Identification and analysis of MHC class II DRB1 (Patr-DRB1) alleles in chimpanzees.

The MHC-DRB1 gene is known to display the most extensive allelic polymorphisms among MHC class II genes. We attempted the selective identification of chimpanzee (Pan troglodytes) DRB1 (Patr-DRB1) alleles using the polymerase chain reaction (PCR) technique in three steps: first, we performed Patr-DRB1*02 lineage-specific 8-kb PCR for *02 lineage detection in each chimpanzee; second, we performed 620-bp PCR for amplification of full-length exon 2; and finally, we carried out an insert check using the pattern of microsatellite repeat length variability. In the genomic DNA of 23 chimpanzees, nine Patr-DRB1 alleles containing two new alleles were detected. Our approach provides a relatively effective method of identifying Patr-DRB1 alleles in individual chimpanzees and should also contribute to our understanding of the features of MHC molecules in non-human primates.

Sequence analysis of the MHC class II DPB1 gene in chimpanzees (Pan troglodytes).

The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.

Evidence for natural selection in the HAVCR1 gene: high degree of amino-acid variability in the mucin domain of human HAVCR1 protein.

The family of genes encoding T-cell immunoglobulin and mucin-domain containing proteins (Tim), which are cell-surface molecules expressed in CD4(+) T helper cells, has important roles in the immune system. Here, we report three unusual patterns of genetic variation in the human hepatitis A virus cellular receptor 1 gene (HAVCR1) that are similar to patterns observed in major histocompatibility complex loci. First, levels of polymorphism in exon 4 of HAVCR1 were exceptionally high in humans (nucleotide diversity (pi)=45.45 x 10(-4)). Second, nonsynonymous substitutions and insertion/deletion variants were more frequent than synonymous substitutions in that exon (10 out of 12 variants). The rate of the mean number of nucleotide substitutions at nonsynonymous sites to synonymous sites at HAVCR1-exon 4 is >1 (P(A)/P(S)=1.92 and pi(A)/pi(S)=2.23). Third, levels of divergence among human, chimp, and gorilla sequences were unusually high in HAVCR1-exon 4 sequences. These features suggest that patterns of variation in HAVCR1 have been shaped by both positive and balancing natural selection in the course of primate evolution. Evidence that the effects of natural selection are largely restricted to the mucin domain of HAVCR1 suggests that this region may be of particular evolutionary and epidemiological interest.

Gene arrangement at the Rhesus blood group locus of chimpanzees detected by fiber-FISH.

The Rhesus (Rh) blood group system in humans is encoded by two genes with high sequence homology. These two genes, namely, RHCE and RHD, have been implied to be duplicated during evolution. However, the genomic organization of Rh genes in chimpanzees and other nonhuman primates has not been precisely studied. We analyzed the arrangement of the Rh genes of chimpanzees (Pan troglodytes) by two-color fluorescence in situ hybridization on chromatin DNA fibers (fiber-FISH) using two genomic DNA probes that respectively contain introns 3 and 7 of human RH genes. Among the five chimpanzees studied, three were found to be homozygous for the two-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5'). Although a similar gene arrangement can be detected in the RH gene locus of typical Rh-positive humans, the distance between the two genes in chimpanzees was about 50 kb longer than that in humans. The remaining two chimpanzees were homozygous for a four-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5') - Rh (3'<--5') - Rh (3'<--5') within a region spanning about 300 kb. This four-Rh-gene type has not been detected in humans. Further analysis of other great apes showed different gene arrangements: a bonobo was homozygous for the three-Rh-gene type; a gorilla was heterozygous for the one-Rh- and two-Rh-gene types; an orangutan was homozygous for the one-Rh-gene type. Our findings on the intra- and interspecific genomic variations in the Rh gene locus in Hominoids would shed further light on reconstructing the genomic pathways of Rh gene duplication during evolution.

New chimpanzee MHC class I alleles cloned by polymerase chain reaction.

We describe polymerase chain reaction (PCR) cloning of full-length Patr-A, -B and -Cw locus alleles from a chimpanzee using one sense/antisense primer pair. Of the six alleles cloned here, two have not previously been described. Comparison of nucleotide sequences between the two novel Patr alleles and various HLA alleles suggests that the new Patr-A allele belongs to the A1/A3/A11 family, whereas the new Patr-Cw allele is not closely related to any known HLA-Cw allele.

Mitochondrial 16S rRNA sequence diversity of hominoids.

We determined nucleotide sequences of the 16S rRNA gene of mitochondrial DNA (mtDNA) (about 1.6 kb) for 35 chimpanzee, 13 bonobo, 10 gorilla, 16 orangutan, and 23 gibbon individuals. We compared those data with published sequences and estimated nucleotide diversity for each species. All the ape species showed higher diversity than human. We also constructed phylogenetic trees and networks. The two orangutan subspecies were clearly separated from each other, and Sumatran orangutans showed much higher nucleotide diversity than Bornean orangutans. Some gibbon species did not form monophyletic clusters, and variation within species was not much different from that among species in the subgenus Hylobates.

Effects of atropine sulfate on rat harderian glands: correlation between morphological changes and porphyrin levels.

We examined the correlation between histopathological changes and porphyrin levels in the Harderian glands of male rats after treatment with atropine sulfate. After a single administration of atropine sulfate (250 mg/kg/day), the porphyrin levels in the Harderian glands gradually increased, beginning from 2 hr after administration, and at 36 hr reached a maximum level, which was about 7 times higher than that of the control animals. Histopathologically, the Harderian glands showed marked luminal dilatation and a brownish pigment accumulation in the lumina 6 hr after a single dose. Under daily repeated administrations of atropine sulfate (250 mg/kg/day), the highest porphyrin levels in the Harderian glands were observed 24 hr after the third dose, and were about 9 times higher than those of the control animals. However, beginning from one week after the initial dose, much lower peak porphyrin levels were observed 6 hr after each dose. The maximum porphyrin levels were only twice as high as those of the control animals, and they returned to the control levels 24 hr after each atropine dose. Histological examinations of the Harderian glands revealed that repeated administrations of atropine sulfate induced the same histopathological changes observed after a single atropine administration, and that no aggravated dilation of the lumina or pigmentation in the lumina appeared after such repeated administrations. The degree and incidence of the histopathological changes observed correlated well with the porphyrin levels. Some animals showed a degeneration of the glandular epithelium after 4 weeks of treatment, and the frequency increased slightly after 13 weeks of treatment. The present study suggests that atropine suppresses the expulsion of secretory materials, including porphyrin, from the glandular lumen of the Harderian glands, and thereafter an excessive accumulation of porphyrin induces luminal dilatation. These changes were gradually reduced by repeated administrations. The degeneration of the glandular epithelium after repeated administration might be a consequence of retention of an excessive accumulation of porphyrin.

Species-specific TT viruses and cross-species infection in nonhuman primates.

Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively; P = 0.038). These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.

Difference in sensitivity of inner cell mass and trophectoderm to X-irradiation in mouse blastocysts.

Pregnant B6C3F1 mice were exposed to a single whole body X-irradiation on day 4 (73-74 hr postconception) of gestation. In experiment 1, they were sacrificed at 2, 4, 6, or 9 hr after a dose of 2 Gy, and their embryos were removed and examined with light and electron microscopy. In experiment 2, dose-response effects of irradiation on the embryos were examined 4 hr after doses of 0-4 Gy. In experiment 3, DNA fragmentation (a marker of apoptosis) was observed by 3'-OH nick-end labeling technique. In inner cell mass (ICM) and trophectoderm (TE) of blastocysts exposed to 2 Gy, cells with cytoplasmic degeneration, or dead cells phagocytosed by their neighboring cells, were found. Although morphological features of these dying cells did not reveal typical characteristics of apoptosis such as nuclear condensation and membrane blebbing, DNA fragmentation was detected by nick-end labeling technique. The degenerated cytoplasm consisted of aggregating ribosomes. Degenerated cells began to increase from 2 hr after irradiation and reached maximal at 4 hr in both ICM and TE. The incidences of degenerated cells in ICM were higher than those in TE at any time point. These findings provide evidence that cell death observed in blastocysts after X-irradiation is apoptotic and sensitivity of the two groups of cells (ICM and TE) to X-rays is different.

Maintenance of constant blood acetylcholine content before and after feeding in young chimpanzees.

We have shown that acetylcholine (ACh) is present in the blood of various species of mammals using a specific, sensitive radioimmunoassay. In the present study, the effect on blood and plasma ACh levels of feeding after overnight fasting was studied in one male and five female 4- to 7-year-old chimpanzees. The mean basal ACh concentrations of the blood and plasma were 3143 +/- 380 and 184 +/- 10 pg/ml (+/-SEM, n = 6), respectively. Feeding each chimpanzee 500 g boiled sweet potatoes as breakfast at 1000 h and tap water given ad libitum did not affect the ACh content of the blood and plasma, and constant values of the blood and plasma ACh contents were observed for 4 h after the feeding. Hematocrit and plasma acetylcholinesterase (AChE) activity were also insensitive to feeding. No correlation was observed between plasma AChE activity and either blood or plasma ACh content. The results of the present study indicate that the blood ACh of chimpanzees is distributed mainly in the blood cell fraction, and that the blood ACh content is not regulated directly by cholinergic nerve activity or by plasma AChE activity.

Developmental disturbance of rat cerebral cortex following prenatal low-dose gamma-irradiation: a quantitative study.

Pregnant rats were exposed to a single whole-body gamma-irradiation on Day 15 of gestation at a dose of 0.27, 0.48, 1.00, or 1.46 Gy. They were allowed to give birth and the offspring were killed at 6 or 12 weeks of age for microscopic and electron microscopic examinations of the cerebrum. Their body weight, brain weight, cortical thickness, and numerical densities of whole cells and synapses in somatosensory cortex were examined. Growth of the dendritic arborization of layer V pyramidal cells was also examined quantitatively with Golgi-Cox specimens. A significant dose-related reduction in brain weight was found in all irradiated groups. Neither gross malformation nor abnormality of cortical architecture was observed in the groups exposed to 0.27 Gy. A significant change was found in thickness of cortex in the groups exposed to 0.48 Gy or more. Cell packing density increased significantly in the group exposed to 1.00 Gy. Significant reduction in the number of intersections of dendrites with the zonal boundaries were found in the groups exposed to 0.27 Gy or more. There was no difference in the numerical density of synapses in layer I between the control and irradiated groups. These results suggested that doses as low as 0.27 Gy could cause a morphologically discernible change in the mammalian cerebrum.

Polydactyly Nagoya, Pdn: A new mutant gene in the mouse.

A new hereditary polydactyly (gene symbol Pdn) was found in the course of breeding JCL : ICR mice. The genetic analysis indicated that the polydactyly was an autosomal dominant trait. The homozygotes died within two days after birth. The homozygous fetuses or newborn had 1-3 extra-digits both in te fore- and hindlimbs on the preaxial side. They occasionally showed exencephaly, cleft palate, open eyelid, short tibia and fibula or deformed sternum. The heterozygotes had one extra-digit preaxial side. They occasionally showed exencephaly, heterozygotes had one extra-digit preaxially in the hindlimb and an enlarged first digit on the forelimb which often showed bifurcated distal phalanx. A tab on the postaxial side of the forelimb was found in all homozygotes and in some heterozygotes.

Cathepsin B and collagenolytic cathepsin in the aqueous humor of patients with Behçet's disease.

Cathepsin B and collagenolytic cathepsin activities were studied biochemically in human aqueous humor. The enzyme activities were found to be significantly high in the aqueous humor of the patients with Behçet's disease, but not detectable in those with senile cataract, retinoblastoma, and maxillar carcinoma. These data suggest that cathepsin B and collagenolytic cathepsin have a certain role in the inflammatory collagenolytic process of Behçet's disease.

The distribution and some properties of collagenolytic cathepsin in the bovine eye.

In vitro degradation of insoluble vitreous collagen by the action of collagenolytic cathepsin was studied biochemically. Among bovine ocular tissues, the uvea and the retina showed relatively high collagenolytic activity. The ciliary body revealed the highest specific activities of both cathepsin B and collagenolytic cathepsin. Leupeptin and p-chloromercuribenzoate inhibited both cathepsin B and collagenolytic cathepsin in the ciliary body lysosomes. Pepstatin inhibited cathepsin D, but did not affect cathepsin B and collagenolytic cathepsin. It is suggested that distribution and properties of collagenolytic cathepsin are similar to those of cathepsin B in the bovine eye.

The presence of collagenolytic cathepsin in uveal lysosomes of bovine eye.

Collagenolytic cathepsin, which can liberate soluble hydroxyproline-containing products from insoluble vitreous collagen with maximum activity at pH 3.5, was biochemically studied in uveal lysosomes of bovine eye. Collagen solubilization was proportional to both enzyme concentration and incubation time. When the enzyme was heated, no reaction was observed. Collagen solubilization by uveal lysosomal extract was almost unaffected by Ca2+ ion, cysteine, beta-mercaptoethanol, and ethylenediaminetetraacetic acid, but inhibited about one-third by pepstatin. The possible role of collagenolytic cathepsin in vitreous liquefaction was considered.