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Biomedical subjects

A L Hughes

Publications and source records attributed to A L Hughes.

At least 19 recordsLinked to original sources
PubMed2007

Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).

At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to determine the morphological and functional ancestral state of their placentas, as it relates to evolutionary relationships among species in this important taxonomic group.

Animals↗
PubMed2006

Cloning of rhesus monkey LILRs.

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.

Amino Acid Sequence↗
PubMed2001

Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum.

It has been proposed that the virulent human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck. In order to test this hypothesis, we estimated the effective population size of this species from the patterns of nucleotide substitution at 23 nuclear protein-coding loci, using a variety of methods based on coalescent theory. Both simple methods and phylogenetically based maximum-likelihood methods yielded the conclusion that the effective population size of this species has been of the order of at least 10(5) for the past 300,000-400,000 years.

Alleles↗
PubMed2001

Distinction between differentiation, cell cycle, and apoptosis signals in PC12 cells by the nerve growth factor mutant delta9/13, which is selective for the p75 neurotrophin receptor.

The common neurotrophin receptor p75(NTR) (low affinity nerve growth factor receptor) participates in the high-affinity binding with the TrkA nerve growth factor (NGF) receptor, may mediate apoptosis, and may signal independently in a cell-specific manner. The potential of p75(NTR) to signal independently of TrkA was investigated with an NGF mutant protein (NGFdelta9/13) that binds poorly to TrkA (Woo et al. [1995] J. Biol. Chem. 270:6278-6285). The NGFdelta9/13 mutant does not activate TrkA autophosphorylation and fails to stimulate the normal NGF-induced growth arrest, demonstrating that TrkA activation is required to arrest PC12 cells at the NGF-activated G1/S cell cycle checkpoint. However, apoptosis is successfully blocked and cell survival is promoted by the NGFdelta9/13 mutant in naive PC12 cells after serum withdrawal, suggesting that p75(NTR) can signal for survival autonomously of TrkA. Annexin V binding, an indication of apoptotic plasma membrane disruption, is inhibited by both NGF and the NGFdelta9/13 mutant after serum deprivation. Both NGF and the NGFdelta9/13 mutant inhibit the rapid apoptotic internucleosomal DNA cleavage of PC12 cells upon serum deprivation. Furthermore, the level of caspase3-like activity that is rapidly activated by serum withdrawal from PC12 cells is reduced by both the NGFdelta9/13 protein and NGF. Finally, upon serum withdrawal, both NGF and the NGFdelta9/13 mutant activate nuclear translocation of the transcriptional factor NF-kappaB (nuclear factor kappaB), a process involved in cell survival. These results are consistent with p75(NTR) inhibition of caspase-mediated apoptosis in PC12 cells. The different physiologic responses elicited by NGFdelta9/13 indicate the potential for individual signaling by the two NGF receptors and also demonstrate the utility of NGF mutants for receptor-selective signal transduction.

Animals↗
PubMed2001

Molecular evolution of viral fusion and matrix protein genes and phylogenetic relationships among the Paramyxoviridae.

Phylogenetic relationships among the Paramyxoviridae, a broad family of viruses whose members cause devastating diseases of wildlife, livestock, and humans, were examined with both fusion (F) and matrix (M) protein-coding sequences. Neighbor-joining trees of F and M protein sequences showed that the Paramyxoviridae was divided into the two traditionally recognized subfamilies, the Paramyxovirinae and the Pneumovirinae. Within the Paramyxovirinae, the results also showed groups corresponding to three currently recognized genera: Respirovirus, Morbillivirus, and Rubulavirus. The relationships among the three genera of the Paramyxovirinae were resolved with M protein sequences and there was significant bootstrap support (100%) showing that members of the genus Respirovirus and the genus Morbillivirus were more closely related to each other than to members of the genus Rubulavirus. Both F and M phylogenies showed that Newcastle disease virus (NDV) was more closely related to the genus Rubulavirus than to the other two genera but were consistent with the proposal (B. S. Seal et al., 2000, Virus Res. 66, 1-11) that NDV be classified as a separate genus within the Paramyxovirinae. Both F and M phylogenies were also consistent with the proposal (L. Wang et al., 2000, J. Virol 74, 9972-9979) that Hendra virus be classified as a new genus closely related and basal to the genus Morbillivirus. Rinderpest was most closely related to measles and a more derived virus than to canine distemper virus, phocine distemper virus, or dolphin morbillivirus.

Databases, Nucleic Acid↗
PubMed2001

Evolution of the integrin alpha and beta protein families.

A phylogenetic analysis of vertebrate and invertebrate alpha integrins supported the hypothesis that two major families of vertebrate alpha integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the alpha PS1 and alpha PS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including alpha PS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate alpha PS1 and alpha PS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of beta integrins did not clearly resolve whether vertebrate beta integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate beta 4 may have done so. In general, the phylogeny of neither alpha nor beta integrins showed a close correspondence with patterns of alpha--beta heterodimer formation or other functional characteristics. One major exception to this trend involved alpha L, alpha M, alpha X, and alpha D, a monophyletic group of immune cell-expressed alpha integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their beta integrin partners.

Animals↗
PubMed2001

The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.

CD8(+) cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.

Acute Disease↗
PubMed2001

Evolutionary change of predicted cytotoxic T cell epitopes of dengue virus.

Peptides bound by human class I major histocompatibility complex molecules and presented to cytotoxic T cells (CTL) were predicted by a computer algorithm, and changes in these predicted CTL epitopes (pCTLE) were reconstructed by maximum parsimony in a phylogeny of complete genotypes of dengue virus, West Nile virus (WNV), and Japanese encephalitis virus (JEV). In a clade including dengue virus serotypes 1 and 3 (D1V and D3V), pCTLE were lost over evolutionary time at a greater rate than new ones were gained. A similar but less pronounced trend was seen in the other dengue serotypes but not in WNV and JEV. The loss of pCTLE predicted to be restricted by HLA-B(*)2705 was particularly pronounced in D1V and D3V. Since dengue is endemic in humans, while WNV and JEV are not, these results are consistent with CTL-driven selection on dengue viruses, particularly, D1V and D3V.

Algorithms↗
PubMed2001

Cloning of rhesus monkey killer-cell Ig-like receptors (KIRs) from early pregnancy decidua.

To define the killer-cell Ig-like receptors (KIR) molecules expressed within the decidua in the rhesus monkey, an early pregnancy rhesus decidual library was screened with an oligonucleotide located within the KIR D2 domain. We obtained 26 full-length clones which represented 11 mRNAs. The cDNAs encode proteins containing 2 or 3 Ig domains, and short or long cytoplasmic domains. The long domains contained immunotyrosine inhibitory motifs (ITIM) motifs indicating inhibitory function. Three clones contained single or dinucleotide deletions within the D1 domain, which would result in proteins truncated within this domain. Phylogenetic analysis of the Ig domains supports a D0 - D2 organization of rhesus KIR2DL4.1, similar to human KIR2DL4 and KIR2DL5. The rhesus KIR2DL4.1 transcript contains a long cytoplasmic tail homologous with human KIR2DL4.

Amino Acid Motifs↗
PubMed2001

Gene duplication and the structure of eukaryotic genomes.

A simple method for understanding how gene duplication has contributed to genomic structure was applied to the complete genomes of Caenorhabditis elegans, Drosophila melanogaster, and yeast Saccharomyces cerevisiae. By this method, the genes belonging to gene families (the paranome) were identified, and the extent of sharing of two or more families between genomic windows was compared with that expected under a null model. The results showed significant evidence of duplication of genomic blocks in both C. elegans and yeast. In C. elegans, the five block duplications identified all occurred intra-chromosomally, and all but one occurred quite recently. In yeast, by contrast, 39 duplicated blocks were identified, and all but one of these was inter-chromosomal. Of these 39 blocks, 28 showed evidence of ancient duplication, possibly as a result of an ancient polyploidization event. By contrast, three blocks showed evidence of very recent duplication, while seven others showed a mixture of ancient and recent duplication events. Thus, duplication of genomic blocks has been an ongoing feature of yeast evolution over the past 200--300 million years.

Animals↗
PubMed2001

Pattern and timing of gene duplication in animal genomes.

Duplication of genes, giving rise to multigene families, has been a characteristic feature of the evolution of eukaryotic genomes. In the case of vertebrates, it has been proposed that an increase in gene number resulted from two rounds of duplication of the entire genome by polyploidization (the 2R hypothesis). In the most extensive test to date of this hypothesis, we compared gene numbers in homologous families and conducted phylogenetic analyses of gene families with two to eight members in the complete genomes of Caenorhabditis elegans and Drosophila melanogaster and the available portion of the human genome. Although the human genome showed a higher proportion of recent gene duplications than the other animal genomes, the proportion of duplications after the deuterostome-protostome split was constant across families, with no peak of such duplications in four-member families, contrary to the expectation of the 2R hypothesis. A substantial majority (70.9%) of human four-member families and four-member clusters in larger families showed topologies inconsistent with two rounds of polyploidization in vertebrates.

Animals↗
PubMed2001

Ancient genome duplications did not structure the human Hox-bearing chromosomes.

The fact that there are four homeobox (Hox) clusters in most vertebrates but only one in invertebrates is often cited as evidence for the hypothesis that two rounds of genome duplication by polyploidization occurred early in vertebrate history. In addition, it has been observed in humans and other mammals that numerous gene families include paralogs on two or more of the four Hox-bearing chromosomes (the chromosomes bearing the Hox clusters; i.e., human chromosomes 2, 7, 12, and 17), and the existence of these paralogs has been taken as evidence that these genes were duplicated along with the Hox clusters by polyploidization. We tested this hypothesis by phylogenetic analysis of 42 gene families including members on two or more of the human Hox-bearing chromosomes. In 32 of these families there was evidence against the hypothesis that gene duplication occurred simultaneously with duplication of the Hox clusters. Phylogenies of 14 families supported the occurrence of one or more gene duplications before the origin of vertebrates, and of 15 gene duplication times estimated for gene families evolving in a clock-like manner, only six were dated to the same time period early in vertebrate history during which the Hox clusters duplicated. Furthermore, of gene families duplicated around the same time as the Hox clusters, the majority showed topologies inconsistent with their having duplicated simultaneously with the Hox clusters. The results thus indicate that ancient events of genome duplication, if they occurred at all, did not play an important role in structuring the mammalian Hox-bearing chromosomes.

Animals↗
PubMed2001

Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata.

For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population of Petunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5' region of S(19) allele was derived from an ancestor allele closely related to S(2), whereas its 3' region was derived from an ancestor allele closely related to S(8). A similar evolutionary relationship was found among S(3), S(12), and S(15) alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of the S(1) allele identified in this work is completely identical to that of the previously identified S(1) allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity, S(6) and S(9) alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed.

Alleles↗
PubMed2001

Simultaneous positive and purifying selection on overlapping reading frames of the tat and vpr genes of simian immunodeficiency virus.

Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of the tat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in the tat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vpr reading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on the vpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.

Animals↗
PubMed2000

Mediation of nerve growth factor-driven cell cycle arrest in PC12 cells by p53. Simultaneous differentiation and proliferation subsequent to p53 functional inactivation.

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G(1)/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G(1) arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

Animals↗
PubMed2000

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.

AIDS Vaccines↗
PubMed2000

Adaptive diversification within a large family of recently duplicated, placentally expressed genes.

The pregnancy-associated glycoproteins (PAG) are putative peptide-binding proteins and products of a large family of genes whose expression is localized to the placental surface epithelium of artiodactyl species. We have tested the hypothesis that natural selection has favored diversification of these genes by examining patterns of nucleotide substitution in a sample of 28 closely related bovine, caprine, and ovine family members that are expressed only in trophoblast binucleate cells. Three observations were made. First, in codons encoding highly variable domains of the proteins, there was a greater accumulation of both synonymous and nonsynonymous mutations than in the more conserved regions of the genes. Second, in the variable regions, the mean number of nonsynonymous nucleotide substitutions per site was significantly greater than the mean number of synonymous substitutions per site. Third, nonsynonymous changes affecting amino acid charge occurred more frequently than expected under random substitution. This unusual pattern of nucleotide substitution implies that natural selection has acted to diversify these PAG molecules at the amino acid level, which in turn suggests that these molecules have undergone functional diversification. We estimate that the binucleate cell-expressed PAG originated 52 +/- 6 million years ago, soon after the divergence of the ruminant lineage. Thus, rapid functional diversification of PAG expressed in trophoblast binucleate cells seems to have been associated with the origin of this unique placental adaptation.

Amino Acid Sequence↗