Functional genomics does not have to be limited to a few select organisms.
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Animals↗
Biomedical subjects
D L Crawford
Publications and source records attributed to D L Crawford.
Left side first: a different approach for teaching laparoscopic Nissen fundoplication.
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Dissection↗
Anaerobic biodegradation of pentachlorophenol in mixtures containing cadmium by two physiologically distinct microbial enrichment cultures.
Anaerobic biodegradation of pentachlorophenol (PCP), in mixtures containing cadmium (Cd), by sulfidogenic (SRB) and methanogenic (MET) enrichment cultures, was studied. Removal of 91-93% of PCP occurred in both SRB- and MET-enriched cultures, in the absence of Cd, within 82 days. The presence of soluble Cd initially decreased the rate of PCP removal by the enrichment cultures, but PCP removal rates improved as the Cd precipitated. GC-MS, 14C-PCP, and 13C-PCP studies confirmed mineralization of PCP by both enrichment cultures, as well as the incorporation of PCP carbon into specific phospholipid fatty acids (PLFAs) of the cell membranes of PCP-degrading anaerobes. This is the first report on anaerobic biodegradation of PCP by SRB- and MET-enriched cultures in the presence, with simultaneous precipitation, of the toxic heavy metal Cd, and of the incorporation of PCP carbons into specific PLFAs of the anaerobic bacterial cells.
Anaerobiosis↗
C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells.
Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.
Amino Acid Motifs↗
Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes.
We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.
Blotting, Southern↗
Thatch biodegradation and antifungal activities of two lignocellulolytic Streptomyces strains in laboratory cultures and in golf green turfgrass.
The use of lignocellulolytic Streptomyces spp. as biological agents, to enhance thatch degradation in turf and to slow its rate of accumulation while controlling fungal growth in the thatch layer, was studied. In flask scale studies, two lignocellulolytic Streptomyces violaceusniger (= hygroscopicus) strains (YCED9 and WYE53) decomposed thatch (> 30% dry weight) over a 12-week incubation period. Biodegradation was accompanied by production of extracellular cellulases, xylanases, and peroxidases. The accumulation of the polymeric, water-soluble lignin degradation intermediate acid, precipitable polymeric lignin (APPL), was also observed. Residual thatch from 12-week-old cultures had an increased lignin-to-carbohydrate ratio, an indication that although lignin was metabolized, carbohydrates were preferential carbon sources for these actinomycetes. A spore-containing soluble dry powder formulation was used as an inoculum in an in situ field experiment. This formulation was maintained in storage at 4 degrees C for over two years without viability loss. Results from the golf green experiment showed that although treated thatch layers in established greens were not appreciably reduced over the course of one summer, the Streptomyces were active and maintained their populations within the thatch, while fungal growth was suppressed as compared to controls. The results show that treatment of turfgrass with these Streptomyces may be useful for the long-term control of fungal populations within the thatch. Longer field studies are required to assess the long-term potential for also controlling thatch build-up and fungal pathogens.
2,4-Dichlorophenoxyacetic Acid↗
Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles.
A previous phylogenetic analysis among 15 taxa of the teleost fish Fundulus suggested that there should be thermal-adaptive differences in heart metabolism among populations. To test this hypothesis, the rate of oxygen consumption and the activities of all 11 glycolytic enzymes were measured in isolated heart ventricle from two populations of Fundulus heteroclitus. Heart ventricular metabolism is greater in a northern population versus a southern population of these fish. Analysis of the amount of glycolytic enzymes indicates that 87% of the variation in cardiac metabolism within and between populations is explained by the variation in three enzymes (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase). These enzymes are the same three enzymes that were predicted to be important based on previously determined phylogenetic patterns of expression. Our data indicate that near-equilibrium enzymes, as well as classically defined rate-limiting enzymes, can also influence metabolism.
Acclimatization↗
Isolation and analysis of three peroxide sensor regulatory gene homologs ahpC, ahpX and oxyR in Streptomyces viridosporus T7A--a lignocellulose degrading actinomycete.
Increased lignolytic peroxidase activity has been demonstrated with the addition of sublethal doses of toxic H2O2 in Streptomyces viridosporus T7A. Until now, the effect of H2O2 at the molecular level has not been well characterized. Here, for the first time we report the isolation and analysis of three peroxide-induced gene homologs from S. viridosporus T7A; ahpC and ahpX (encoding alkyl hydroxyperoxidase subunits) and oxyR (encoding oxygen stress regulatory protein). The genome organization of these stress related genes were found to be divergently adjacent to each other. The protein sequence analysis of the oxyR homolog revealed a helix-turn-helix DNA-binding motif characteristic to the LysR of regulatory proteins induced by H2O2. The nucleotide sequence analysis of the intergenic region between ahpC and oxyR revealed that they shared a core T-n11-A, a signature protein-binding region of LysR family members. Based on similarities in sequence analysis, genetic organization, and the induction of lignin peroxidase activity upon exposure to hydrogen peroxide, we hypothesize a peroxide induction mechanism for the regulation of oxidative lignin biodegradation by S. viridosporus, possibly via use of OxyR which is also involved in regulating the peroxide stress response in this actinomycete.
Actinomycetales↗
Inexpensive retraction technique for advanced laparoscopic procedures.
The described technique offers surgeons an inexpensive simple way to hold intraperitoneal structures in position for long periods with minimal trauma.
Anastomosis, Surgical↗
Functional analyses of natural variation in Sp1 binding sites of a TATA-less promoter.
Within the lactate dehydrogenase-B (LdhB) proximal promoter is a region with multiple in vivo footprinted sites that resembles the binding site for the transcription factor SP1. Like many sequences that regulate transcription rate, these Sp1 binding sites are well conserved among species of the teleost fish Fundulus. The only exception is in the northern population of F. heteroclitus, where there are many changes in the Sp1 binding sites. These changes affect footprinting patterns, measures of promoter strength, and are associated with the adaptive increase in Ldh-B transcription rates. Reported here is data that demonstrates that Fundulus hepatocyctes have an SP1-like protein; in comparison to human SP1 protein, it has similar specificity and size and a greater affinity for the consensus Sp1 site. This Fundulus hepatocyte SP1-like protein as well as the human SP1 protein binds the Ldh-B Sp1 sites. Sequence variation in the northern Sp1 region eliminates the "preferred" Sp1 binding site, yet these northern Sp1 sites have significantly greater affinity for the SP1 protein than either the Sp1 sites from southern F. heteroclitus ( approximately 1.6-fold) or the consensus Sp1 site (GGGCGG; approximately 1.8-fold). Furthermore, the Ldh-B Sp1 sites also bind non-SP1 proteins, and the extent of binding is affected by the sequence variation in the proximal promoter. These data suggest that natural variation in Sp1 sites affect binding of transcription factors and may effect a modest change in transcription rates.
Animals↗
Laparoscopic common bile duct exploration.
Laparoscopic cholecystectomy has become the procedure of choice for laparoscopically skilled surgeons when dealing with both chronic and acute cholecystitis. When choledocholithiasis is encountered in the treatment of these patients the skilled laparoscopist has several treatment options available to treat the patient in one stage and avoid the morbidity of endoscopic sphincterotomy. Although still controversial, laparoscopic common bile duct exploration has been shown to be safe, applicable, and cost-effective in the treatment of choledocholithiasis. This report details several laparoscopic treatment alternatives for choledocholithiasis.
Cholangiography↗
In vitro and in vivo antagonism of pathogenic turfgrass fungi by Streptomyces hygroscopicus strains YCED9 and WYE53.
Disease prevention is a current practice used to minimize fungal diseases of turfgrasses in lawns and golf greens. Prevention is accomplished through fungicide applications, and by periodic thatch removal. During the development of a microbial biodethatch product utilizing the lignocellulose-degrading Streptomyces hygroscopicus strains YCED9 and WYE53, we demonstrated using in vitro plate antagonism bioassays that both strains are antagonists of various turfgrass fungal pathogens. These activities were present when the cultures were growing on thatch, as demonstrated by antifungal antagonism bioassays with culture filtrates. Experiments conducted using a growth chamber demonstrated that a bio-dethatch formulation containing spores of strains YCED9 and WYE53 in a zeolite carrier, provided protection for Kentucky bluegrass seedlings against turfgrass pathogens, including Pythium ultimum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia homeocarpa, Gaeumannomyces graminis and Microdochium nivale. Results showed that by integrating the use of the S. hygroscopicus YCED9/WYE53 bio-dethatch formulation into routine turf management practices, it should be possible to both minimize thatch build-up while also controlling fungal turfgrass diseases by way of the antifungal biocontrol activity of these strains. This in turn would help control fungal pathogens in turfgrass while minimizing the need for routine chemical fungicide applications.
Fungi↗
Evolutionary analysis of TATA-less proximal promoter function.
Many molecular studies describe how components of the proximal promoter affect transcriptional processes. However, these studies do not account for the likely effects of distant enhancers or chromatin structure, and thus it is difficult to conclude that the sequence variation in proximal promoters acts to modulate transcription in the natural context of the whole genome. This problem, the biological importance of proximal promoter sequence variation, can be addressed using a combination of molecular and evolutionary analyses. Provided here are molecular and evolutionary analyses of the variation in promoter function and sequence within and between populations of Fundulus heteroclitus for the lactate dehydrogenase-B (Ldh-B) proximal promoter. Approximately one third of the Ldh-B proximal promoter contains interspersed regions that are functionally important: (1) they bind transcription factors in vivo, (2) they effect a change in transcription as assayed by transient transfection into two different fish cell lines, and (3) they bind purified transcription factors in vitro. Evolutionary analyses that compare sequence variation in these functional regions versus the nonfunctional regions indicate that the changes in the Ldh-B proximal promoter sequences are due to directional selection. Thus, the Ldh-B proximal promoter sequence variations that affect transcriptional processes constitute a phenotypic change that is subject to natural selection, suggesting that proximal promoter sequence variation affects transcription in the natural context of the whole genome.
Animals↗
Magnesium deficiency-induced osteoporosis in the rat: uncoupling of bone formation and bone resorption.
Magnesium (Mg) intake has been linked to bone mass and/or rate of bone loss in humans. Experimental Mg deficiency in animal models has resulted in impaired bone growth, osteopenia, and increased skeletal fragility. In order to assess changes in bone and mineral homeostasis that may be responsible, we induced dietary Mg deficiency in adult Simonsen albino rats for 16 weeks. Rats were fed either a low Mg diet (0.002 percent) or a normal control Mg diet (0.063 percent). Blood was obtained at baseline, 4 weeks, 8 weeks, 12 weeks and 16 weeks in both groups for serum Mg, calcium, PTH, and 1.25(OH)2-vitamin D determinations. Femora were harvested at 4 weeks and 16 weeks for mineral analysis and histomorphometry. Serum Mg fell in the Mg depleted group to 0.6 mg/dl (mean) by 16 weeks (controls = 2.0 mg/dl). The serum calcium (Ca) concentration was higher in the Mg depleted animals at 16 weeks, 10.8 mg/dl (controls = 8.9 mg/dl). Serum PTH concentration fell progressively in the Mg deficient rats to 30 pg/ml by week 16 (control = 96 pg/ml). Serum concentration of 1.25(OH)2-vitamin D also fell progressively in the Mg deficient animals by 16 weeks to 14 pg/ml (control = 30 pg/ml). While the percent ash weights of Ca and phosphorus in the femur were not different at any time point, the percent ash weight of Mg progressively fell to 0.54 percent vs control (0.74 percent) by 16 weeks. The percent ash weight of potassium also fell progressively in the Mg deficient group to approximately 30 percent of control by 16 weeks. Histomorphometric analyses showed a significant drop in trabecular bone volume in Mg deficient animals by 16 weeks (percent BV/TV = 13.2 percent vs 17.3 percent in controls). Evaluation of the endosteal bone surface features showed significantly greater bone resorption in the Mg depleted group as reflected in increased number of tartrate-resistant positive osteoclasts/mm bone surface (7.8 vs 4.0 in controls) and an elevated percent of bone surface occupied by osteoclasts (percent OcS/BS = 12.2 percent vs 6.7 percent in controls. This increased resorption occurred in the presence of an inappropriate lowered bone forming surface relative to controls; a decreased number of osteoblasts per mm bone surface (0.23 vs 0.94 in control) and a decrease in percent trabecular surface lined by osteoid (percent OS/BS = 0.41 vs 2.27 percent in controls) were also noted. Our findings demonstrate a Mg-deficiency induced uncoupling of bone formation and bone resorption resulting in a loss of bone mass. While the fall in PTH and/or 1.25(OH)2-D may explain a decrease in osteoblast activity, the mechanism for increased osteoclast activity is unclear. These data suggest that Mg deficiency may be a risk factor for osteoporosis.
Animals↗
Divergent Hsc70 binding properties of mitochondrial and cytosolic aspartate aminotransferase. Implications for their segregation to different cellular compartments.
Cytosolic Hsc70 discriminates between the homologous mitochondrial and cytosolic isozymes of aspartate aminotransferase, binding exclusively the mitochondrial form. By screening a library of synthetic peptides spanning the sequence of the mitochondrial enzyme, we have identified binding sites in this polypeptide that interact with Hsc70. These potential binding sites are scattered over the entire sequence and map to secondary structure elements, particularly the alpha-helix, that are partly exposed on the surface of the native protein. Several peptides corresponding to analogous positions in the cytosolic enzyme sequence do not bind to Hsc70. Phylogenetic analyses suggest that Hsc70 binding sequences have diverged as a consequence of biochemical specialization ensuring differential interaction of each isozyme with the cellular machinery in charge of protein folding and translocation.
Amino Acid Sequence↗
Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A.
Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce an Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases.
Amino Acid Sequence↗
Laparoscopic repair and groin hernia surgery.
Over the past 15 years, laparoscopic herniorrhaphy has made the transition from an experimental to a proven procedure. With increasing laparoscopic skills in the surgical community, many surgeons are now faced with the question of when to recommend laparoscopic herniorrhaphy to their patients. A surgeon's best hernia repair is the one with which they have had the greatest experience. This results in the lowest recurrence and complication rate in his or her hands. Certainly, simple, unilateral hernias and bilateral hernias can be repaired with either anterior or laparoscopic techniques. Many times, laparoscopic herniorrhaphy is too much surgery for a young patient with a unilateral hernia. In such a case, repair is best performed with the patient under local anesthesia. Also, young patients in whom it is advantageous to avoid mesh should not undergo laparoscopic herniorrhaphy. The authors prefer laparoscopic TEP herniorrhaphy in patients with recurrent hernias, bilateral hernias, and unilateral hernias with a suspected contralateral hernia. There is also a consensus that patients with multiple recurrent hernias in whom a preperitoneal repair is appropriate are best served with a laparoscopic repair. Surgeons without advanced laparoscopic skills or without the time to develop the skills necessary to perform laparoscopic herniorrhaphy should consider referring patients with recurrent hernias to surgeons with experience in TEP. TEP is preferable to TAPP because of its lower complication and recurrence rates and in the authors' hands is the "best repair." TAPP should be reserved for patients with prior lower abdominal wall incisions that make the dissection of the peritoneum from the underside of the incision impossible. Patients who cannot tolerate general anesthesia or who have had extensive lower abdominal surgery should not undergo laparoscopic herniorrhaphy. Complication and recurrence rates, although initially higher than traditional repairs, have now fallen to equal or lower levels at centers experienced in laparoscopic techniques. Prospective randomized trials prove that when patients are selected properly and surgeons are adequately trained and proctored, laparoscopic herniorrhaphy can be performed with acceptably low incidences of recurrence and complications.
Adolescent↗
Spontaneous renojejunal fistula.
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Female↗