Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids.

Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 µM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations.

Cues to androgens and quality in male gibbon songs.

Animal vocal signals may provide information about senders and mediate important social interactions like sexual competition, territory maintenance and mate selection. Hence, it is important to understand whether vocal signals provide accurate information about animal attributes or status. Gibbons are non-human primates that produce loud, distinctive and melodious vocalizations resembling more those of birds than of other non-human primates. Wild gibbons are characterized by flexibility in social organization (i.e., pairs and multimale units) as well as in mating system (i.e., monogamy and polyandry). Such features make them a suitable model to investigate whether the physiology (hormonal status) and socio-demographic features find their correspondence in the structure of their songs. By combining male solo song recordings, endocrine outputs using non-invasive fecal androgen measures and behavioral observations, we studied 14 groups (10 pair-living, 4 multimale) of wild white-handed gibbons (Hylobates lar) residing at Khao Yai National Park, Thailand. We collected a total of 322 fecal samples and recorded 48 songs from 18 adult animals. Our results confirmed inter-individuality in male gibbon songs, and showed a clear correlation between androgen levels and song structures. Gibbons with higher androgen levels produced calls having higher pitch, and similarly adult individuals produced longer calls than senior males. Thus, it is plausible that gibbon vocalizations provide receivers with information about singers' attributes.

Evolutionary breakpoints in the gibbon suggest association between cytosine methylation and karyotype evolution.

Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15-18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.

Hominoid seminal protein evolution and ancestral mating behavior.

Hominoid mating systems show extensive variation among species. The degree of sexual dimorphism in body size and canine size varies among primates in accordance with their mating system, as does the testes size and the consistency of ejaculated semen, in response to differing levels of sperm competition. To investigate patterns of evolution at hominoid seminal proteins and to make inferences regarding the mating systems of extinct taxa, we sequenced the entire coding region of the prostate-specific transglutaminase (TGM4) gene in human, chimpanzee, bonobo, western lowland gorilla, eastern lowland gorilla, orangutan, and siamang, including multiple humans, chimps, and gorillas. Partial DNA sequence of the coding regions was also obtained for one eastern lowland gorilla at the semenogelin genes (SEMG1 and SEMG2), which code for the predominant proteins in semen. Patterns of nucleotide variation and inferred protein sequence change were evaluated within and between species. Combining the present data with previous studies demonstrates a high rate of amino acid substitutions, and low intraspecific variation, at seminal proteins in Pan, presumably driven by strong sperm competition. Both gorilla species apparently possess nonfunctional TGM4, SEMG1, and SEMG2 genes, suggesting that gorillas have had low sperm competition, and therefore their current polygynous mating system, for a long time before their divergence. Similarly, orangutans show longstanding stasis at TGM4, which may be interpreted as evidence for an unchanging mating system for most of their evolution after their divergence from African apes. In contrast to the great apes, the data from humans could be interpreted as evidence of fluctuations between different mating systems or alternatively as a relaxed functional constraint in these proteins. It is our hope that this study is a first step toward developing a model to predict ancestral mating systems from extant molecular data to complement interpretations from the fossil record.

Sexual swellings in wild white-handed gibbon females (Hylobates lar) indicate the probability of ovulation.

Conspicuous sexual swellings in the females of some primate species have been a focus of scientific interest since Darwin first wrote about them in 1871. To understand these visual signals, research focused on exaggerated sexual swellings of Old World primates. However, some primate species develop much smaller sexual swellings and it is as yet unclear if these smaller swellings can serve similar functions as those proposed for exaggerated swellings, i.e. advertising fertility to attract mates. We studied the temporal patterns of sexual swellings, timing of ovulation and female reproductive status in wild white-handed gibbons (Hylobates lar) at Khao Yai National Park, Thailand, where this species has a variable social organization. We established fecal progestogen profiles in fifteen cycles of eight cycling females and, to detect swellings outside the menstrual cycle, five pregnant and six lactating females. In 80% of menstrual cycles, ovulation and maximum swelling phase (duration: Ø 9.3 days; 42.8% of cycle length), overlapped tightly. The probability of ovulation peaked on day 3 of the maximum swelling period. Nevertheless, the temporal relationship between maximum swelling and probability of ovulation varied from day -1 to day 13 of the swelling period and three times ovulations fell outside the maximum swelling phase. The different swellings phases occurred in similar proportions in cycling and pregnant, but not lactating females, which were rarely swollen. Despite their smaller size, gibbons' sexual swellings probably serve functions similar to those suggested for exaggerated swellings by the graded-signal hypothesis, which predicts that sexual swellings indicate the probability of ovulation, without allowing males to pinpoint its exact time.

Loss of urate oxidase activity in hominoids and its evolutionary implications.

We have determined and compared the promoter, coding, and intronic sequences of the urate oxidase (Uox) gene of various primate species. Although we confirm the previous observation that the inactivation of the gene in the clade of the human and the great apes results from a single CGA to TGA nonsense mutation in exon 2, we find that the inactivation in the gibbon lineage results from an independent nonsense mutation at a different CGA codon in exon 2 or from either one-base deletion in exon 3 or one-base insertion in exon 5, contrary to the previous claim that the cause is a 13-bp deletion in exon 2. We also find that compared with other organisms, the primate functional Uox gene is exceptional in terms of usage of CGA codons which are prone to TGA nonsense mutations. Nevertheless, we demonstrate rather strong selective constraint against nonsynonymous sites of the functional Uox gene and argue that this observation is consistent with the fact that the Uox gene is unique in the genome and evolutionarily conserved not only among animals but also among eukaryotes. Another finding that there are a few substitutions in the cis-acting element or CAAT-box (or both) of primate functional Uox genes may explain the lowered transcriptional activity. We suggest that although the inactivation of the hominoid Uox gene was caused by independent nonsense or frameshift mutations, the gene has taken a two-step deterioration process, first in the promoter and second in the coding region during primate evolution. It is also argued that the high concentration of uric acid in the blood of humans and nonhuman primates has developed molecular coevolution with the xanthine oxidoreductase in purine metabolism. However, it remains to be answered whether loss of Uox activity in hominoids is related to protection from oxidative damage and the prolonged life span.

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus.

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA[S]) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium. In S. typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore. hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

Galanin immunoreactivity within the primate basal forebrain: evolutionary change between monkeys and apes.

Galanin immunoreactivity (GAL-ir) is differentially expressed within the basal forebrain of monkeys and humans. Most monkey magnocellular basal forebrain neurons colocalize GAL-ir. In contrast, virtually no human magnocellular basal forebrain neurons express GAL-ir. Rather, an extrinsic galaninergic fiber plexus innervates these neurons in humans. The present study examined the expression of GAL-ir within the basal forebrain of apes to establish the phylogenetic level at which this transformation occurs. The staining patterns of GAL-ir within the basal forebrain of both lesser (gibbons) and great (chimpanzee and gorilla) apes were compared to that previously observed within monkeys and humans. All apes displayed a pattern of basal forebrain GAL-ir indistinguishable from humans. GAL-ir was not expressed within ape basal forebrain magnocellular neurons as seen in monkeys. Rather like humans, a dense collection of GAL-ir fibers was seen in close apposition to magnocellular perikarya. In addition, a few GAL-ir parvicellular neurons were scattered within the ape basal forebrain. These data indicate that the evolutionary change in the expression of GAL-ir within the primate basal forebrain occurs at the branch point of monkeys and apes.