Development and first-in-human testing of FLUME urinary catheter with protected tip and relocated drainage holes.

Purpose: Evaluation of a catheter design which protects its tip with the retaining balloon when inflated, and has eyeholes at the base of the balloon to improve drainage. Materials and methods: Preclinical tests included assessment of retaining balloon performance, and microbiological blockage. Clinical testing evaluated short-term use and safety in hospital (stage 1) or the patient's usual residence (stage 2). Results: The retaining balloon supported static loads of 0.7kg, with reduced trauma when modelling forced evulsion. In vitro time to blockage with P. Mirabilis was significantly slower for FLUME compared with latex Foley catheters, but not the silicone Foley. Stage 1 testing (10 patients) confirmed balloon inflation, drainage, retention and removal, with no serious adverse events caused by catheterisation; one balloon failed to inflate, one patient could not be catheterised. Of five patients at stage 2, one had the catheter for 28 days without complication, one experienced spontaneous balloon deflation (14th day) and three needed early removal (blood clot, bypassing, difficulty connecting the drainage bag). Bacterial profiles of two FLUME catheters retained at least 2 weeks matched the Foley catheters. Acquired catheter colouration (two FLUME, one Foley) was not associated with biochemical change in the material. Conclusion: FLUME catheter performed well in preclinical blockage and balloon tests. Tests in 15 patients confirmed basic function and additional training was not needed for staff familiar with Foley catheterisation. Clinical issues commonly seen with catheters included failed catheterisation, clot blockage and bypassing. In addition, an unintended balloon deflation and a failure of bag connection occurred. Plain language summary: This article describes a new catheter design which aims to improve patient comfort and safety, and maximise bladder drainage, by protecting the bladder from the exposed catheter tip and by locating the drainage holes better. Various tests were done to check the catheter retaining balloon was safe and how well the catheter did when exposed to bacteria that could block it. The catheter was also used in people for the first time, to check it could be put in safely and functioned as intended. The results showed the FLUME catheter did well in the balloon and blockage tests. Tests in 15 patients confirmed basic function and showed placement was easy for staff familiar with conventional catheters. There were some clinical issues typical of urinary catheters and some possible improvements were identified.

Sequences, phylogeny and evolution of mitochondrial delta-1-pyrroline-5-carboxylate dehydrogenases (ALDH4A1). Evidence for a second locus (ALDH4A2) in Drosophila.

ALDH4A1 genes encode mitochondrial enzymes of delta-1-pyrroline-5-carboxylate metabolism, generating glutamate from either proline or ornithine. Analyses were undertaken of several vertebrate and invertebrate genomes using reported human and mouse ALDH4A1 amino acid sequences. ALDH4A1 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure, previously reported for mouse and human ALDH4A1. The human ALDH4A1 gene contained 15 coding exons and was more highly expressed in human liver and kidney cortex. Vertebrate ALDH4A1 mitochondrial leader sequences exhibited diverse sequences. Phylogeny studies supported the appearance of the ALDH4A1 gene in invertebrate evolution which has been conserved and retained throughout subsequent vertebrate evolution as a single ALDH4A1 gene. Exceptions included polyploidy observed for the Atlantic salmon (Salmo salar) and African toad (Xenopus laevis) genes. An examination of ALDH4A1 sequences from related Drosophila species supported the appearance of a second ALDH4A gene (ALDH4A2) and time dependent evolutionary changes over the past 50 million years for both genes.

Evolution of aldehyde dehydrogenase genes and proteins in diploid and allotetraploid Xenopus frog species.

At least 19 human aldehyde dehydrogenase (ALDH) genes and enzymes have been studied among vertebrate organisms. BLAT and BLAST analyses were undertaken of Xenopus tropicalis (western clawed frog) and Xenopus laevis (African clawed frog) genomes which are related diploid (N = 20) and allotetraploid (N = 36) species, respectively. The corresponding ALDH genes and proteins within these Xenopus genomes were identified and studied. Evidence is presented for tetraploid copies of 10 Xenopus laevis ALDH genes, whereas another 7 identified ALDH genes were diploid in nature. Xenopus laevis and Xenopus tropicalis ALDH amino acid sequences were highly homologous with the human enzymes, with the exception of the mitochondrial signal peptide sequences. Amino acids performing catalytic and structural roles were conserved and identified based on previous reports of 3D structures for the corresponding mammalian enzymes.

Development of a more clinically relevant bladder and urethral model for catheter testing.

This paper sets out the rationale and design for a more physiologically representative bladder/urethral model than the current rigid funnel industry standard. We suggest this flexible model can better serve as a basis for evaluating new catheters. We investigated the parameters and the validation tests required to construct and test with more flexible materials. The protocol includes static and impact force tests, while measuring internal retention balloon pressure. We have designed a flexible elastomer bladder floor and urethral model and tested it with a Foley balloon catheter. The tests showed that the model could hold the catheter with static loads up to 0.7 kg, but that the catheter was pulled out by impact forces from dropped weights. The Foley catheter design and its associated standards make no provision to mitigate urethral injury in the surprisingly common event of removal with a filled balloon. Our design is a more realistic model for testing retention and extraction characteristics of a urethral catheter. Validation tests have confirmed the feasibility of measuring urethral dilation and balloon pressure, both under traction and during progressive deflation. We suggest this model improves upon the current standard tests and will enable the design of safer catheters.

Polyploidy among salmonid aldehyde dehydrogenase genes and proteins.

Bioinformatic analyses of salmon (Salmo salar) ALDH amino acid sequences supported the presence of at least 30 ALDH genes, which is more than for any other higher vertebrate and is greater than the 19 human ALDH genes currently reported. These included 8 polyploid ALDH genes and proteins: ALDH1A2 (chromosomes 11 and 26); ALDH1L2 (chromosomes 7 and 17); ALDH2, encoding mitochondrial ALDH2 (chromosomes 2 and 5); ALDH3A2 (chromosomes 4, 9 and 20), for which evidence for 5 genes was obtained; ALDH3B1 (chromosomes 3, 6 and 24); ALDH4A1 (chromosomes 12 and 22); ALDH6A1 (chromosomes 1, 6 and 15); and ALDH18A1 (chromosomes 19 and 28). In contrast, 7 salmon ALDH gene families (ALDH1A1, ALDH1A3, ALDH5, ALDH7, ALDH8, ALDH9 and ALDH16) possessed only one gene family member. Phylogenetic studies of salmon and rainbow trout ALDH3A2 genes and proteins suggested that salmonid gene tetraploidy has occurred in at least 2 distinct stages of ALDH3A2 gene evolution.

Ceramide Synthase 6: Comparative Analysis, Phylogeny and Evolution.

Ceramide synthase 6 (CerS6, also known as LASS6) is one of the six members of ceramide synthase gene family in humans. Comparisons of CerS6 amino acid sequences and structures as well as of CerS6 gene structures/locations were conducted using data from several vertebrate genome projects. A specific role for the CerS6 gene and protein has been identified as the endoplasmic reticulum C14- and C16-ceramide synthase. Mammalian CerS6 proteins share 90⁻100% similarity among different species, but are only 22⁻63% similar to other CerS family members, suggesting that CerS6 is a distinct gene family. Sequence alignments, predicted transmembrane, lumenal and cytoplasmic segments and N-glycosylation sites were also investigated, resulting in identification of the key conserved residues, including the active site as well as C-terminus acidic and serine residues. Mammalian CerS6 genes contain ten exons, are primarily located on the positive strands and transcribed as two major isoforms. The human CERS6 gene promoter harbors a large CpG island (94 CpGs) and multiple transcription factor binding sites (TFBS), which support precise transcriptional regulation and signaling functions. Additional regulation is conferred by 15 microRNA (miRNA) target sites identified in the CERS6 3'-UTR region. Phylogenetic analysis of the vertebrate CerS1⁻6 gene families relationships supports a major role for the CerS6 enzyme that is strongly conserved throughout vertebrate evolution.

Comparative studies of vertebrate iduronate 2-sulfatase (IDS) genes and proteins: evolution of A mammalian X-linked gene.

IDS is responsible for the lysosomal degradation of heparan sulfate and dermatan sulfate and linked to an X-linked lysosomal storage disease, mucopolysaccharidosis 2 (MPS2), resulting in neurological damage and early death. Comparative IDS amino acid sequences and structures and IDS gene locations were examined using data from several vertebrate genome projects. Vertebrate IDS sequences shared 60-99% identities with each other. Human IDS showed 47% sequence identity with fruit fly (Drosophila melanogaster) IDS. Sequence alignments, key amino acid residues, N-glycosylation sites and conserved predicted secondary and tertiary structures were also studied, including signal peptide, propeptide and active site residues. Mammalian IDS genes usually contained 9 coding exons. The human IDS gene promoter contained a large CpG island (CpG46) and 5 transcription factor binding sites, whereas the 3'-UTR region contained 5 miRNA target sites. These may contribute to IDS gene regulation of expression in the brain and other neural tissues of the body. An IDS pseudogene (IDSP1) was located proximally to the IDS gene on the X-chromosome in primate genomes. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate IDS gene. These suggested that IDS has originated in an invertebrate ancestral genome and retained throughout vertebrate evolution and conserved on marsupial and eutherian X-chromosomes, with the exception of rat Ids on chromosome 8.

Comparative and evolutionary studies of mammalian arylsulfatase and sterylsulfatase genes and proteins encoded on the X-chromosome.

At least 19 sulfatase genes have been reported on the human genome, including four arylsulfatase (ARS) genes (ARSD; ARSE; ARSF; ARSH) and a sterylsulfatase (STS) gene located together on the X-chromosome. Bioinformatic analyses of mammalian genomes were undertaken using known human STS and ARS amino acid sequences to study the evolution of these genes and proteins encoded on eutherian and marsupial genomes. Several domain regions and key residues were conserved including signal peptides, active site residues, metal (Ca2+) and substrate binding sequences, transmembranes and N-glycosylation sites. Phylogenetic analyses describe the relationships and potential origins of these genes during mammalian evolution. Primate ARSH enzymes lacked signal peptide sequences which may influence their biological functions. CpG117 and CpG92 were detected within the 5' region of the human STS and ARSD genes, respectively, and miR-205 within the 3'-UTR for the human STS gene, using bioinformatic methods A proposal is described for a primordial invertebrate STS-like gene serving as an ancestor for unequal cross over events generating the gene complex on the eutherian mammalian X-chromosome.

Comparative and evolutionary studies of ALDH18A1 genes and proteins.

Vertebrate ALDH18A1 genes encode a bifunctional mitochondrial enzyme, catalyzing a 2-step conversion of glutamate to glutamyl semialdehyde, subsequently converted into proline, ornithine and arginine. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH18A1 amino acid sequences. G5K (glutamyl kinase) and GPR (glutamyl phosphate reductase) domain sequences were identified for all vertebrate and invertebrate genomes examined, whereas bacterial sequences encoded separate enzymes. Vertebrate ALDH18A1 (also called P5CS) sequences were highly conserved throughout vertebrate evolution. A mechanism for generating two major vertebrate ALDH18A1 isoforms is proposed with 'a' isoform containing Asn239-Val240 with wide tissue expression, whereas the 'b' isoform lacking the dipeptide has been reported in gut tissues. Phylogenetic analyses describe the relationships and potential origins of the ALDH18A1 gene during vertebrate and invertebrate evolution and a proposal for generating the bifunctional vertebrate and invertebrate ALDH18A1 gene from a bacterial operon (proBA) encoding G5K and GPR. A more recent Aldh18a1 gene duplication event has apparently occurred with a primordial rat genome.

Mammalian Glutamyl Aminopeptidase Genes (ENPEP) and Proteins: Comparative Studies of a Major Contributor to Arterial Hypertension.

Glutamyl aminopeptidase (ENPEP) is a member of the M1 family of endopeptidases which are mammalian type II integral membrane zinc-containing endopeptidases. ENPEP is involved in the catabolic pathway of the renin-angiotensin system forming angiotensin III, which participates in blood pressure regulation and blood vessel formation. Comparative ENPEP amino acid sequences and structures and ENPEP gene locations were examined using data from several mammalian genome projects. Mammalian ENPEP sequences shared 71-98% identities. Five N-glycosylation sites were conserved for all mammalian ENPEP proteins examined although 9-18 sites were observed, in each case. Sequence alignments, key amino acid residues and predicted secondary and tertiary structures were also studied, including transmembrane and cytoplasmic sequences and active site residues. Highest levels of human ENPEP expression were observed in the terminal ileum of the small intestine and in the kidney cortex. Mammalian ENPEP genes contained 20 coding exons. The human ENPEP gene promoter and first coding exon contained a CpG island (CpG27) and at least 6 transcription factor binding sites, whereas the 3'-UTR region contained 7 miRNA target sites, which may contribute to the regulation of ENPEP gene expression in tissues of the body. Phylogenetic analyses examined the relationships of mammalian ENPEP genes and proteins, including primate, other eutherian, marsupial and monotreme sources, using chicken ENPEP as a primordial sequence for comparative purposes.

Effects of Best Management Practice on Ecological Condition: Does Location Matter?

Best management practices (BMPs) are increasingly being promoted as a solution to the potentially adverse effects agriculture can have on aquatic systems. However, the ability of BMPs to improve riverine systems continues to be questioned due to equivocal empirical evidence linking BMP use with improved stream conditions, particularly in regard to ecological conditions. Explicitly viewing BMP location in relation to hydrological pathways may, however, assist in establishing stronger ecological linkages. The goal of this study was to assess the association between water chemistry, benthic macroinvertebrate community structure, and the number and location of agricultural BMPs in a catchment. Macroinvertebrate and water samples were collected in 30 small (<12 km(2)) catchments exhibiting gradients of BMP use and location in the Grand River Watershed, Southern Ontario, Canada. Stepwise regression analysis revealed that concentrations of most stream nutrients declined in association with greater numbers of BMPs and particularly when BMPs were located in hydrologically connected areas. However, BMPs were significantly associated with only one metric (%EPT) describing macroinvertebrate community structure. Furthermore, variance partitioning analysis indicated that less than 5% of the among site variation in the macroinvertebrate community could be attributed to BMPs. Overall, the implemented BMPs appear to be achieving water quality improvement goals but spatial targeting of specific BMP types may allow management agencies to attain further water quality improvements more efficiently. Mitigation and rehabilitation measures beyond the BMPs assessed in this study may be required to meet goals of enhanced ecological condition.

Evolution of Vertebrate Solute Carrier Family 9B Genes and Proteins (SLC9B): Evidence for a Marsupial Origin for Testis Specific SLC9B1 from an Ancestral Vertebrate SLC9B2 Gene.

SLC9B genes and proteins are members of the sodium/lithium hydrogen antiporter family which function as solute exchangers within cellular membranes of mammalian tissues. SLC9B2 and SLC9B1 amino acid sequences and structures and SLC9B-like gene locations were examined using bioinformatic data from several vertebrate genome projects. Vertebrate SLC9B2 sequences shared 56-98% identity as compared with ∼50% identities with mammalian SLC9B1 sequences. Sequence alignments, key amino acid residues and conserved predicted transmembrane structures were also studied. Mammalian SLC9B2 and SLC9B1 genes usually contained 11 or 12 coding exons with differential tissue expression patterns: SLC9B2, broad tissue distribution; and SLC9B1, being testis specific. Transcription factor binding sites and CpG islands within the human SLC9B2 and SLC9B1 gene promoters were identified. Phylogenetic analyses suggested that SLC9B1 originated in an ancestral marsupial genome from a SLC9B2 gene duplication event.

Aldehyde dehydrogenase homologous folate enzymes: Evolutionary switch between cytoplasmic and mitochondrial localization.

Cytosolic and mitochondrial 10-formyltetrahydrofolate dehydrogenases are products of separate genes in vertebrates but only one such gene is present in invertebrates. There is a significant degree of sequence similarity between the two enzymes due to an apparent origin of the gene for the mitochondrial enzyme (ALDH1L2) from the duplication of the gene for the cytosolic enzyme (ALDH1L1). The primordial ALDH1L gene originated from a natural fusion of three unrelated genes, one of which was an aldehyde dehydrogenase. Such structural organization defined the catalytic mechanism of these enzymes, which is similar to that of aldehyde dehydrogenases. Here we report the analysis of ALDH1L1 and ALDH1L2 genes from different species and their phylogeny and evolution. We also performed sequence and structure comparison of ALDH1L enzymes possessing aldehyde dehydrogenase catalysis to those lacking this feature in an attempt to explain mechanistic differences between cytoplasmic ALDH1L1 and mitochondrial ALDH1L2 enzymes and to better understand their functional roles.

Comparative and evolutionary studies of vertebrate ALDH1A-like genes and proteins.

Vertebrate ALDH1A-like genes encode cytosolic enzymes capable of metabolizing all-trans-retinaldehyde to retinoic acid which is a molecular 'signal' guiding vertebrate development and adipogenesis. Bioinformatic analyses of vertebrate and invertebrate genomes were undertaken using known ALDH1A1, ALDH1A2 and ALDH1A3 amino acid sequences. Comparative analyses of the corresponding human genes provided evidence for distinct modes of gene regulation and expression with putative transcription factor binding sites (TFBS), CpG islands and micro-RNA binding sites identified for the human genes. ALDH1A-like sequences were identified for all mammalian, bird, lizard and frog genomes examined, whereas fish genomes displayed a more restricted distribution pattern for ALDH1A1 and ALDH1A3 genes. The ALDH1A1 gene was absent in many bony fish genomes examined, with the ALDH1A3 gene also absent in the medaka and tilapia genomes. Multiple ALDH1A1-like genes were identified in mouse, rat and marsupial genomes. Vertebrate ALDH1A1, ALDH1A2 and ALDH1A3 subunit sequences were highly conserved throughout vertebrate evolution. Comparative amino acid substitution rates showed that mammalian ALDH1A2 sequences were more highly conserved than for the ALDH1A1 and ALDH1A3 sequences. Phylogenetic studies supported an hypothesis for ALDH1A2 as a likely primordial gene originating in invertebrate genomes and undergoing sequential gene duplication to generate two additional genes, ALDH1A1 and ALDH1A3, in most vertebrate genomes.

Comparative genomics, molecular evolution and computational modeling of ALDH1B1 and ALDH2.

Vertebrate ALDH2 genes encode mitochondrial enzymes capable of metabolizing acetaldehyde and other biological aldehydes in the body. Mammalian ALDH1B1, another mitochondrial enzyme sharing 72% identity with ALDH2, is also capable of metabolizing acetaldehyde but has a tissue distribution and pattern of activity distinct from that of ALDH2. Bioinformatic analyses of several vertebrate genomes were undertaken using known ALDH2 and ALDH1B1 amino acid sequences. Phylogenetic analysis of many representative vertebrate species (including fish, amphibians, birds and mammals) indicated the presence of ALDH1B1 in many mammalian species and in frogs (Xenopus tropicalis); no evidence was found for ALDH1B1 in the genomes of birds, reptiles or fish. Predicted vertebrate ALDH2 and ALDH1B1 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for human ALDH2. Studies of ALDH1B1 sequences supported the hypothesis that the ALDH1B1 gene originated in early vertebrates from a retrotransposition of the vertebrate ALDH2 gene. Given the high degree of similarity between ALDH2 and ALDH1B1, it is surprising that individuals with an inactivating mutation in ALDH2 (ALDH2*2) do not exhibit a compensatory increase in ALDH1B1 activity. We hypothesized that the similarity between the two ALDHs would allow for dominant negative heterotetramerization between the inactive ALDH2 mutants and ALDH1B1. Computational-based molecular modeling studies examining predicted protein-protein interactions indicated that heterotetramerization between ALDH2 and ALDH1B1 subunits was highly probable and may partially explain a lack of compensation by ALDH1B1 in ALDH2(∗)2 individuals.

Comparative studies of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1: evidence for a eutherian mammalian origin for the GPIHBP1 gene from an LY6-like gene.

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) functions as a platform and transport agent for lipoprotein lipase (LPL) which functions in the hydrolysis of chylomicrons, principally in heart, skeletal muscle and adipose tissue capillary endothelial cells. Previous reports of genetic deficiency for this protein have described severe chylomicronemia. Comparative GPIHBP1 amino acid sequences and structures and GPIHBP1 gene locations were examined using data from several mammalian genome projects. Mammalian GPIHBP1 genes usually contain four coding exons on the positive strand. Mammalian GPIHBP1 sequences shared 41-96% identities as compared with 9-32% sequence identities with other LY6-domain-containing human proteins (LY6-like). The human N-glycosylation site was predominantly conserved among other mammalian GPIHBP1 proteins except cow, dog and pig. Sequence alignments, key amino acid residues and conserved predicted secondary structures were also examined, including the N-terminal signal peptide, the acidic amino acid sequence region which binds LPL, the glycosylphosphatidylinositol linkage group, the Ly6 domain and the C-terminal α-helix. Comparative and phylogenetic studies of mammalian GPIHBP1 suggested that it originated in eutherian mammals from a gene duplication event of an ancestral LY6-like gene and subsequent integration of exon 2, which may have been derived from BCL11A (B-cell CLL/lymphoma 11A gene) encoding an extended acidic amino acid sequence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13205-011-0026-4) contains supplementary material, which is available to authorized users.

Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36).

Platelet glycoprotein 4 (CD36) (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3]) is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs) and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53-100% identity as compared with 29-32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 'short loops'; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA) contributing to a high gene expression level (6.6 times average). Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate SCARB1 and SCARB2 genes. These suggested that CD36 originated in an ancestral genome and was subsequently duplicated to form three vertebrate CD36 gene family members, SCARB1, SCARB2 and CD36.

Comparative Structures and Evolution of Vertebrate Carboxyl Ester Lipase (CEL) Genes and Proteins with a Major Role in Reverse Cholesterol Transport.

Bile-salt activated carboxylic ester lipase (CEL) is a major triglyceride, cholesterol ester and vitamin ester hydrolytic enzyme contained within pancreatic and lactating mammary gland secretions. Bioinformatic methods were used to predict the amino acid sequences, secondary and tertiary structures and gene locations for CEL genes, and encoded proteins using data from several vertebrate genome projects. A proline-rich and O-glycosylated 11-amino acid C-terminal repeat sequence (VNTR) previously reported for human and other higher primate CEL proteins was also observed for other eutherian mammalian CEL sequences examined. In contrast, opossum CEL contained a single C-terminal copy of this sequence whereas CEL proteins from platypus, chicken, lizard, frog and several fish species lacked the VNTR sequence. Vertebrate CEL genes contained 11 coding exons. Evidence is presented for tandem duplicated CEL genes for the zebrafish genome. Vertebrate CEL protein subunits shared 53-97% sequence identities; demonstrated sequence alignments and identities for key CEL amino acid residues; and conservation of predicted secondary and tertiary structures with those previously reported for human CEL. Phylogenetic analyses demonstrated the relationships and potential evolutionary origins of the vertebrate CEL family of genes which were related to a nematode carboxylesterase (CES) gene and five mammalian CES gene families.

Comparative studies of vertebrate lipoprotein lipase: a key enzyme of very low density lipoprotein metabolism.

Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins.

Comparative studies of vertebrate aldehyde dehydrogenase 3: sequences, structures, phylogeny and evolution. Evidence for a mammalian origin for the ALDH3A1 gene.

Mammalian ALDH3 genes (ALDH3A1, ALDH3A2, ALDH3B1 and ALDH3B2) encode enzymes of peroxidic and fatty aldehyde metabolism. ALDH3A1 also plays a major role in anterior eye tissue UV-filtration. BLAT and BLAST analyses were undertaken of several vertebrate genomes using rat, chicken and zebrafish ALDH3-like amino acid sequences. Predicted vertebrate ALDH3 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure as reported by Liu et al. [27] for rat ALDH3A1. Phylogeny studies of human, rat, opossum, platypus, chicken, xenopus and zebrafish ALDH3-like sequences supported three hypotheses: (1) the mammalian ALDH3A1 gene was generated by a tandem duplication event of an ancestral vertebrate ALDH3A2 gene; (2) multiple mammalian and chicken ALDH3B-like genes were generated by tandem duplication events within genomes of related species; and (3) vertebrate ALDH3A and ALDH3B genes were generated prior to the appearance of bony fish more than 500 million years ago.