Molecular phylogeny of the hexagrammid fishes using a multi-locus approach

Ideally, organisms are grouped into monophyletic assemblages reflecting their evolutionary histories. Single (molecular) markers can reflect the evolutionary history of the marker, rather than the species in question, therefore, phylogenetic relationships should be inferred from adequate sampling of characters. Because the use of multiple loci greatly improves the resolving power of the molecular assay, we constructed a molecular phylogeny of the family Hexagrammidae based on six loci, including two mitochondrial and four nuclear loci. The resulting molecular phylogeny, from the combined data, was significantly different from the morphological topology suggested by Shinohara [Memoirs of the Faculty of Fisheries, Hokkaido University 41 (1994) 1]. Our data support a monophyletic assemblage for the genera Hexagrammos and Pleurogrammus. However, other taxa traditionally included in the family Hexagrammidae did not form a monophyletic assemblage. The monotypic genus Ophiodon was more closely associated with cottids than with other hexagrammids. Our data concur with the morphological topology in that the genera Zaniolepis and Oxylebius formed a monophyletic clade, which was distinct and basal to the remaining hexagrammids, seven cottids and one agonid.     

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries

BACKGROUND: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis. METHODS: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.     

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.     

Phenotypic variation resulting from a deficiency of epidermal growth factor receptor in mice is caused by extensive genetic heterogeneity that can be genetically and molecularly partitioned

The timing of lethality caused by homozygosity for a null allele of the epidermal growth factor receptor (Egfrtm1Mag) in mice is strongly dependent on genetic background. Initial attempts to genetically map background modifiers using Swiss-derived, outbred CD-1 mice were unsuccessful. To investigate the genetic architecture contributing to survival of Egfrtm1Mag homozygous embryos, the genetic variability segregating within the outbred population was partitioned by surveying viability of Egfrtm1Mag mutants using intercrosses between 129S6/SvEvTAC-Egfrtm1Mag and nine Swiss-derived, inbred strains: ALR/LtJ, ALS/LtJ, APN, APS, ICR/HaRos, NOD/LtJ, NON/LtJ, SJL/J, and SWR/J. The observations showed that these strains support varying levels of survival of Egfrtm1Mag homozygous embryos, suggesting that genetic heterogeneity within the CD-1 stock contributed to the original lack of Egfrtm1Mag modifier detection. Similar to the Swiss-derived intercrosses, nine congenic strains, derived from 129S6/SvEvTAC, AKR/J, APN, BALB/cJ, BTBR-T+ tf/tf, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ inbred backgrounds, also supported varying levels of survival of Egfrtm1Mag mutants. By intercrossing the congenic lines to create hybrid F1 embryos, different genetic backgrounds were found to have complementary modifiers. Analysis of the congenic lines argues against heterosis of outbred backgrounds contributing to Egfrtm1Mag phenotypic variability. A detailed analysis of the crosses suggests that modifiers function at three distinct stages of development. One class of modifiers supports survival of Egfrtm1Mag homozygous embryos to mid-gestation, another class supports development through the mid-gestation transition from yolk-sac to placental-derived nutrient sources, and a third class supports survival through later stages of gestation. Data from microarray analysis using RNA from wild-type and Egfrtm1Mag mutant placentas support the existence of extensive genetic heterogeneity and suggest that it can be molecularly partitioned. This method should be generally useful to partition heterogeneity contributing to other complex traits.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Glycation increases human annulus fibrosus stiffness in both experimental measurements and theoretical predictions

One of the primary age-related changes to collagenous tissues is the increased concentration of advanced glycation endproducts (AGEs). Although AGEs have been shown to increase the mechanical stiffness of many tissues, their influence on the mechanical properties of the annulus fibrosus has not been measured experimentally. In previous theoretical work, we hypothesized that the mechanical influence of AGEs on the annulus could be represented in an additive strain energy function with a separate crosslinking term, but the material coefficients associated with this term were not correlated with AGE concentration. In the current study, we measured the tensile stress-strain response of the human annulus in the axial direction both before and after glycation with methylglyoxal. Using nonlinear regression, the strain energy function was simultaneously applied to these new data and to data from a wide range of experimental protocols reported in the literature to determine values for the material coefficients appearing in the constitutive equation. Nonenzymatic collagen crosslinking induced a statistically significant change in annular material properties. Furthermore, the concentration of AGEs correlated positively with the material coefficients found in the terms of the strain energy function that we associate with collagen crosslinking. These data suggest that AGEs contribute to age-related disc stiffening as well as validate the hypothesis that biochemical constituents can be related mathematically to tissue behavior. In the future, this structurally guided constitutive relationship may provide further insight into the structure-function relationships of the annulus fibrosus.     

TCDD as a biological response modifier for Mitomycin C: oxygen tension affects enzyme activation, reactive oxygen species and cell death

TCDD was assessed as a biological response modifier for increasing MMC cytotoxicity through aryl hydrocarbon receptor (AhR) activation and increasing levels of bioreductive enzymes. Human MCF-7 cells were exposed to TCDD, MMC and combinations thereof under aerobic or hypoxic conditions. Cytotoxicity, enzyme activities (NQO1, XO, XDH, CYPR, CYP1A, GST and UGT) and intracellular reactive oxygen species (ROS) were subsequently measured. Under aerobic conditions, TCDD alone had no significant toxicity but combinations of TCDD and MMC significantly increased cell death. LD50 values were: MMC alone, 0.89 +/- 0.04 microM; TCDD co-treatment, 0.26 +/- 0.007 microM (P = 0.008 vs. MMC alone) and TCDD pre-treatment, 0.04 +/- 0.01 microM (P = 0.003 vs. MMC alone). Under hypoxia, TCDD itself caused significant cell death, likely due to increased ROS, but no combinations of MMC/TCDD altered the LD50 of MMC. Significant changes in enzyme activities were caused by TCDD under aerobic but not hypoxic conditions while MMC decreased the activity of its activating enzymes regardless of oxygen tension. Greater toxicity of MMC/TCDD combinations in aerobic culture, were most likely mediated by increased levels of bioreductive enzymes caused through AhR activation. Data presented herein also demonstrate that low oxygen tension decreases AhR activation and signaling and increases the inherent toxicity of TCDD.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.