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Biomedical subjects

D P Clark

Publications and source records attributed to D P Clark.

At least 19 recordsLinked to original sources
PubMed2001

A novel RNase G mutant that is defective in degradation of adhE mRNA but proficient in the processing of 16S rRNA precursor.

Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA. Consequently, defects in RNase G result in elevation of AdhE levels. Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation. We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng. Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430. DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430. This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2). However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants. We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced. These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.

Alcohol Dehydrogenase↗
PubMed2001

Novel alcohol dehydrogenase activity in a mutant of Salmonella able to use ethanol as sole carbon source.

We selected a mutant of Salmonella enterica serovar Typhimurium that is capable of growing in air on ethanol as sole carbon and energy source. This adhI mutant expressed high levels of a novel alcohol dehydrogenase (AdhI) that uses ethanol, 1-propanol and 2-propanol as substrates. The fermentative AdhE alcohol dehydrogenase was not expressed aerobically in the adhI mutant. Anaerobically, both the novel AdhI enzyme and the AdhE were expressed simultaneously in the adhI mutant. However, the adhI mutant showed no alteration in the composition of the fermentation products. In addition we found that both the parental Salmonella and its alcohol using adhI mutant expressed substantial levels of a dye-linked aldehyde dehydrogenase that is presumably responsible for conversion of acetaldehyde to acetate. This contrasts with the situation in Escherichia coli where mutants able to grow on ethanol express high aerobic levels of the AdhE enzyme, which performs both the alcohol dehydrogenase and aldehyde dehydrogenase reactions.

2-Propanol↗
PubMed2001

Human telomerase reverse transcriptase expression in Diff-Quik-stained FNA samples from thyroid nodules.

Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.

Actins↗
PubMed2001

Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli.

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0. 5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB(glc). Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.

Alleles↗
PubMed2001

Cryptosporidium parvum infection requires host cell actin polymerization.

The intracellular protozoan parasite Cryptosporidium parvum accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.

Actin-Related Protein 2↗
PubMed2000

Acetaldehyde dehydrogenase activity of the AdhE protein of Escherichia coli is inhibited by intermediates in ubiquinone synthesis.

Defects in the acd gene (which may be allelic to ubiH) result in the inactivation of the coenzyme A-linked acetaldehyde dehydrogenase activity of the multifunctional AdhE protein of Escherichia coli. This activity is restored by addition of ubiquinone-0 to cell extracts. However, the alcohol dehydrogenase activity of the AdhE protein is not decreased by an acd mutation. Abolition of ubiquinone biosynthesis by mutation of ubiA or ubiF does not affect either the acetaldehyde dehydrogenase or the alcohol dehydrogenase activity of AdhE. Guaiacol (2-methoxyphenol), which resembles the intermediate that builds up in ubiH mutants, except in lacking the octaprenyl side-chain, was found to inhibit ethanol metabolism in vivo, presumably via inhibition of acetaldehyde dehydrogenase. In vitro assays confirmed that guaiacol inhibited acetaldehyde dehydrogenase. This suggests that the acetaldehyde dehydrogenase activity of AdhE is specifically inhibited by intermediates of ubiquinone synthesis that accumulate in acd mutants and that this inhibition may be relieved by ubiquinone.

Alcohol Dehydrogenase↗
PubMed2000

Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.

Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.

Actins↗
PubMed2000

Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid.

Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the adhE(+) structural gene was cloned into expression vectors under the control of the lacZ and trp(c) promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from adhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.

Aerobiosis↗
PubMed2000

Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12.

The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene. Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase. Tryptophan, indole, and several indole derivatives had no effect.

Artificial Gene Fusion↗
PubMed2000

Comparison of the fermentative alcohol dehydrogenases of Salmonella typhimurium and Escherichia coli.

The adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (Genbank accession number U68173) was cloned and sequenced. The Salmonella AdhE protein has 619/878 (70%) amino acid residues identical to the AdhE protein of Escherichia coli. Salmonella AdhE was synthesized only anaerobically. It was present in higher amounts when cells were grown on reduced substrates such as sorbitol, instead of glucose. Growth on glucuronate, which generated no net nicotinamide-adenine dinucleotide reduced (NADH) during metabolism, showed the lowest AdhE levels. Analysis of fermentation products by in vivo nuclear magenetic resonance showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate. The Salmonella enzyme had a lower Michaelis-Menten constant (Km) for alcohol substrates than AdhE of E. coli although both enzymes displayed a similar Km for nicotinamide-adenine dinucleotide (NAD+). Although AdhE of E. coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to eight carbons.

Alcohol Dehydrogenase↗
PubMed1999

Liposuction of the abdomen. An analysis of form.

This article presupposes that the surgeon has complete command of the process or craft of liposuction. This consideration of form does not presume to issue a commentary upon the ultimate fitness of the form; rather, form confines itself with shape. The final question that matters is, "has the surgeon used the craft to successfully alter the three-dimensional mass into an aesthetically pleasing end."

Abdomen↗
PubMed1999

Mohs micrographic surgery referral patterns: the University of Missouri experience.

BACKGROUND: Mohs micrographic surgery (MMS) provides a higher cure rate for nonmelanoma skin cancer (NMSC) than other forms of therapy. The American Academy of Dermatology has published recommended guidelines for MMS referral. However, factors other than the location, size, and type of NMSC may often affect the referral process. OBJECTIVE: To tabulate and analyze the rates of referral of NMSC for MMS by the dermatology clinics within the University of Missouri system. Data obtained for every biopsy-proven basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) diagnosed at each of our four dermatology clinics during the 3-year period of October 1993-September 1996 were cross-referenced with our Mohs surgery clinic logbook to identify which patients had been referred for MMS. RESULTS: During the study period, 19.2% of NMSC patients diagnosed were referred for MMS. However, there was significant disparity in referral rates among our four clinics. When the skewed data from our Veterans Administration Hospital clinic were discounted, the overall referral rate from the other clinics was found to be 30.8%. CONCLUSION: Our finding of a 30.8% referral rate of NMSC for MMS (27. 4% for SCC and 32.9% for BCC) within our university dermatology system is similar to the rates found in previous studies by the Mayo Clinic and Brooke Army Medical Center. MMS referral patterns are affected by many factors besides whether the NMSC meets MMS criteria, including the preference of each individual referring physician, patient, and involved insurance carrier.

Basal Cell Carcinoma↗
PubMed1999

Human telomerase reverse transcriptase (hTERT) gene expression in FNA samples from thyroid neoplasms.

BACKGROUND: Although fine-needle aspiration (FNA) is the most sensitive method for the detection of thyroid carcinoma, it cannot provide a definitive diagnosis of malignancy in 60% of the patients operated on for suspicious lesions. Recently, human telomerase reverse transcriptase (hTERT) has been found to be a diagnostic marker of malignancy. We therefore sought to determine whether hTERT gene expression could serve as an adjunct to FNA in the differential diagnosis of thyroid nodules. METHODS: Twenty-four FNA samples from thyroid nodules that were suspected of malignancy were collected. RNA was extracted, and hTERT gene expression was examined by RT-PCR. Cytologic and histologic examinations were also performed. RESULTS: Two of three follicular, three of three Hürthle cell, and eight of eight papillary thyroid carcinomas had corresponding FNA samples that were positive for hTERT. One of two Hürthle cell adenomas was hTERT positive. FNA samples from three follicular adenomas and five hyperplastic nodules were negative for hTERT. Positive and negative predictive values were 93% and 90%, respectively. CONCLUSIONS: The detection of hTERT gene expression in thyroid FNA samples holds promise as a diagnostic marker in the distinction of benign from malignant thyroid lesions. Its application could alter the surgical management of these patients.

Adenocarcinoma, Follicular↗
PubMed1999

New insights into human cryptosporidiosis.

Cryptosporidium parvum is an important cause of diarrhea worldwide. Cryptosporidium causes a potentially life-threatening disease in people with AIDS and contributes significantly to morbidity among children in developing countries. In immunocompetent adults, Cryptosporidium is often associated with waterborne outbreaks of acute diarrheal illness. Recent studies with human volunteers have indicated that Cryptosporidium is highly infectious. Diagnosis of infection with this parasite has relied on identification of acid-fast oocysts in stool; however, new immunoassays or PCR-based assays may increase the sensitivity of detection. Although the mechanism by which Cryptosporidium causes diarrhea is still poorly understood, the parasite and the immune response to it probably combine to impair absorption and enhance secretion within the intestinal tract. Important genetic studies suggest that humans can be infected by at least two genetically distinct types of Cryptosporidium, which may vary in virulence. This may, in part, explain the clinical variability seen in patients with cryptosporidiosis.

Animals↗
PubMed1999

Human telomerase reverse transcriptase (hTERT) gene expression in thyroid neoplasms.

Ten percent of fine-needle aspirations (FNAs) of the thyroid are deemed "indeterminate" or "suspicious" for malignancy by the cytopathologist, but most of these lesions are benign. Therefore, additional markers of malignancy may prove to be a useful adjunct. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been found to be reactivated in immortalized cell lines. Reverse transcription-PCR of the hTERT gene revealed expression in 15 (79%) of 19 malignant thyroid neoplasms, including 6 of 6 follicular carcinomas and 9 of 13 papillary carcinomas. In contrast, hTERT gene expression was detected in only 5 (28%) of 18 benign thyroid nodules, including 2 of 7 follicular adenomas and 3 of 11 hyperplastic nodules. All five benign thyroids exhibiting hTERT gene expression had lymphocytic thyroiditis. No normal thyroids exhibited hTERT gene expression. Telomerase enzyme activity was examined in all 37 nodules and was found to correlate with hTERT gene expression in 35 (95%) nodules. The two cases in which telomerase activity and hTERT expression results were discrepant were in two papillary carcinomas that were telomerase activity negative and hTERT positive. Finally, we have demonstrated that hTERT gene expression can be measured in in vivo FNA samples. These results suggest that hTERT expression may be more accurate than telomerase activity in distinguishing benign from malignant and may be measured in FNA samples from suspicious thyroid lesions.

Biomarkers, Tumor↗
PubMed1998

A clinicopathologic analysis of AIDS-related cryptosporidiosis.

OBJECTIVE: To characterize the histology of AIDS-associated cryptosporidiosis and identify features that explain the clinical variability. DESIGN: A retrospective analysis of HIV-positive individuals with cryptosporidiosis who underwent endoscopy at the Johns Hopkins Hospital between 1985 and 1996. METHODS: The histologic features (intensity of Cryptosporidium infection, inflammation, mucosal damage, copathogens) of gastrointestinal biopsies from 37 HIV-positive individuals with cryptosporidiosis were systematically graded. These histologic features were correlated with the severity of the diarrheal illness obtained from a patient chart review. RESULTS: Histologic features associated with Cryptosporidium infection include a neutrophilic infiltrate in the stomach, villus blunting in the duodenum, cryptitis and epithelial apoptosis in the colon, and reactive epithelial changes in the stomach and duodenum. The nature and intensity of the inflammatory response varied widely; however, duodenal biopsies from a subset of patients (37%) revealed marked acute inflammation that was associated with concomitant cytomegalovirus infection. Although duodenal infection was common (93% of individuals), infection of other sites was variable (gastric cryptosporidiosis in 40% and colonic cryptosporidiosis in 74%). Widespread infection of the intestinal tract, which included both the large and small intestine, was associated with the most severe diarrheal illness. CONCLUSIONS: Cryptosporidium infection produces histologic evidence of gastrointestinal mucosal injury. The inflammatory response to the infection is variable, and may be modified by copathogens such as cytomegalovirus. The clinical manifestations are influenced, in part, by the anatomic distribution of the infection, with extensive infections involving both small and large intestines producing the most severe illness.

AIDS-Related Opportunistic Infections↗
PubMed1998

A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol.

Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E. coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.

Acetic Acid↗