ABSTRACT
We applied a multiscale modeling approach that involves the statistical-mechanical three-dimensional reference interaction site model with the Kovalenko-Hirata closure approximation (3D-RISM-KH molecular theory of solvation) as well as density functional theory (DFT) of electronic structure to study the role of water in aggregation of the asphaltene model compound 4,4'-bis(2-pyren-1-yl-ethyl)-2,2'-bipyridine (PBP) [X. Tan, H. Fenniri and M. R. Gray, Energy Fuels, 2008, 22, 715]. The solvation free energy and potential of mean force predicted by 3D-RISM-KH reveal favorable pathways for disaggregation of PBP dimers in pure versus water-saturated chloroform solvent. The water density distribution functions elucidate hydrogen bonding preferences and water bridge formation between PBP monomers. The ΔG(298) values of -5 to -7 kcal mol(-1) for transfer of water molecules in chloroform to a state interacting with PBP molecules are in agreement with experimental results. Geometry optimization and thermochemistry analysis of PBP dimers with and without water bridges using WB97Xd/6-31G(d,p) predict that both PBP dimerization and dimer stabilization by water bridges are spontaneous (ΔG(298) < 0). The (1)H NMR chemical shifts of PBP monomers and dimers predicted using the gauge-independent atomic orbital method and polarizable continuum model for solvation in chloroform are in an excellent agreement with the experimental results for dilute and concentrated PBP solutions in chloroform, respectively [X. Tan, H. Fenniri and M. R. Gray, Energy Fuels, 2009, 23, 3687]. The DFT calculations of PBP dimers with explicit water show that bridges containing 1-3 water molecules lead to stabilization of PBP dimers. Additional water molecules form hydrogen bonds with these bridges and de-shield the PBP protons, negating the effect of water on the (1)H(C3) NMR chemical shift of PBP, in agreement with experiment. The ΔG(298) results show that hydrogen bonding to water and water-promoted polynuclear assembly bridging is as important as π-π interactions for asphaltene aggregation.
ABSTRACT
During the last century, much of the United States experienced warming temperatures and changes in amount and intensity of precipitation. Changes in future climate conditions present additional risk to water and watershed managers. The most recent release of U.S. EPA's BASINS watershed modeling system includes a Climate Assessment Tool (CAT) that provides new capabilities for assessing impacts of climate change on water resources. The BASINS CAT provides users with the ability to modify historical climate and conduct systematic sensitivity analyses of specific hydrologic and water quality endpoints to changes in climate using the BASINS models (Hydrologic Simulation Program - FORTRAN (HSPF)). These capabilities are well suited for addressing questions about the potential impacts of climate change on key hydrologic and water quality goals using the watershed scale at which most important planning decisions are made. This paper discusses the concepts that motivated the CAT development effort; the resulting capabilities incorporated into BASINS CAT; and the opportunities that result from integrating climate assessment capabilities into a comprehensive watershed water quality modeling system.
Subject(s)
Climate , Environmental Monitoring , Software , Water Pollutants/analysis , Water Supply , Calibration , Models, Theoretical , United States , Water MovementsABSTRACT
AIMS: To isolate bacteria capable of cleaving aliphatic carbon-sulfur bonds as potential biological upgrading catalysts for the reduction of molecular weight and viscosity in heavy crude oil. METHODS AND RESULTS: Thirty-one bacterial strains isolated from enrichment cultures were able to biotransform model compounds representing the aliphatic sulfide bridges found in asphaltenes. Using gas chromatography and mass spectrometry, three types of attack were identified: alkyl chain degradation, allowing use as a carbon source; nonspecific sulfur oxidation; and sulfur-specific oxidation and carbon-sulfur bond cleavage, allowing use as a sulfur source. Di-n-octyl sulfide degradation produced octylthio- and octylsulfonyl-alkanoic acids, consistent with terminal oxidation followed by beta-oxidation reactions. Utilization of dibenzyl sulfide or 1,4-dithiane as a sulfur source was regulated by sulfate, indicating a sulfur-specific activity rather than nonspecific oxidation. Finally, several isolates were also able to use dibenzothiophene as a sulfur source, and this was the preferred organic sulfur substrate for one isolate. CONCLUSIONS: The use of commercially available alkyl sulfides in enrichment cultures gave isolates that followed a range of metabolic pathways, not just sulfur-specific attack. SIGNIFICANCE AND IMPACT OF THE STUDY: These results give new insight into biodegradation of organosulfur compounds from petroleum and for biotreatment of such compounds in chemical munitions.
Subject(s)
Bacteria/metabolism , Chemical Industry , Industrial Microbiology/methods , Petroleum , Sulfides/metabolism , Aerobiosis , Biodegradation, Environmental , Bioreactors , Carbon , Molecular Weight , Sulfur , ViscosityABSTRACT
Twenty-five patients with definite bites by Badumna spp. spiders were recruited prospectively. Bites occurred across Australia, 92% from October to March, and most during daylight. Pain occurred in all cases, was severe in seven cases, with a median duration of 5 min. Other effects included: puncture marks (24%), swelling (12%) and erythema (68%). Minor systemic effects occurred in 4 bites. No patient had a necrotic ulcer (0%; 97.5% confidence interval 0-14%). Black house spiders (Badumna spp.) are unlikely to cause necrosis, contrary to reports of suspected bites. Patients can be reassured and do not require any further investigation or treatment.
Subject(s)
Insect Bites and Stings/complications , Pain/etiology , Spider Bites/etiology , Spider Venoms/adverse effects , Spiders , Adolescent , Adult , Aged , Animals , Australia/epidemiology , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Necrosis , Prospective Studies , SeasonsABSTRACT
Spermatozoa and spermiogenesis of the prithine filistatid spider Wandella orana are described. The spider produces coenospermia, i.e. sperm aggregations that include several single sperm cells commonly surrounded by a secretion sheath. One sectioned coenospermium in W. orana contains at least five spermatozoa. During copulation many coenospermia are transferred into the female. Coenospermia are regarded as a peculiar transfer form of sperm which occurs in early derivative spiders such as Liphistiomorphae and Mygalomorphae. The only exception which was found in Araneomorphae until now was the filistatine spider Filistata insidiatrix. Our observation is the second case and supports the view that Filistatidae represent an early derivative taxon. Furthermore, the individual sperm cells show characteristics which also may be regarded as being plesiomorphic. There is a cone-shaped acrosomal vacuole, a very long acrosomal filament, a rather stout nucleus and a small implantation fossa. The axoneme shows the 9x2+3 pattern of microtubules which is synapomorphic in Megoperculata (Uropygi, Amblypygi and Araneae). The finding of coenospermia in two distant taxa of Filistatidae may have consequences for phylogenetic and systematic considerations.
Subject(s)
Phylogeny , Spermatogenesis/physiology , Spermatozoa/ultrastructure , Spiders/cytology , Acrosome/ultrastructure , Animals , Cell Nucleus/ultrastructure , Male , Microscopy, Electron , Sperm Head/ultrastructure , Spermatogenesis/genetics , Spiders/genetics , Testis/ultrastructure , Vas Deferens/ultrastructureABSTRACT
STUDY OBJECTIVE: WNT7A gene mutations were evaluated as a potential cause for Müllerian duct derivative abnormalities in human females. The WNT gene family encodes glycoproteins that serve as signaling molecules during early development. The WNT7A gene has been previously identified as necessary for normal murine Müllerian duct development. WNT7A mutant mice display several Müllerian duct derivative abnormalities. DESIGN: Molecular genetic analysis of female patients with Müllerian duct derivative abnormalities. SETTING: Medical center-based academic research institution. PARTICIPANTS: 40 women with developmental abnormalities of the uterus and vagina and 12 normal controls. INTERVENTIONS: Polymerase chain reaction DNA amplification from human genomic DNA and denaturing gradient gel electrophoresis analysis of amplified DNA fragments. MAIN OUTCOME MEASURES: Presence or absence of WNT7A gene mutations in analyzed DNA fragments. RESULTS: No mutations were found in the WNT7A gene in any patient or control tested. CONCLUSIONS: WNT7A mutations are an unlikely cause of Müllerian duct derivative abnormalities in humans.
Subject(s)
Mullerian Ducts/abnormalities , Mutation , Proto-Oncogene Proteins/genetics , Case-Control Studies , Female , Humans , Polymerase Chain Reaction , Wnt ProteinsABSTRACT
The aetiology of anomalous embryonic and fetal development of the female reproductive tract, ranging from common uterine abnormalities to the somewhat rare congenital absence of the uterus and vagina (CAUV), is unknown. Some have proposed that abnormal galactose metabolism might cause CAUV. An association between CAUV and the N314D allele of the galactose-1-phosphate uridyl transferase (GALT) gene has been proposed as aetiological. We tested this hypothesis further by performing a case-control molecular study analysing 32 patients with CAUV for the presence of the N314D allele. These patients were compared with 138 normal controls. No association between CAUV and the N314D polymorphism was found (P = 0.32). It is unlikely that either maternal or fetal GALT enzyme activity could affect paramesonephric duct development, because neither galactosaemic subjects nor their children have an increased incidence of uterine anomalies.
Subject(s)
Polymorphism, Genetic , UTP-Hexose-1-Phosphate Uridylyltransferase/genetics , Uterus/abnormalities , Vagina/abnormalities , Case-Control Studies , DNA Mutational Analysis , Female , Gene Frequency , Humans , MaleABSTRACT
The kinetics of biodegradation of polycyclic aromatic hydrocarbons (PAHs) by a mixed culture were determined in a creosote-contaminated soil and in a pristine soil. A competitive-inhibition model was able to represent the kinetics of degradation of PAHs from the creosote-contaminated soil, from the lag phase through to active degradation, but not data from pristine soil with the same PAHs alone and in mixtures. The presence of phenanthrene introduced a lag phase of 4.5 days in the degradation of fluoranthene and 5 days for chrysene. Rapid degradation of pyrene followed a lag phase of circa 5 days, regardless of the presence of other PAHs. These results show that even when kinetics of PAH degradation by mixed cultures appear to follow competitive-inhibition kinetics, the underlying mechanisms may be more complex.
Subject(s)
Polycyclic Aromatic Hydrocarbons/metabolism , Soil Pollutants/metabolism , Biodegradation, Environmental , Chrysenes/metabolism , Creosote/metabolism , Fluorenes/metabolism , Kinetics , Phenanthrenes/metabolism , Pyrenes/metabolismABSTRACT
Analysis of spider webs from limestone arches in New South Wales, Australia showed levels of lead and zinc to be several times higher at Jenolan Caves than reference sites at Abercrombie and Wombeyan Caves. The high concentrations at Jenolan were attributed to emissions from motor vehicles that travel through the arch. Analysis of water-soluble ions in webs from Jenolan and Abercrombie Caves showed large differences between the sites, which could be explained by differences in the geology and biology of the cave environments. Spider webs proved useful indicators of environmental chemistry and undoubtedly have applications beyond the cave environments studied here.
Subject(s)
Environmental Monitoring , Environmental Pollutants/analysis , Metals, Heavy/analysis , Spiders , Animals , New South Wales , Vehicle EmissionsABSTRACT
Possible effects of habitat fragmentation on the population genetics of a species of wolf spider (Lycosidae) from remnant Callitris woodland in the wheat belt of central western New South Wales in Australia are examined. Single-strand conformational analysis of mitochondrial cytochrome oxidase (subunit 1) was used to characterize the haplotypes of 295 spiders in six blocks each of four woodland sites. DNA sequences were collected from 119 of these spiders to confirm haplotype scoring, allow phylogeny estimation and permit calculation of sequence-based statistics. Intra-block tests do not suggest widespread effects of fragmentation. Genetic diversity is high in all blocks, with 25 haplotypes being identified. Nucleotide diversity is relatively low, as all of the haplotypes are closely related. One block had a significantly low value for the Ewens/Watterson test of neutrality and one block's value was nearly significantly high. Two blocks had nearly significant values of the Harpending Raggedness Index testing for recent population bottlenecks. No other intrablock tests approach significance. Interpopulation comparisons show significant nonhomogeneity of haplotype frequencies globally and in all pairwise comparisons. Relationships between woodland blocks based on haplotype frequencies are discordant with geographical proximity. Haplotype distribution patterns suggest that population structuring existed prior to fragmentation. We develop two measures of genetic distinctiveness to identify subpopulations of interest for conserving evolutionary processes in a species' regional population. One is based on the sum of pairwise FST values and one on the spatial distribution of genetic variation. High values of the measure suggest a subpopulation might have been recently perturbed and low values that it is relatively undisturbed. The two measures identify different blocks as being of particular interest.
Subject(s)
Genetic Variation , Genetics, Population , Spiders/genetics , Animals , Base Sequence , DNA, Mitochondrial , Ecosystem , Electron Transport Complex IV/genetics , Environment , Haplotypes/genetics , Molecular Sequence Data , New South Wales , Phylogeny , TriticumABSTRACT
BACKGROUND: Spider bite is a subject of much medical mythology with prevalent fears that spiders cause severe envenoming, with neurotoxic effects or necrotic ulcers. Clinical experience and small studies suggest otherwise, but this has not been confirmed by prospective studies of bites by identified spiders. AIM: To describe the clinical effects of bites by accurately identified spiders, and determine whether early clinical features and circumstances can predict spider type. DESIGN: Prospective follow-up study. METHODS: Patients were recruited from admissions to two emergency departments (n=48) and referrals from three state poison information centres (n=1426), over 27 months. Overall, there were 750 people with definite spider bites where the spiders were immediately collected and expertly identified. RESULTS: Significant effects occurred in 44 bites (6%), including 37 (of 56) redback spider bites (Latrodectus hasselti) with significant pain lasting >24 h. Of these, only 6 (11%) received antivenom. One severe neurotoxic envenoming by an Australian funnelweb spider required antivenom. No definite spider bites resulted in necrotic ulcers (0%, 99%CI 0-0.7%). There were no early allergic reactions and secondary infection occurred in seven cases (0.9%, 95%CI 0.4-1.9%). Circumstances and early clinical effects were strongly associated with taxonomic spider identification, with positive predictive values >0.95 for common groups of spiders. CONCLUSIONS: Australian spider bite caused minor effects in most cases and is unlikely to cause necrotic ulcers, allergic reactions or infection. Redback spider bite (widow spider) caused prolonged pain, and antivenom could have been used more frequently. The circumstances and early clinical features of spider bites may allow early appropriate advice and treatment of spider bite without taxonomic identification.
Subject(s)
Spider Bites/diagnosis , Spiders/classification , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Australia , Child , Child, Preschool , Expert Testimony , Female , Follow-Up Studies , Humans , Infant , Male , Middle Aged , Prospective Studies , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To determine the types of spiders causing bites and the clinical effects of their bites in tropical Northern Territory (north of the town of Katherine). DESIGN: A prospective study of confirmed and suspected spider-bites and a retrospective analysis of data from a standardised, local database of spider- and snake-bites. Confirmed spider-bites were those in which there was a clear history of the bite, and the captured spider was identified by an arachnologist. SETTING: Emergency department of a teaching hospital. SUBJECTS AND DATA SOURCE: All subjects with confirmed or suspected spider-bite presenting to the Emergency Department or referred from August 1999 to August 2000, or identified from the database. RESULTS: Thirty-four subjects had a confirmed spider-bite from an identified spider: 25 in the prospective group and nine in the retrospective group. The spiders were Sparassidae (huntsman spider) (12 bites), Missulena pruinosa (northern mouse spider) (7), Latrodectus (widow spider) (4), Araneidae (orb-weaver) (4), Salticidae (jumping spider) (4), Nemesidae (trapdoor spider) (1), Conothele (1) and Selenocosmia (whistling spider) (1). Clinical effects were local pain in 97% (severe in 29%), redness in 47% and swelling in 24% of cases. Systemic effects occurred in three victims, two of whom were bitten by M. pruinosa. There were no cases of confirmed necrotic arachnidism. CONCLUSIONS: None of the spider-bites resulted in severe effects. Compared with data from other parts of Australia, different species were involved and latrodectism was uncommon. Our study highlighted the importance of correctly identifying the spider species.
Subject(s)
Spider Bites/epidemiology , Spiders/classification , Adolescent , Adult , Aged , Animals , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Northern Territory/epidemiology , Prospective Studies , Retrospective Studies , Spider Bites/pathologyABSTRACT
Molecular genetic techniques were used to determine if mutations in the genes encoding anti-Müllerian hormone (AMH) (also known as Müllerian inhibiting substance (MIS)) and its receptor (AMHR) are commonly present in patients with congenital absence of the uterus and vagina (CAUV). Twenty-two CAUV patients and 96 control subjects from diverse ethnic groups were studied after obtaining informed consent. Genomic DNA samples prepared from leukocytes were digested separately with several different restriction enzymes, and the resultant fragments were analyzed for restriction fragment melting polymorphisms (RFMPs) by denaturing gradient gel electrophoresis (DGGE). Electrophoretic mobility of DNA fragments which were 200-700 base pairs in length was compared using polyacrylamide gels that included linear gradients of denaturing solvents designed to separate DNA fragments according to sequence-dependent variation in thermal stability. Two RFMPs were found in the AMH gene in both patients and normal control subjects. One RFMP in the AMHR gene was present at low frequencies in both patients and normal control subjects. No RFMPs specific to CAUV patients were found in either gene. Because no mutations or rare DNA sequence polymorphisms were detected in the AMH and the AMHR genes in this group of CAUV patients, it is unlikely that either gene commonly has an etiologic role in CAUV.
Subject(s)
Glycoproteins , Growth Inhibitors/genetics , Receptors, Peptide/genetics , Testicular Hormones/genetics , Urogenital Abnormalities/genetics , Uterus/abnormalities , Vagina/abnormalities , Anti-Mullerian Hormone , DNA/analysis , Female , Humans , Mullerian Ducts/growth & development , Mutation , Polymorphism, Restriction Fragment Length , Receptors, Transforming Growth Factor betaABSTRACT
OBJECTIVE: Imperforate hymen is an uncommon anomaly of the reproductive tract, occurring in approximately 0.1% of newborn females. The familial occurrence of imperforate hymen in a child, her mother, and her mother's monozygotic twin is reported. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): Three affected family members. MAIN OUTCOME MEASURE(S): Karyotype and pedigree analysis. RESULT(S): The proband, presenting with peritonitis, was evaluated at age 12 for imperforate hymen because this condition was diagnosed in her mother at age 14. At age 14, the mother's monozygotic twin was asymptomatic except for primary amenorrhea and was also demonstrated to have imperforate hymen. No other reproductive system abnormalities were known to be present in the remaining family members. Chromosomal structural analysis confirmed that the mother of the proband had no chromosomal abnormalities. CONCLUSION(S): The occurrence of imperforate hymen in two consecutive generations of a family is consistent with a dominant mode of transmission, either sex-linked or autosomal. Previously reported examples of siblings with imperforate hymen suggested a recessive mode of inheritance. Taken together, these cases suggest that imperforate hymen can be caused by mutations in several genes. This case highlights the importance of evaluating all family members of affected patients. Familial examples of other developmental anomalies of the female reproductive tract also suggest a multifactorial genetic etiology.
Subject(s)
Diseases in Twins , Genes, Dominant , Hymen/abnormalities , Vaginal Diseases/genetics , Adult , Female , Humans , Karyotyping , Middle Aged , Pedigree , Twins, Monozygotic/geneticsABSTRACT
The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.
Subject(s)
Environmental Pollutants/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Pseudomonas fluorescens/metabolism , Binding, Competitive , Biological Transport, Active , Energy Metabolism , Kinetics , Mutation , Phenanthrenes/metabolism , Plasmids/genetics , Pseudomonas fluorescens/geneticsABSTRACT
OBJECTIVES: Denaturing gradient gel electrophoresis can detect single base sequence differences in deoxyribonucleic acid and methylation differences in small cloned fragments of deoxyribonucleic acid. We previously detected cell type-specific melting differences by denaturing gradient gel electrophoresis in paired leukocyte and sperm cell samples of deoxyribonucleic acid. We proposed that these differences were caused by differential methylation and that blotting strategies using denaturing gradient gel electrophoresis might be useful in detecting in vivo variations in methylation patterns. STUDY DESIGN: Genomic deoxyribonucleic acid from leukocytes and sperm cells of 35 male subjects was analyzed by denaturing gradient gel electrophoresis after digestion by 4-bp site enzymes and Msp I and its methylation-sensitive isoschizomer Hpa II. Some fragments were amplified by polymerase chain reaction. RESULTS: Cell type-specific melting polymorphisms were detected in all genes from all subjects. Analysis of Msp I/Hpa II sites demonstrated that differences noted correlated with the methylation state. Cell type-specific differences were absent in fragments amplified by polymerase chain reaction. CONCLUSIONS: The denaturing gradient gel electrophoresis blotting technique is a fast and comprehensive method for comparing in vivo methylation differences.
Subject(s)
DNA/genetics , Polymorphism, Genetic/genetics , Alleles , DNA/isolation & purification , Electrophoresis, Gel, Two-Dimensional , Female , Genome , Humans , Leukocytes/chemistry , Male , Methylation , Nucleic Acid Hybridization , Polymerase Chain Reaction , Restriction Mapping , Spermatozoa/chemistry , Substrate SpecificityABSTRACT
Surfactants have been proposed as a promising method to enhance bioremediation of hydrophobic compounds in contaminated soils. However, the results of effects of surfactants on bioremediation are not consistent. This study showed that Triton X-100 at low concentration (0.024 mM or 0.09 CMC) inhibited the rate of growth of either a Mycobacterium sp. or a Pseudomonas sp. on solid anthracene as sole carbon source. Recovery of microbial growth rate could be achieved by dilution of surfactants, while addition of more surfactant gave an immediate decrease in growth rate. No inhibition of growth by Triton X-100 was observed with growth on glucose. The surfactant sorbed onto the surfaces of both the cells and the anthracene particles, which could inhibit uptake of anthracene. The results were consistent with the hypothesis that inhibition of microbial adhesion of cells to anthracene was responsible for the inhibition of growth by Triton X-100.
Subject(s)
Anthracenes/metabolism , Mycobacterium/growth & development , Pseudomonas/growth & development , Surface-Active Agents/pharmacology , Cell Division/drug effects , Dose-Response Relationship, Drug , Glucose/metabolism , Micelles , Mycobacterium/drug effects , Mycobacterium/metabolism , Octoxynol/metabolism , Octoxynol/pharmacology , Pseudomonas/drug effects , Pseudomonas/metabolism , Soil Microbiology , Surface-Active Agents/metabolismABSTRACT
Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Factor VIII/genetics , Nucleic Acid Denaturation , Point Mutation/genetics , Blotting, Southern , DNA/analysis , DNA Mutational Analysis/methods , Exons/genetics , Female , Gene Duplication , Hemophilia A/genetics , Humans , Male , Mutagenesis, Insertional , Pedigree , Polymorphism, Genetic/genetics , Sequence DeletionABSTRACT
OBJECTIVE: Several lines of evidence suggest that expression of the WT1 transcription factor gene is necessary for normal development of the renal and male reproductive systems. Female patients with severe reproductive tract developmental defects were examined for WT1 gene mutations. STUDY DESIGN: The WT1 gene was analyzed in 25 patients with congenital absence of the uterus and vagina for mutations. Genomic deoxyribonucleic acid prepared from blood leukocytes was subjected to Southern blot analysis and denaturing gradient gel electrophoresis. RESULTS: Common WT1 gene deoxyribonucleic acid sequence polymorphisms were found in both normal control subjects and patients with congenital absence of the uterus and vagina. No deoxyribonucleic sequence differences or mutations likely to cause congenital absence of the uterus and vagina were detected in the patients. CONCLUSIONS: The absence of WT1 gene mutations in patients with congenital absence of the uterus and vagina supports the hypothesis that WT1 expression is required only for later urogenital development, after the mesonephric and paramesonephric ducts have already formed.
Subject(s)
DNA-Binding Proteins/genetics , Genes, Wilms Tumor/physiology , Mullerian Ducts/growth & development , Transcription Factors/genetics , Uterus/abnormalities , Vagina/abnormalities , Base Sequence , DNA/genetics , Electrophoresis , Female , Humans , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , WT1 ProteinsABSTRACT
In this study, potential alterations in hepatic blood flow, plasma protein binding, hepatic tissue binding, and enzyme activities induced by LD iv infusion of lidocaine (LD) were evaluated using a chronically instrumented dog model. Four conscious female mongrel dogs (19.0-23.5 kg) were each given, on days 1 and 10, a 5-min infusion of a mixture of unlabeled LD at approximately 2 mg/kg and 14C-labeled LD at approximately 25 microCi and, on day 8, a 12-h constant rate iv infusion of LD (approximately 76 microg/kg/min). During LD infusion, there was a 11-79% increase in total hepatic blood flow, mainly due to a 1.6-9.2-fold increase in hepatic arterial flow. Despite similar blood clearance (27.5 +/- 6.0 mL/min/kg vs 27.5 +/- 3.5 mL/min/kg), volume of distribution at steady state (1.38 +/- 0.08 L/kg vs 1.36 +/- 0.17 L/kg), and free fraction values of LD between days 1 and 10 (p > 0.05), intrinsic clearance values were consistently reduced (1224 +/- 859 mL/ min/kg vs 285 +/- 104 mL/min/kg; p = 0.034). Furthermore, hepatic tissue uptake of LD and/or its metabolites was less on day 10 than on day 1 (39.7 +/- 14.5 micromol vs 30.1 +/- 15.1 micromol; p = 0.072). The extent of N-dealkylation of LD to MEGX was unaltered, whereas sequential biotransformation of MEGX was impaired. Hence, these findings suggested that LD infusion led to a reduction of hepatic intrinsic clearance, although the change was not significant enough to alter its conventional kinetic parameters.