Owl monkey MHC-DRB exon 2 reveals high similarity with several HLA-DRB lineages

One hundred and ten novel MHC-DRB gene exon 2 nucleotide sequences were sequenced in 96 monkeys from three owl monkey species (67 from Aotus nancymaae, 30 from Aotus nigriceps and 13 from Aotus vociferans). Owl monkeys, like humans, have high MHC-DRB allele polymorphism, revealing a striking similarity with several human allele lineages in the peptide binding region and presenting major convergence with DRB lineages from several Catarrhini (humans, apes and Old World monkeys) rather than with others New World monkeys (Platyrrhini). The parallelism between human and Aotus MHC-DRB reveals additional similarities regarding variability pattern, selection pressure and physicochemical constraints in amino acid replacements. These observations concerning previous findings of similarity between the Aotus immune system molecules and their human counterparts affirm this specie’s usefulness as an excellent animal model in biomedical research.Experiments carried out in this work complied with current Colombian Ministry of Health law and regulations governing animal care and handling.An erratum to this article can be found at     

Population densities and geographic distribution of night monkeys (Aotus nancymai and Aotus vociferans) (cebidae: Primates) in Northeastern Peru

Population densities of two species of night (or owl) monkeys (Aotus nancymai and Aotus vociferans) were estimated using transect census methods. Densities of Aotus nancymai were approximately 46.3 individuals/km² in lowland forests and 24.2 individuals/km² in highland forests. For Aotus vociferans densities were 33.0 individuals/km² in lowland forests and 7.9 individuals/km² in highland forests. A. vociferans occurs north of the Río Amazonas-Marañon and A. nancymai south of it, except for the northern enclave between the Ríos Tigre and Pastaza. The two species are nowhere sympatric. However, the two known karyotypic forms of A. vociferans occupy the same habitats throughout the Peruvian range of the species.     

Chromosomal distribution of interstitial telomeric sequences in nine neotropical primates (Platyrrhini): possible implications in evolution and phylogeny

To localize interstitial telomeric sequences (ITSs) and to test whether their pattern of distribution could be linked to chromosomal evolution, we hybridized telomeric sequence probes (peptide nucleic acid, PNA) on metaphases of New World monkeys: Callithrix argentata, Callithrix jacchus, Cebuella pygmaea, Saguinus oedipus, Saimiri sciureus, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae), Lagothrix lagotricha (Atelidae) and Callicebus moloch (Pithecidae), characterized by a rapid radiation and a high rate of chromosomal rearrangements. Our analysis of the probe signal localization allowed us to show in all the species analysed, as normally, the telomeric location at the terminal ends of chromosomes and unexpected signal distributions in some species. Indeed, in three species among the nine studied, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae) and Lagothrix lagotricha (Atelidae), we showed a high variability in terms of localization and degree of amplification of interstitial telomeric sequences, especially for the ones found at centromeric or pericentromeric positions (het‐ITS). A comparative analysis, between species, of homologous chromosomes to human syntenies, on which we have found positive interspersed PNA signals, allowed us to explain the observed pattern of ITS distribution as results of chromosomal rearrangements in the neotropical primates analysed. This evidence permitted us to discuss the possible implication of ITSs as phylogenetic markers for closely related species. Moreover, reviewing previous literature data of ITSs distribution in Primates and in the light of our results, we suggest an underestimation of ITSs and highlight the importance of the molecular cytogenetics approach in characterizing ITSs, which role is still not clarified.     

Reference strand conformational analysis (RSCA) is a valuable tool in identifying MHC-DRB sequences in three species of Aotus monkeys

The Aotus monkey has been of great value in the pre-clinical study of malaria vaccine candidates. Several components of this primate’s immune system have been studied and they display great similarity to their human counterparts. Cloning and sequencing studies have revealed extensive sequence polymorphisms in Aotus MHC-DRB with very high similarities to several human allelic lineages, grouping at least nine distinct MHC-DRB lineages. As the efficacy of peptide vaccines in this animal model may be strongly influenced by exon 2 MHC-DRB polymorphism, the availability of a reliable and rapid MHC-DRB typing method for three species of Aotus (Aotus nancymaae, Aotus vociferans and Aotus nigriceps) is necessary. Reference strand conformational analysis (RSCA) was used here for differentiating the distinctive Aotus MHC-DRB sequences’ mobility using five fluorescently labelled references proved to be very useful for resolving closely related sequences, establishing the number of sequences transcribed in a particular monkey and their identity. The RSCA method’s reliability in terms of identifying Aotus MHC-DRB sequences will facilitate evaluating individual responsiveness to vaccines and prompt studies associating susceptibility/resistance to infectious agents or auto-immune disease, for which Aotus monkeys may be considered to be an appropriate animal model.     

Sequence and diversity of T-cell receptor alpha V, J, and C genes of the owl monkey Aotus nancymaae

 We cloned and sequenced TcR alpha chain cDNA of three healthy Aotus nancymaae monkeys. Fifteen different TRAJ segments and 9 different TRAV genes were identified in the 29 rearrangements analyzed. As expected from the greater phylogenetic distance, A. nancymaae TRA gene sequences diverged more from the human sequences than those of the chimpanzee or the rhesus macaque. However, no Aotus TRAJ segment or TRAV gene was found which lacked a human counterpart. These counterparts were AJ02, AJ05, AJ09, AJ15, AJ22, AJ23, AJ28, AJ30, AJ32, AJ34, AJ37, AJ40, AJ42, AJ45, AJ52 and AV2S1, AV2S3, AV3S1, AV8S1, AV12S1, AV15S1, ADV21S1/DV5, AV22S1S and AV23S1, respectively. In most cases the identity of amino acid sequences between corresponding Aotus and human genes was greater than 80%. This marked conservation of TRA gene sequences indicates a close structural relationship of Aotus and human TcR and demonstrates that the TcR repertoire in primates is remarkably stable. The results support the concept of using Aotus monkeys, which are susceptible to infection with the human malaria parasite Plasmodium falciparum, as an animal model for the evaluation of molecularly defined malaria vaccine candidates. Received: 15 January 1998 / Revised: 23 March 1998     

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites

 The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the β-pleated sheets and by frequent exchange of fixed sequence motifs in the α-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. Received: 10 August 1999 / Revised: 11 October 1999     

Aboveground phytochemical responses to belowground herbivory in poplar trees and the consequence for leaf herbivore preference

Belowground (BG) herbivory can influence aboveground (AG) herbivore performance and food preference via changes in plant chemistry. Most evidence for this phenomenon derives from studies in herbaceous plants but studies in woody plants are scarce. Here we investigated whether and how BG herbivory on black poplar (Populus nigra) trees by Melolontha melolontha larvae influences the feeding preference of Lymantria dispar (gypsy moth) caterpillars. In a food choice assay, caterpillars preferred to feed on leaves from trees that had experienced attack by BG herbivores. Therefore, we investigated the effect of BG herbivory on the phytochemical composition of P. nigra trees alone and in combination with AG feeding by L. dispar caterpillars. BG herbivory did not increase systemic AG tree defences like volatile organic compounds, protease inhibitors and salicinoids. Jasmonates and salicylic acid were also not induced by BG herbivory in leaves but abscisic acid concentrations drastically increased together with proline and few other amino acids. Leaf coating experiments with amino acids suggest that proline might be responsible for the caterpillar feeding preference via presumptive phagostimulatory properties. This study shows that BG herbivory in poplar can modify the feeding preference of AG herbivores via phytochemical changes as a consequence of root‐to‐shoot signaling.     

Illuminance preferences of nocturnal primates

Laboratory tests reveal a preference for illuminances in a broad range of night light by individuals belonging to four species of nocturnal primates (Aotus trivirgatus, Galago crassicaudatus, Galago senegalensis, andNycticebus coucang). In volitional tests the animals altered the light level very frequently, in one case as often as every 7 sec. In these tests the animals tended to avoid total darkness and extremely dim light as well as moderately bright light. These avoided levels, particularly extremely dim light and darkness, inhibited locomotor activity. The greater bush babies preferred dimmer light for manipulative activities than for locomotion. Nocturnal primates differ from nocturnal rodents in being much more highly motivated to seek variety and frequent stimulus change when in deprivation conditions, and in their avoiding and being markedly inhibited by darkness.     

Chromosome comparison among five species of platyrrhini (Alouatta caraya,Aotus azarae,Callithrix jacchus,Cebus apella,andSaimiri sciureus)

Karyotypes of 82 individuals from five Platyrrhini species (Alouatta caraya, Aotus azarae, Callithrix jacchus, Cebus apella, andSaimiri sciureus) were studied and compared using a G-banding technique. Cytogenetic analysis showed full chromosome or full arm homologies among these geographically neighbouring species. A small number of chromosomal rearrangements (inversions, deletions, and translocations) could be detected among these taxa. These five species are closely related in chromosomal evolution. An interesting correspondance was found betweenCebus apella chromosomes and those of the other four species.Alouatta caraya andCebus apella are the closest species.Callithrix jacchus andAotus azarae would have the most separated karyotypes.     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.

On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.     

Identification,characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 → Ile, Ile325 → Val and Val409 → Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 → Met and Val 409 → Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.     

Owl monkeys (Aotus) are highly divergent in mitochondrial Cytochromec Oxidase (COII) sequences

The evolutionary history and differentiation of the owl or night monkeys (Aotus),remain poorly resolved. Variation in pelage and skeletal morphology is relatively minor across their broad range, but cytogenetic studies have revealed that at least 12 karyotypically distinct forms exist, with 2Nchromosome numbers ranging from 46 to 58. We obtained DNA samples from three putative species- A. lemurinus, A. nancymae,and A. azarae-and five karyotypes (I, II, III, IV and VI), amplified the mitochondrial cytochrome coxidase subunit II gene (COII) via PCR and sequenced it. Comparisons of the sequences indicate relatively high levels of sequence divergence among the putative species (4.3 to 6.3%), and suggest that the taxa are genetically quite distinct and have likely experienced extended periods of isolated evolution. The levels of COII divergence represent approximately one- third of the levels found between divergent platyrrhine genera, such as Aotus, Saimiriand Callimico.Using estimates of substitution rates of COII evolution in hominoid primates,the estimated date of divergence for the AotusCOII sequences is 3.6 million years. The AotusCOII data support the existence of multiple species of Aotus,with origins predating late Pleistocene climatic events. Although A. nancymaeand A. azaraeboth live south of the Amazon River and have been considered members of the same species group, phylogenetic analysis of the COII sequences does not support a close relationship between them.     

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O‐formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20^oC) induces N‐formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N‐formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below –20^oC.     

DNA fingerprinting in three species of neotropical primates

DNA fingerprinting allows the simultaneous detection of a large number of hypervariable loci consisting of highly polymorphic tandem repeat units that are extensively dispersed in the genome. With the 33.6 human minisatellite probe, hypervariable fragments were detected, for the first time, in the genome of three different species of wild-caught neotropical primates: Aotus infulatus, Aotus azarae, and Cebus apella. As in the human, these species were highly polymorphic, showing distinctive, individual-specific patterns. Estimates of relatedness within each group were calculated from interspecific comparisons based on the number of shared fragments between individuals. This work shows that the 33.6 human minisatellite probe can be very useful for increasing our understanding of population dynamics and behavior of these species in their natural habitat. © 1996 Wiley-Liss, Inc.     

The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids

To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinaciaoleracea L.) leaves, rape (Brassicanapus L.) seeds and Cuphea lanceolata Ait. seeds. The action of two major C. lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography. In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C. lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated. Both ACP isoforms from C. lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts. Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments. No preference of the medium-chain thioesterase for one specific ACP isoform was observed. The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C. lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction. Received: 23 June 1997 / Accepted: 23 October 1997     

AVPR1A Sequence Variation in Monogamous Owl Monkeys (Aotus azarai) and Its Implications for the Evolution of Platyrrhine Social Behavior

The arginine vasopressin V1a receptor gene (AVPR1A) has been implicated in increased partner preference and pair bonding behavior in mammalian lineages. This observation is of considerable importance for studies of social monogamy, which only appears in a small subset of primate taxa, including the Argentinean owl monkey (Aotus azarai). Thus, to investigate the possible influence of AVPR1A on the evolution of social behavior in owl monkeys, we sequenced this locus in a wild population from the Gran Chaco. We also assessed the interspecific variation of AVPR1A in platyrrhine species that represent a set of phylogenetically and behaviorally disparate taxa. The resulting data revealed A. azarai to have a unique genic structure for AVPR1A that varies in coding sequence and microsatellite repeat content relative to other primate and mammalian species. Specifically, one repetitive region that has been the focus in studies of human AVPR1A diversity, “RS3,” is completely absent in A. azarai and all other platyrrhines examined. This finding suggests that, if AVPR1A modulates behavior in owl monkeys and other neotropical primates, it does so independent of this region. These observations have also provided clues about the process by which the range of social behavior in the Order Primates evolved through lineage-specific neurogenetic variation.     

Fatty acid activation and utilization by Alistipes finegoldii,a representative Bacteroidetes resident of the human gut microbiome

Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram-negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium-chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long-chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2-position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long-chain acyl-ACP. The ability to assimilate a broad-spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl-acyl carrier protein (ACP) synthetases. Acyl-ACP synthetase 1 had a substrate preference for medium-chain fatty acids and synthetase 2 had a substrate preference for long-chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium- and long-chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.     

Luteinizing hormone-releasing hormone in olfactory bulbs of primates

This immunohistochemical study of luteinizing hormone-releasing hormone (LHRH) in the olfactory bulbs in primates was undertaken in order to see whether there was an LHRH innervation in these species similar to that found in rodents. One old world (Macaca fascicularis) and two new world (Saimiri sciureus and Aotus trivirgatus) monkeys were studied. Aotus trivirgatus was of particular interest as it is noctural and so presumably more dependent upon olfactory cues. Animals were perfused with fixative, olfactory bulbs removed and sectioned, and tissues reacted immunocytochemically using LR1 (Benoit) antiserum to LHRH. Some LHRH innervation was found in the olfactory bulbs of all three species, comprising a few LHRH neurons and many fibers that ramified within the bulbs. The accessory bulb (not present as a distinct entity in old world primates) had more LHRH innervation than did the main olfactory bulb. Aotus trivirgatus had the greatest representation of LHRH of the three species. The layer of the olfactory bulb with the greatest number of LHRH fibers was the external plexiform layer. This is also true in rodents. There is evidence that LHRH has a role in the mediation of olfactory cues in reproductive behavior in rodents. It is not known how LHRH functions within the olfactory system in primates. However, the fact that it is distributed similarly in the two groups suggests that it may serve a similar function.     

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.     

Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA)

The substrate selectivity of several microbial lipases has been examined in the esterification of the conjugated linoleic acid (CLA) isomers cis-9,trans-11-, cis-9,cis-11-, trans-9,trans-11- and trans-10,cis-12-octadecadienoic acid with n-butanol in n-hexane. Lipases from Candida cylindracea and Mucor miehei had a preference for the cis-9,trans-11-octadecadienoic acid, while Chirazyme L-5, a Candida antarctica lipase A, accepted the trans-9,trans-11-fatty acid with a high selectivity. Moreover, lipase from Candida cylindracea and Chirazyme L-5 catalysed the esterification of the cis-9,trans-11-octadecadienoic acid with n-butanol faster than the corresponding reaction of the trans-10,cis-12-fatty acid.