot analysis and radioimmunoassay. Effects of ADM and ET-1 on the number of RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA expression levels and immunoreactive ADM levels in the medium were increased by hypoxia in all three human RPE cell lines (ARPE-19, D407, and F-0202). Immunoreactive ET was detected in the cultured media of D407 cells and ARPE-19 cells and identified as ET-1 by reversed-phase high performance liquid chromatography. Hypoxia treatment for 48 hours increased immunoreactive ET levels approximately 1.3-fold in the cultured media of D407, but not ARPE-19 cells. Hypoxia decreased the number of ARPE-19 cells and F-0202 cells, and the treatment with ADM ameliorated the hypoxia-induced decrease in the cell number. In contrast, exogenously added ET-1 had no significant effects on the number of ARPE-19 cells under normoxia and hypoxia. CONCLUSIONS: Hypoxia increased the expression of ADM in all three human RPE cell lines, whereas the induction of ET-1 by hypoxia was found only in D407 cells. ADM induced by hypoxia may have protective roles against hypoxic cell damage in RPE cells.

Secretion of inhibin A and inhibin B throughout pregnancy and the early postpartum period in chimpanzees.

Plasma concentrations of inhibin A and inhibin B during pregnancy and early lactation in chimpanzees were determined by enzyme-linked immunosorbent assay (ELISA). Plasma samples were taken from five pregnant chimpanzees at 6-9, 10, 20 and 25 weeks of pregnancy, and following parturition. Throughout pregnancy and the early postpartum period, circulating inhibin A and inhibin B concentrations remained low, at similar levels to those during the normal menstrual cycle in chimpanzees. Concentrations of inhibin A in the placental homogenate were high enough to be measured by the ELISA and by bioassay, whereas circulating inhibin bioactivities in late pregnancy were too low to be measured. Plasma concentrations of FSH remained low with no significant changes throughout pregnancy and the postpartum period. Plasma concentrations of oestradiol-17beta and progesterone at 25 weeks of pregnancy were much higher than normal menstrual cycle levels. It was concluded that in chimpanzees the levels of circulating inhibin A and inhibin B remained low throughout pregnancy and the early postpartum period, and that the concentrations of bioactive dimeric inhibin did not increase towards the end of pregnancy. The suppression of circulating FSH levels during pregnancy is suggested to be controlled by steroid hormones that increased significantly in late pregnancy, and the present findings further suggest that the secretory pattern and role of inhibin during pregnancy in chimpanzees may be different from that in human and other primates.

Expression of tyrosinase-related protein 2/DOPAchrome tautomerase in the retinoblastoma.

Tyrosinase-related protein 2 (TRP-2), also known as DOPAchrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/DOPAchrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.

Microphthalmia-associated transcription factor (MITF): multiplicity in structure, function, and regulation.

Microphthalmia-associated transcription factor (MITF) regulates the differentiation and development of melanocytes and retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Heterozygous mutations in the MITF gene cause auditory-pigmentary syndromes. MITF consists of at least five isoforms, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, differing at their N-termini and expression patterns. Here we show a remarkable similarity between the N-terminal domain of MITF-A and cytoplasmic retinoic acid-binding proteins. To date, four isoform-specific first exons have been identified in the MITF gene: exons 1A, 1H, 1B, and 1M in the 5' to 3' direction, each of which encodes the unique N-terminus of a given isoform. The 5'-flanking regions of these isoform-specific exons are termed A, H, B, and M promoters, respectively. Among these promoters, the M promoter has received particular attention, because it is functional only in melanocyte-lineage cells and is upregulated by Wnt signaling via the functional LEF-1-binding site. Moreover, the M promoter is upregulated by other transcription factors, PAX3, SOX10, and CREB. The activity and degradation of MITF-M are regulated by extracellular signals via protein phosphorylation, such as c-Kit signaling. Together, multiple signals appear to converge on the M promoter as well as on MITF proteins, leading to the proper regulation of MITF-M in melanocytes and other MITF isoforms in many cell types.

Regulation of pigment cell-specific gene expression by MITF.

Melanocyte and retinal pigment epithelium (RPE) specifically express tyrosinase and other melanin-producing enzymes. Microphthalmia-associated transcription factor (Mitf), encoded at the mouse microphthalmia locus, regulates the development and survival of many cell types, including melanocyte and RPE. MITF, the human homolog of Mitf, consists of at least four isoforms with distinct amino-termini, referred to as A, MITF-C, MITF-H, and MITF-M. MITF-M is exclusively expressed in melanocytes and melanoma cells, and thus represents the melanocyte lineage-specific isoform. In contrast, other isoforms are expressed in many cell types so far examined. These isoforms appear to function as transcriptional activators of the melanogenesis genes, as assessed by transient transfection assays in cultured cells. Functional significance of Mitf-M in melanocyte differentiation was verified by the molecular lesion of black-eyed white Mitf(mi-bw) mice, which lack melanocytes but have normal RPE. The Mitf gene of this mutant has the insertion of an L1 retrotransposable element in the intron between exon 3 and exon 4, leading to complete repression of Mitf-M mRNA expression. Taken together, these results suggest that melanogenesis in melanocyte and RPE is regulated by separate Mitf/MITF isoforms. Recent findings on the multiplicity of MITF isoforms are summarized.

Identification of a composite enhancer of the human tyrosinase-related protein 2/DOPAchrome tautomerase gene.

The human tyrosinase-related protein 2 (TRP-2) gene promoter contains a cis-regulatory element (positions -447 to -416), termed DOPAchrome tautomerase distal enhancer 1 (DDE1). DDE1 functions as an enhancer in cultured melanoma cells and its core element includes a potential binding site for transcription factors containing a high-mobility-group domain. This core element is bound in vitro by multiple nuclear proteins, which are preferentially expressed in melanoma cells. DDE1 represents a composite enhancer that may be involved in melanocyte-specific transcription of the human TRP-2 gene.

Entamoeba dispar, but not E. histolytica, detected in a colony of chimpanzees in Japan.

Chimpanzees (Pan troglodytes) residing in the Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho, were surveyed for the presence of intestinal parasites. Stool samples from 107 chimpanzees were examined by microscopy after formalin-ether sedimentation. Of these animals, 100 were infected with at least 1 species of ameba. The positivity rates recorded were as follows: Entamoeba coli, 88%; E. histolytica/E. dispar, 48%; E. hartmanni, 15%; Iodamoeba buetschlii, 8%; Endolimax nana, 4%; and Entamoeba chattoni, 2%. Polymerase chain reaction (PCR) analysis to distinguish between E. histolytica and E. dispar was performed on these samples. E. dispar DNA was detected in 60 of 107 samples (56%), including 9 that had been microscopically determined to be negative for E. histolytica/ E. dispar. In contrast, no E. histolytica DNA was detected in the 107 samples. Zymodeme analysis indicated that 10 isolates were E. dispar. When 104 chimpanzees were examined serologically for E. histolytica infection, 1 sample was scored as positive by indirect hemagglutination and another was found to be positive by an indirect fluorescent antibody test. However, both specimens were borderline-positive and were clearly negative in other tests, suggesting that they might be false-positives. These results demonstrate that the pathogenic E. histolytica was absent in this colony, regardless of the high degree of prevalence of other amebas. For an accurate diagnosis, PCR analysis is recommended in addition to microscopic examination.

Adrenomedullin in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells. METHODS: Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-gamma, tumor necrosis factor-alpha, interleukin (IL)1beta, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-gamma or IL-beta increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it. CONCLUSIONS: Human RPE cells produced and secreted ADM. IFN-gamma and IL-1beta induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.

Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters.

Microphthalmia-associated transcription factor (MITF) affects the development of many types of cells, including melanocytes and retinal pigment epithelium (RPE). MITF consists of at least three isoforms, MITF-A, MITF-H and MITF-M, differing at their amino-termini and expression patterns. Here, we characterize the structural organization of the human MITF gene. The gene contains at least four isoform-specific first exons, exons 1A, 1H, 1B and 1M in the 5' to 3' direction, each of which encodes the unique amino-terminus of a given isoform, including newly identified MITF-B. The 5'-flanking regions of these isoform-specific exons are termed promoters A, H, B and M, respectively, which showed different promoter activities, as judged by transient transfection assay. Promoter A directs the expression of a reporter gene in RPE, cervical cancer and melanoma cells, whereas promoter M is functional only in melanoma cells. Promoter H showed the significant activity in RPE and cervical cancer cells but not in melanoma cells. In contrast, the 1.7 kb 5'-flanking region of exon 1B showed no noticeable promoter activity in these cell lines. Therefore, alternative promoters provide the MITF gene with the diversity in transcriptional regulation and the capability of generating structurally different protein isoforms.

Increased expression of adrenomedullin mRNA in the tissues of intraocular and orbital tumors.

PURPOSE: To explore the possible involvement of adrenomedullin in the pathophysiology of intraocular and orbital tumors. METHODS: Competitive reverse transcription-polymerase chain reaction was used to determine adrenomedullin mRNA levels in the tissues from 40 consecutive patients (40 eyes) undergoing vitrectomy, orbital tissue biopsy, or enucleation for various ocular diseases, including intraocular (n = 4) and orbital (n = 3) tumors, proliferative vitreoretinopathy (n = 8), proliferative diabetic retinopathy (n = 8), age-related macular degeneration (n = 4), preretinal macular fibrosis (n = 9), and acute retinal necrosis (n = 4). RESULTS: Adrenomedullin mRNA levels in the tissues obtained from patients with intraocular or orbital tumors were significantly higher than those of patients with proliferative vitreoretinopathy (P <.05), proliferative diabetic retinopathy (P <.05), preretinal macular fibrosis (P <.005), and acute retinal necrosis (P <.01). CONCLUSION: Adrenomedullin may play a role in the pathophysiology of intraocular and orbital tumors.

Induction of melanocyte-specific microphthalmia-associated transcription factor by Wnt-3a.

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

Changes in plasma concentrations of inhibin A and inhibin B throughout sexual maturation in the male chimpanzee.

Profiles of circulating plasma inhibin A and inhibin B during sexual maturation in male chimpanzees were investigated by using two-site enzyme-linked immunoassay (ELISA). Plasma concentrations of testosterone and pituitary gonadotropins were also measured. Concentrations of inhibin B, testosterone, luteinizing hormone (LH) and prolactin increased with age throughout prepuberty to adulthood, whereas inhibin A level was low and there were no age-related changes in concentrations of either inhibin A and follicle-stimulating hormone (FSH). Inhibin B showed an inverse correlation with FSH in adult (7 years or order) but not in immature (6 years or younger) male chimpanzees. There was no correlation between plasma levels of FSH and testosterone throughout the period of sexual maturation. However, testosterone levels were positively correlated with inhibin B levels. These results suggest that circulating inhibin B is involved in the regulation of FSH secretion after puberty in adult male chimpanzees, and also that circulating inhibin B is an important form of inhibin as a marker of Sertoli cell function in adult male chimpanzees.

Elevated adrenomedullin in the vitreous of patients with proliferative vitreoretinopathy.

PURPOSE: To explore the possible involvement of adrenomedullin in the pathophysiology of proliferative vitreoretinopathy. METHODS: The radioimmunoassay technique was used to determine immunoreactive adrenomedullin concentrations in the vitreous fluid from 41 eyes of 41 patients undergoing vitrectomy for a variety of retinal conditions, including proliferative vitreoretinopathy, proliferative diabetic retinopathy, age-related macular degeneration, and macular hole. RESULTS: Immunoreactive adrenomedullin levels in the vitreous of patients with proliferative vitreoretinopathy were significantly higher than those of patients with proliferative diabetic retinopathy (P < .005), age-related macular degeneration (P < .001), and macular hole (P < .0001). CONCLUSION: Adrenomedullin may be involved in the pathophysiology of proliferative vitreoretinopathy.

Molecular cloning of cDNA encoding a novel microphthalmia-associated transcription factor isoform with a distinct amino-terminus.

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein, and plays an important role in the development of various cell types, such as neural-crest-derived melanocytes and optic-cup-derived retinal pigment epithelium. Three isoforms of MITF with distinct amino-termini have been described. These include melanocyte lineage-specific MITF-M, heart-type MITF-H, and the recently identified MITF-A. Here we identify a fourth isoform, MITF-C, with a unique amino-terminus of 34 amino acid residues, which shares about 43% sequence identity with putative transactivation segments of two previously identified leukemogenic factors, ENL and AF-9. Reverse transcription-polymerase chain reaction analysis revealed that MITF-C mRNA is expressed in many cell types, including retinal pigment epithelium, but is undetectable in melanocyte-lineage cells. In contrast, MITF-A and MITF-H mRNAs are coexpressed in all cell types examined. Transient cotransfection assays suggested that MITF-C, like other MITF isoforms, functions as a transcriptional activator of certain target genes, but its transactivation specificity for the target promoters is different from those of other MITF isoforms. Therefore, isoform multiplicity provides MITF with differential expression patterns as well as functional diversity.

Implications of isoform multiplicity of microphthalmia-associated transcription factor in the pathogenesis of auditory-pigmentary syndromes.

Microphthalmia-associated transcription factor (MITF) is the human homolog of a basic helix-loop-helix-leucine zipper protein (Mitf), encoded by the mouse microphthalmia locus. Mutations in the MITF gene have been identified in some patients with Waardenburg syndrome type 2 (WS2), which is a dominantly inherited disorder, characterized by varying combinations of sensorineural hearing loss and pigmentary disturbances. Furthermore, mice with mutations at the Mitf locus are associated with various phenotypes, such as white coat color, small eyes, a deficiency in mast cells, and osteopetrosis. Thus, MITF/Mitf may play an important role in differentiation of melanocytes and some other cell types. Recently we have identified two MITF isoforms with extended amino-termini, MITF-A and MITF-H. Both isoforms possess unique amino-termini that are different from the amino-terminus of the originally identified melanocyte-specific MITF (MITF-M). MITF-M mRNA is exclusively expressed in melanocytes and pigmented melanoma cells, whereas MITF-A and MITF-H mRNA are widely expressed in many cell types, including retinal pigment epithelium. Transient transfection assays suggested that these isoforms possess differential transactivation capacity. It is therefore conceivable that the previously identified mutations may alter the functions of not only MITF-M but also MITF-A and MITF-H. Possible implications of the MITF isoform multiplicity in the pathogenesis of auditory-pigmentary disorders are discussed.

A big gene linked to small eyes encodes multiple Mitf isoforms: many promoters make light work.

Among more than 80 different loci related to mouse coat color, microphthalmia-associated transcription factor (Mitf) encoded at the mouse microphthalmia locus is one of the most exciting molecules that regulates the development and survival of many cell types, including melanocyte, retinal pigment epithelium (RPE), and mast cells. Mitf and its human homolog MITF consist of at least three isoforms, referred to as Mitf-A/MITF-A, the heart-type Mitf-H/MITF-H, and the melanocyte lineage-specific Mitf-M/MITF-M, respectively. These isoforms differ in the amino-terminal domains but share a transactivation domain and a basic helix-loop-helix and leucine-zipper structure that is required for DNA binding and dimerization. MITF-M is exclusively expressed in melanocytes and melanoma cells, but not in other cell types, including RPE cells. In contrast, MITF-A mRNA is widely expressed in many cell types. These three isoform mRNAs are possibly generated by differential usage of the gene promoters and by alternative splicing. We predict that the entire MITF gene spans about 200 kb of DNA. Like MITF-M, MITF-A is able to activate the two melanogenesis gene promoters, tyrosinase and tyrosinase-related protein 1. These results suggest that melanogenesis may be regulated by different MITF isoforms in melanocyte and RPE. Possible implications of the multiplicity in Mitf/MITF isoforms are discussed.