Pharmacokinetic/pharmacodynamic analysis of adjunctive perampanel in subjects with partial-onset seizures.

OBJECTIVES: Explore perampanel pharmacokinetics (PK) in all subjects (aged ≥12 years) vs adolescents (aged ≥12 to ≤17 years) with partial-onset seizures (POS) and identify factors explaining between-subject variability in efficacy using a population PK/pharmacodynamic (PD) analysis. MATERIALS & METHODS: Population PK analysis was performed using nonlinear mixed-effect modeling with data from phase II/III randomized, double-blind, placebo-controlled studies of adjunctive perampanel in POS. Perampanel exposure was predicted for all subjects and adolescents. Population PK/PD analyses were performed using data from phase III studies to explore the relationship between perampanel exposure and 28-day average seizure frequency and responder probability. RESULTS: Pooled perampanel PK data from 1318 subjects were described by a one-compartment disposition model. In the absence of antiepileptic drugs (AEDs) affecting perampanel PK, estimated perampanel apparent clearance (CL/F) was 0.668 L/h (all subjects) and 0.682 L/h (adolescent subjects). Co-administration of carbamazepine and oxcarbazepine/phenytoin reduced perampanel exposure. Gender, Asian race (excluding Japanese or Chinese), and increasing alanine aminotransferase lowered perampanel CL/F, but differences were small and not considered clinically relevant. Adolescent outcomes were similar to the total population. Based on PK/PD data from 1748 subjects, percent reduction in 28-day average seizure frequency from baseline and responder probability increased with increasing perampanel exposure; concomitant CYP3A-inducing AEDs lowered perampanel exposure but did not impact the slope for responder probability. CONCLUSIONS: These results are consistent with previous analyses but expand on these through inclusion of a larger number of patients from different ethnic groups, and demonstrate that outcomes were similar between adults and adolescents.

Molecular cloning and phylogeny of HERV-E family that is expressed in Japanese monkey (Macaca fuscata) tissues.

More than 50 copies of HERV-E family have been estimated to exist in the human genome. Here, we examined the expression pattern and their relationships of the HERV-E in Japanese monkey tissues by RT-PCR and sequence analysis. The env gene of HERV-E family was expressed in monkey tissues (testis, prostate, kidney, thymus, intestine and stomach) except for cerebellum, pancreas and ovary, exhibiting that they may have transcriptional potential. Phylogenetic analysis of the HERV-E env family from Japanese monkey tissues and our previous data could be divided into two distinctive groups (I and II). They were integrated into the genomes of anthropoids and have evolved at the rate of 0.3% nucleotide differences per MYr through evolutionary divergence in primate evolution. Divergence times of the two groups were estimated as 11.6 MYr for group I and 41.6 MYr for group II. Those HERV-E sequences were extensively proliferated in the genome of humans and great apes. These data will contribute to further studies on the transcriptional potential of the HERV-E family in the Japanese monkey genome and to biomedical knowledge related to human diseases.

Nucleotide sequence analyses of human complement 6 (C6) gene suggest balancing selection.

The sixth complement component (C6) has a common charge polymorphism, C6A and C6B, with similar gene frequencies in all major populations. In addition, C6B2 is also found in Japanese populations at a frequency of about 6%. Sequence analyses of the coding region of three human and ape C6 alleles indicated four nonsynonymous and three synonymous changes in C6*B2 relative to C6*A, suggesting that a recombination event occurred between C6*B2 and C6*A to give rise to C6*B. Sequence variation in a 3.86 kb region encompassing exon 3, where the causal base change of the common C6 polymorphism is found, indicated that several single nucleotide polymorphisms (SNPs) were in extensive linkage disequilibrium (LD), with little differentiation among populations. Sliding window estimates of two test statistics for neutrality revealed significant values in a subregion where the replacement coding polymorphism resides, in all three human populations. These results raise the possibility that the two common C6 alleles in human populations are maintained by balancing selection.

Population differences in DNA sequence variation and linkage disequilibrium at the PON1 gene.

Polymorphisms of the promoter region (-108C/T) and the coding region (192Q/R) of the paraoxonase 1 gene (PON1) showed differences in association with cardiovascular disease risk in various populations. To characterize the genetic variation underlying these important polymorphisms, we examined DNA sequence variation both in a 1.3-kb promoter region 16.5 kb from codon 192, and in a 1.7-kb region centered on the 192Q/R polymorphic site of the coding region of PON1, in 30 Africans, 30 Europeans and 64 Japanese. We found 10 polymorphic sites and 11 haplotypes in the 1.3-kb promoter region and 10 biallelic polymorphic sites and 10 haplotypes in the 1.7-kb region. From the PON1 sequences of chimpanzees and an orangutan, the ancestral type of codon 192 was found to be R. The number of pairs of polymorphic sites between the promoter and 1.7-kb regions that were in significant linkage disequilibrium was much higher in a Japanese population than in African and European populations. In addition, the pairs of polymorphic sites in linkage disequilibrium differed among the three populations. These results suggest that some of the population differences in association with risk for coronary heart disease can be explained by population differences in haplotype frequency of PON1 haplotypes.

Endogenous retrovirus HC2 pol fragments in the squirrel monkey: expression, evolution, and phylogeny.

Human endogenous retrovirus HC2 is an incomplete provirus containing the entire gag and pol genes and a 3' LTR, whereas the 5' LTR and env gene are missing. We investigated expression of the HC2 pol gene in the squirrel monkey ( Saimiri sciureus) by RT-PCR. The pol gene was expressed in cerebellum, liver, lung, and spleen of the squirrel monkey, but not in six other tissues tested. RT-PCR products were cloned and sequenced resulting in seven sequences that were analyzed. These sequences showed 73.7-89.2% sequence similarity to HC2 pol genes present in the human genome. No frameshifts or termination codons caused by deletion/insertion or point mutation were found in clones SM-HC27-1 and SM-HC27-4 isolated from squirrel monkey lung tissues. Phylogenetic analysis showed that HC2 pol elements from the squirrel monkey were randomly clustered with those in human genome and the genomes of other nonhuman primates, indicating that substantial evolution of the HC2 elements occurred prior to primate speciation with additional evolution of the elements, independent of each other, after speciation.

Molecular characterization and phylogenetic relationship of HERV-W family in the Macaca fuscata.

A human endogenous retrovirus, HERV-W, has recently been identified on human chromosome 7 and contains a single complete open reading frame putatively encoding an envelope protein. Its env gene was expressed in various tissues, and mainly in human placenta. We investigated env gene of the HERV-W family in the Macaca fuscata (Japanese monkey). The env gene expression was detected in various tissues (testis, prostate, kidney, cerebellum, thymus, placenta, intestine, stomach, ovary) of the Japanese monkey by RT-PCR. Southern blot analysis indicated that the monkey genome contained at least 15 copies of the HERV-W family. Using the PCR approach with the monkey genome, thirteen env fragments of the HERV-W family were identified and analyzed. These env fragments from monkey showed a high degree of sequence similarity (91-94%) to that of human HERV-W (AF072506). Putative amino acid sequences of the env fragments indicated multiple frameshifts and termination codons by deletion/insertion or point mutation in all clones identified in this study. Phylogenetic analysis of the env fragments derived from the monkey genome with those of the human genome showed a random sister relationship.

Isolation and phylogeny of endogenous retrovirus HERV-F family in Old World monkeys. Brief report.

A new human endogenous retroviral family (HERV-F) has recently been identified from human chromosome 7q31.1-q31.3 that was identical to the XA34 cDNA clone isolated from a human glioma cDNA library with an ERV-9 env probe. We investigated pol fragments of the HERV-F family from the Old World monkeys (crab-eating monkey, African green monkey, and baboon) and analyzed these with the HERV-F (Hu-XA34). Fifteen pol fragments of the HERV-F family were detected from the Old World monkeys. They showed a high degree of sequence similarity (81-99%) with that of the HERV-F (Hu-XA34). Phylogenetic analysis of pol fragments with those of the human genome distinctively showed five groups, indicating that HERV-F family could be amplified at least five times after the original integration into the monkey genome or represent integration events independently during primate evolution.

Phylogeny of SINE-R retroposons in Asian apes.

The SINE-R retroposon family was derived from the long terminal repeats (LTRs) of human endogenous retrovirus K (HERV-K) that had been active during the hominoid evolution. The retroposons and HERV-K LTR elements have potential relevance to structural change and genetic variation of the hominoid genome. In our previous study, we found that the SINE-R retroposons were hominoid specific. Here we identified seventeen new SINE-R retroposons (14 from orangutan and 3 from gibbon) from Asian apes and phylogenetically analysed them in comparison with those of the humans and African great apes. None of the retroposons from Asian apes were closely related to SINE-R.C2 that is human specific, and originally identified in the gene for the C2 component of complement, whereas some retroposons (Ch-M10, Ch-M16, Gor-M, Gor-F1, Gor-M6, and Gor-F9) from African great apes showed very close relationship with that of the SINE-R.C2 retroposon. The phylogenetic tree based on the SINE-R retroposons revealed wide overlap of the retroposons across species, suggesting that the SINE-R retroposons have been evolved parallel pattern in the course of hominoid evolution.

[DNA PCR typing of non-human primates]. / DNA-PCR-Typisierung an nicht-menschlichen Primaten.

Human beings and non human primates show similarities in the non coding DNA range too, but up to now there are only a few data. This paper presents first results of a study dealing with a larger spectrum of species and individuals, considering the genetic marker HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC (partionally coding) and VWA, FES, F13B, TH01, CD4, FGA (not coding). The results show that not only the apes can be typed but also Macaca sylvanus as a member of the Cercopithecoidea. In contrast to earlier publications there is an unexpected larger similarity between the allele ranges of the apes studied and those of human beings.

Phylogenetic position of Eulipotyphla inferred from the cDNA sequences of pepsinogens A and C.

Although to date the phylogenetic position of the provisional order Eulipotyphla has been assessed by various molecular markers, it has not been conclusively clarified due to low statistical supporting values and inconsistent results. To clarify the phylogenetic position of Eulipotyphla, we cloned cDNAs for pepsinogens A and C from five mammalian species belonging to four different orders and determined their nucleotide sequences. Molecular phylogenetic analysis based on the 1st and 2nd codon positions of the protein-coding region of cDNA sequences strongly supported the close relationship between Eulipotyphla and Chiroptera. Carnivora was found to be a sister group to these two orders. The monophyly of the order Rodentia and that of the cohort Glires (Rodentia and Lagomorpha) was also shown by the present phylogenetic trees of pepsinogens.

Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques.

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment.

Contrasting patterns of polymorphisms at the ABO-secretor gene (FUT2) and plasma alpha(1,3)fucosyltransferase gene (FUT6) in human populations.

The coding sequences ( approximately 1 kb) of FUT2 [ABO-Secretor type alpha(1,2)fucosyltransferase] and of FUT6 [plasma alpha(1,3)fucosyltransferase] were analyzed for allelic polymorphism by direct sequencing in five populations. The nucleotide diversities of FUT2 estimated from pairwise sequence differences were 0.0045, 0.0042, 0.0042, 0.0009, and 0.0008 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. The nucleotide diversities of FUT6 were 0.0024, 0.0016, 0.0015, 0.0017, and 0.0020 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. At FUT2, excesses in pairwise sequence differences compared to the number of polymorphic sites as indicated by a significantly positive Tajima's D were observed in European-Africans and in Iranians. The data do not fit expectations of the equilibrium neutral model with an infinite number of sites. On the other hand, Tajima's D's at FUT6 in each of the five populations and at FUT2 in Africans, Chinese, and Japanese were not significantly different from zero. F(ST) between the Asians and the others measured at FUT2 was higher than at FUT6. These results suggest that natural selection was responsible for the generation of the FUT2 polymorphism in European-Africans and in Iranians.

Unique occurrence of the 1CF11 carbohydrate epitope in primate saliva.

We examined a large number of individual human and animal saliva samples for the reactivity with ICF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of ICF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with ICF11.

Genomic and spectral analyses of long to middle wavelength-sensitive visual pigments of common marmoset (Callithrix jacchus).

The genetic basis of red-green color vision of common marmoset (Callithrix jacchus) is not fully understood. Here, we have cloned and characterized the three alleles at a locus that encode the long to middle wavelength-sensitive (LWS/MWS) visual pigments of this species. Using in situ hybridization, we localized this locus to the telomeric region of the long arm of X chromosome. The three visual pigments achieve the wavelengths of maximal absorption at 561, 553, and 539 nm and fully explain the red-green color vision of the common marmoset. The 'tri-allelic single-locus X-chromosome' model operates under the unique phenomenon, known as blood chimerism.

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.

Evolution of the X-linked zinc finger gene and the Y-linked zinc finger gene in primates.

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.

Evolution of the ABO blood group gene in Japanese macaque.

We determined 5 sequences of Japanese macaque ABO blood group gene exon 7 (ca. 0.5 kb) and 2 sequences for exon 5 and intron 6 (ca. 1.7 kb). We compared those data with published sequences of other Old World monkey species, and the results suggest that alleles A and B were polymorphic in the ancestral species of macaques, and that B type allele evolved independently in macaque and baboon lineages.

Structural variations of the VWA locus in humans and comparison with non-human primates.

The HUMVWA locus was examined in 160 samples from the Japanese population. A total of 142 fragments were sequenced, and the counterpart sequences were also determined in non-human primates. In humans, 10 different alleles were found; they could be grouped into seven allelic classes based on the total number of repeats. No variation was observed in the alleles 17, 18 and 19, which showed consensus sequence structures and in the allele 14, which showed a different structure. New variation was found in alleles 15, 16, and 20, which had differences occurred in a basic (TCTA)(TCTG)(n) repeat in the 5' side. The counterpart fragments were successfully amplified in three species (chimpanzees, gorilla, and orangutan) out of four kinds of anthropoids, three species (rhesus macaques, Japanese macaques, and green monkey) out of four kinds of old world monkeys, but not in one species of either new world monkey or prosimian. The sizes of the fragments distributed from 92 to 180 bp in non-human primates and showed allelic size differences in four species. The sequence of the 5' flanking region followed by primer sequences in humans and anthropoids, which consisted of 19 bp, was identical in all, but differed from that in old world monkeys. The basic repeat motifs of humans and anthropoids consisted of TCTA, TCTG, and TCCA but that of old world monkeys consisted of TCTG, TCCG and TCCA The structures of humans and anthropoids were essentially similar, but with characteristic difference in each species. Differences in the allelic structures of old world monkeys were complex. Seven different alleles were observed in two rhesus and two Japanese macaques and one type of allele was observed in two green monkeys. Duplication of more than two repeat units of 4 bp was found in an allele of an old world monkey. These data illuminate interesting features of mutational changes in STRs during the long generations and also some insight into evolutional aspects of primates.

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase.

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.

Multiplicities and some enzymatic characteristics of ape pepsinogens and pepsins.

Pepsinogen levels in ape stomachs were comparable to those in macaques and significantly higher than those in the stomachs of other mammals, including carnivores and ruminants. The occurrence of multiple forms of pepsinogens was remarkable. Nine, sixteen, eight, and fourteen pepsinogens were purified or partially purified from the gastric mucosa of a gibbon, orang-utan, gorilla, and chimpanzee, respectively. Most of these were type-A pepsinogens, and only one type-C pepsinogen was identified in each ape. The two types could be readily distinguished by staining for proteolytic activity on polyacrylamide gel electrophoresis (PAGE) in the presence/absence of pepstatin. Type-A pepsinogens were further divided into two subtypes. One subtype, constituting a major group of pepsinogens in apes, exhibited high hemoglobin-digestive activity. The other subtype was specified by a relatively high content of Lys and low hemoglobin-digestive activity. It is likely that pepsinogen-A genes have been duplicated several times as hominoids, including humans, evolved in the primate lineage. The presence of multiple pepsinogens in apes might be advantageous in the efficient digestion of a wide variety of foods.