Molecular phylogeny of the highly diversified catfish subfamily Loricariinae (Siluriformes, Loricariidae) reveals incongruences with morphological classification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5' UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Chimpanzee: A predictive model for the human cytochrome P450 3A subfamily

The human cytochrome P450 3A (CYP3A) subfamily is responsible for most of the metabolism of therapeutic drugs; however, an adequate in vivo model has yet to be discovered. This study begins with an investigation of a controversial topic surrounding the human CYP3As--estrogen regulation. A novel approach to this topic was used by defining expression in the estrogen-responsive endometrium. This study shows that estrogen down-regulates CYP3A4 expression in the endometrium. On the other hand, analogous studies showed an increase in CYP3A expression as age increases in liver tissue. Following the discussion of estrogen regulation, is an investigation of the cross-species relationships among all of the CYP3As was completed. The study compares isoforms from piscines, avians, rodents, canines, ovines, bovines, and primates. Using the traditional phylogenetic analyses and employing a novel approach using exon and intron lengths, the results show that only another primate could be the best animal model for analysis of the regulation of the expression of the human CYP3As. This analysis also demonstrated that the chimpanzee seems to be the best available human model. Moreover, the study showed the presence and similarities of one additional isoform in the chimpanzee genome that is absent in humans. Based on these results, initial characterization of the chimpanzee CYP3A subfamily was begun. While the human genome contains four isoforms--CYP3A4, CYP3A5, CYP3A7, and CYP3A43--the chimpanzee genome has five, the four previously mentioned and CYP3A67. Both species express CYP3A4, CYP3A5, and CYP3A43, but humans express CYP3A7 while chimpanzees express CYP3A67. In humans, CYP3A4 is expressed at higher levels than the other isoforms, but some chimpanzee individuals express CYP3A67 at higher levels than CYP3A4. Such a difference is expected to alter significantly the total CYP3A metabolism. On the other hand, any study considering individual isoforms would still constitute a valid method of study for the human CYP3A4, CYP3A5, and CYP3A43 isoforms. ^

Characterization of cytochrome P450 4F subfamily: Response in traumatic brain injury and gene regulation

CYP4F enzymes metabolize endogenous molecules including arachidonic acid, leukotrienes and prostaglandins. The involvement of these eisosanoids in inflammation has led to the hypothesis that CYP4Fs may modulate inflammatory conditions after traumatic brain injury (TBI). In rat, TBI elicited changes in mRNA expression of CYP4Fs as a function of time in the cerebrum region. These changes in CYP4F mRNA levels inversely correlated with the cerebral leukotriene B4 (LTB4) level following injury at the same time points. TBI also resulted in changes in CYP4F protein expression and localization around the injury site, where CYP4F1 and CYP4F6 immunoreactivity increased in surrounding astrocytes and CYP4F4 immunoreactivity shifted from endothelia of cerebral vessels to astrocytes. The study with rat primary astrocytes indicated that pro-inflammatory cytokines TNFα and IL-1β could affect the transcription of CYP4Fs to a certain degree, whereas the changing pattern in the primary astrocytes appeared to be different from that in the in vivo TBI model.^ In addition, the regulation of CYP4F genes has been an unsolved issue although factors including cytokines and fatty acids appear to affect CYP4Fs expression in multiple models. In this project, HaCaT cells were used as an in vitro cellular model to define signaling pathways involved in the regulation of human CYP4F genes. Retinoic acids inhibited CYP4F11 expression, whereas cytokines TNFα and IL-1β induced transcription of CYP4F11 in HaCaT cells. The induction of CYP4F11 by both cytokines could be blocked by a JNK specific inhibitor, indicating the involvement of the JNK pathway in the up-regulation of CYP4F11. Retinoic acids are known to function in gene regulation through nuclear receptors RARs and RXRs. The RXR agonist LG268 greatly induced transcription of CYP4F11, whereas RAR agonist TTNPB obviously inhibited CYP4F11 transcription, indicating that the down-regulation of CYP4F11 by retinoic acid was mediated by RARs, and that inhibition of CYP4F11 by retinoic acid may also be related to the competition for RXR receptors. Thus, the CYP4F11 gene is regulated by signaling pathways including the RXR pathway and the JNK pathway. In contrast, the regulation mechanism of other CYP4Fs by retinoic acids appears to be different from that of CYP4F11.^

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.

A nonpeptide agonist of the invertebrate receptor for SchistoFLRFamide (PDVDHVFLRFamide), a member of a subfamily of insect FMRFamide-related peptides.

We describe a nonpeptide mimetic analog of an invertebrate peptide receptor. Benzethonium chloride (Bztc) is an agonist of the SchistoFLRFamide (PDVDHVFLRFamide) receptors found on locust oviducts. Bztc competitively displaces [125I-labeled Y1]SchistoFLRFamide binding to both high- and low-affinity receptors of membrane preparations. Bztc mimics the physiological effects of SchistoFLRFamide on locust oviduct, by inhibiting myogenic and induced contractions in a dose-dependent manner. Bztc is therefore recognized by the binding and activation regions of the SchistoFLRFamide receptors. This discovery provides a unique opportunity within insects to finally target a peptide receptor for the development of future pest management strategies.

The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element.

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.

Revision of the North American moths of the subfamily Eucosminae of the family Olethreutidae /

At head of title: Smithsonian Institution. United States National Museum.

Fruits and seeds of genera in the subfamily Faboideae (Fabaceae) /

Includes bibliographical references (p. 1160-1184) and index.