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Biomedical subjects

S E Mitchell

Publications and source records attributed to S E Mitchell.

At least 19 recordsLinked to original sources
PubMed2000

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis.

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.

Animals↗
PubMed2000

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Animals↗
PubMed1999

Suppression of leptin during lactation: contribution of the suckling stimulus versus milk production.

Lactation in the rat is characterized by the suppression of pulsatile LH secretion, a large increase in food intake, and changes in energy balance due to the metabolic drain of milk production. The change in energy balance may be a major component in altering reproductive function. A number of factors may contribute to changing energy balance of a lactating animal; one is leptin, the product of adipose tissue, which is known to act partly as a satiety factor to decrease food intake. The aims of the present study were to determine whether there are changes in leptin levels during lactation, a state of high energy demand, and during periods of acute suckling in the presence or absence of changes in energy demand. Our goals were to determine whether lactation and the suckling stimulus influenced serum leptin levels and whether there was a potential role for leptin in the suppression of LH secretion during lactation. The first experiment was performed during diestrus of the estrous cycle, and chronic lactation, (day 9 post partum) in animals suckling 8 pups. The results showed that leptin levels were significantly decreased in both ovarian intact or ovariectomized lactators; this decrease parallels the suppression of pulsatile LH secretion. Serum insulin levels were not altered in the lactating animals. The second experiment was performed in ovariectomized lactators whose 8 pup litters were removed for 48 h, starting on day 9. On day 11, mothers received no pups or pups that were either nonfostered (resulting in no milk production) or fostered (resulting in milk production). The pups were allowed to suckle for 24 h. Following 24 h of acute suckling, serum leptin, and insulin levels correlated with the energy drain on the mother. The levels of leptin were normal and of insulin were elevated in mothers producing no milk. Conversely, leptin levels were suppressed and insulin levels normal in mothers producing milk. The third experiment used the same groups as described for the second experiment except that serial blood samples were collected for measurement of pulsatile LH secretion following 24 h of acute suckling. The results showed that regardless of whether leptin levels remained normal or were suppressed in response to acute suckling, pulsatile LH secretion was significantly inhibited compared with the nonsuckled control animals. In summary, these data suggest that the metabolic drain of milk production, and not the suckling stimulus itself, is the most likely factor responsible for the suppression of leptin secretion during lactation. Furthermore, although the decreased levels of leptin may be causally related to the inhibition of pulsatile LH secretion during chronic lactation, changes in leptin are not a prerequisite for the suppression of LH secretion in response to suckling.

Animals↗
PubMed1998

Peanut yellow spot virus is a member of a new serogroup of Tospovirus genus based on small (S) RNA sequence and organization.

Peanut yellow spot virus (PYSV) represents a distinct tospovirus species based on serology and nucleic acid hybridization. THe sequence of the S RNA was 2970 nucleotides with 22 nucleotide long inverted repeats (with three mismatches) at the termini. The coding was ambisense with a long open reading frame (ORF) in each strand. The 5'-large ORF (1,400 nucleotides in the viral sense (v) strand) encoded a protein with a predicted size of 53.2 kDa that was identified as the nonstructural (NSs) protein based on 16-21% sequence identity and 42-48% sequence similarity with other tospoviruses. A 3' ORF (741 nucleotides) in the virus complementary (vc) sense encoded a 28.0 kDa protein that was identified as the nucleocapsid (N) gene based on immuno-blot analysis of the in vitro expressed protein with PYSV polyclonal antiserum. The predicted N protein had 24-28% amino acid sequence identity and 44-51% sequence similarity with the members of other serogroups. In contrast to other tospoviruses, a third ORF (204 nucleotides) occurred in the vc strand, which could encode a protein with a predicted size of 7.5 kDa with two strong hydrophobic regions. The low degree of homology of N and NSs protein sequences with other serogroup members coupled with an additional ORF suggests that PYSV should be classified as a distinct species of the Tospovirus genus. This conclusion also is supported by the absence of serological cross reaction with other serogroups, and biological characteristics including thrips transmission, symptoms and host range.

Amino Acid Sequence↗
PubMed1998

Species-specific repeat units in the intergenic spacer of the ribosomal RNA cistron of Anopheles aquasalis Curry.

A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.

Animals↗
PubMed1997

ob gene expression and secretion of leptin following differentiation of rat preadipocytes to adipocytes in primary culture.

Expression of the ob gene and production of leptin have been examined on differentiation of rat fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were obtained from the inguinal fat pad of suckling rats, and following differentiation the cells contained lipid droplets and the mRNAs for both lipoprotein lipase and adipsin were detected by Northern blotting. ob mRNA was not, however, detected on Northern blots, but analysis by RT-PCR indicated that the ob gene was expressed, particularly after differentiation. Measurement of leptin in the culture medium by ELISA showed that the ob gene product was secreted by adipocytes from approximately 4 days after the induction of differentiation. Leptin production was sustained over a 2-week period with a peak at 8-10 days post-induction. Dexamethasone stimulated leptin production, while an inhibition was observed with the beta-adrenoceptor agonist isoprenaline. These results demonstrate that following the differentiation of fibroblastic preadipocytes to adipocytes in primary culture, leptin is secreted with the cells responding to stimuli which regulate production of the hormone.

Adipocytes↗
PubMed1997

A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7.

A multiplex PCR assay specifically detecting Escherichia coli O157:H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157:H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157:H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55:H7 and E. coli O55:NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.

Adhesins, Bacterial↗
PubMed1997

Laparoscopic cholecystectomy-related bile duct injuries: a health and financial disaster.

OBJECTIVE: This study was designed to evaluate the total costs associated with repair of laparoscopic cholecystectomy (LC)-related bile duct injuries. SUMMARY BACKGROUND DATA: The popularity of LC with both patients and surgeons is such that this procedure now exceeds open cholecystectomy by a ratio of approximately 4 to 10:1. However, costs associated with LC-related injuries, particularly regarding treatment patterns, have up to now not been explored fully. METHODS: The complete hospital and interventional radiology (IR) billing records for 49 patients who have completed treatment for laparoscopic cholecystectomy-related bile duct injuries were divided into 8 categories. These records were totaled for comparison of costs between patient groups that experienced different injuries and treatment patterns. RESULTS: Patients with LC-related bile duct injuries were billed a mean of $51,411 for all care related to repair of their bile duct injury. Patients incurred an average of 32 days of inpatient hospitalization and 10 outpatient care days. Postoperative treatment included long-term chronic biliary intubation averaging 378 days. Two patients (4%) died as a result of their LC-related complications. Patients with bile duct injuries that were recognized immediately at the time of the initial surgery ultimately experienced a total cost for their repair and hospitalization of 43% to 83% less than for patients in whom recognition of the injury was delayed (p < 0.019 to 0.070). In addition, the total hospitalization and outpatient care days was reduced by as much as 76% with early recognition of an iatrogenic injury. CONCLUSIONS: Repair of cholecystectomy-related bile duct injuries can run 4.5 to 26.0 times the cost of the uncomplicated procedure and carries a significant mortality rate. Intraoperative recognition of such an injury with immediate conversion to an open procedure for definitive repair can result in significant cost savings and relates directly to a decreased morbidity, mortality, length of hospitalization, and number of outpatient care days.

Adult↗
PubMed1996

Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence, genome organization and homology to other tospoviruses.

The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22-34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82-86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.

Amino Acid Sequence↗
PubMed1996

Polymerase chain reaction for detecting Escherichia coli O157: H7.

Escherichia coli O157:H7 is known as an important cause of hemorrhagic colitis and hemolytic uremic syndrome. Real-time procedures that are sensitive for detecting small populations of this bacterium in food are lacking and needed. An expression library was constructed by ligation of BamHI-EcoRI DNA fragments of E. coli O157:H7 to plasmid vector pUC19 and transformation of recombinant plasmids to E. coli JM109. A clone that contained a specific DNA fragment of E. coli O157:H7 was identified by colony immunoblot assay using monoclonal antibody MAb 4E8C12 that uniquely links to E. coli O157:H7 and a few other serotypes of verotoxin-producing E. coli. The DNA sequence of the clone consisted of 110 bp of 5' region of enterohemorrhagic E. coli (EHEC) eae gene and a 688 bp DNA fragment adjacent to 5' end of the eae gene, including an unknown function gene encoding 156 amino acids. A pair of oligonucleotide primers was synthesized based on the sequence of the 688 bp fragment. The primers were used in a polymerase chain reaction (PCR) to amplify a target DNA of 633 bp. The primers amplified 1 ng of DNA from 67 strains of E. coli O157:H7, two strains of E. coli O157:NM, and 7 of 11 E. coli O55:H7 and O55:NM strains, but not 50 ng of DNA from 34 strains of 29 other E. coli serotypes and 25 strains of 8 other bacterial species. Annealing temperatures from 60 to 63 degrees C could be used for the PCR without loss of specificity. The minimum amount of target DNA detected by the PCR was 5 pg. When a boiling method and GeneReleaser were used, the PCR was able to detect as few as 25 and 38 CFU of E. coli O157:H7, respectively, in 3 h.

Antibodies, Monoclonal↗
PubMed1996

The complete nucleotide sequence and genome organization of the M RNA segment of peanut bud necrosis tospovirus and comparison with other tospoviruses.

The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3' large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58-59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21-23% identity and 44-47% similarity with those of other members of the genus Bunyavirus. The 5' small ORF (924 nucleotides in the virussense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41-43% identity and 60-63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.

Amino Acid Sequence↗
PubMed1996

Outcome of tunneled hemodialysis catheters placed by radiologists.

PURPOSE: To compare the outcomes of hemodialysis catheters placed by interventional radiologists with those placed by surgeons. MATERIALS AND METHODS: The outcomes were retrospectively analyzed of 237 hemodialysis catheters placed in 140 patients by a radiology service from January 1991 through December 1992. Follow-up data were available for 222 catheters (94%). Catheter secondary patency and freedom from infection were analyzed statistically and by means of life-table analysis. RESULTS: Pneumothorax occurred after the placement of six catheters (2.5%); in two patients, a chest tube was required for decompression. Other short-term complications included air embolism with no clinical sequelae (two procedures) and prolonged oozing from the tunnel (two procedures). Long-term complications included infection and catheter failure. Infection occurred in 26 patients (18%) with 32 catheters (14%) and resulted in removal of 25 catheters. Ninety-three catheters (42%) failed, and 63 catheters (28%) were removed because of failure. CONCLUSION: Hemodialysis catheters placed by radiologists do not have a higher rate of complications or failure than catheters placed by surgeons.

Case-Control Studies↗
PubMed1995

Cloning and nucleotide sequence of a gene upstream of the eaeA gene of enterohemorrhagic Escherichia coli O157:H7.

A DNA segment located immediately upstream of the eaeA gene of enterohemorrhagic Escherichia coli O157:H7 strain HA1 was cloned and sequenced. This segment contained an open reading frame encoding a predicted protein of 156 amino acids. A database search identified similar open reading frames upstream of the eaeA gene in two other bacterial pathogens, i.e. enteropathogenic E. coli and Citrobacter freundii. The predicted amino acid sequence of the enterohemorrhagic E. coli protein shared 96.8% and 94.2% identity with the enteropathogenic E. coli and C. freundii sequences, respectively. Because the open reading frame is located within the locus of enterocyte effacement region of the E. coli chromosome, a 'hot spot' for insertion of virulence factor genes, and shares high sequence homology with attaching and effacing EPEC and C. freundii, this protein may be associated with pathogenicity of E. coli O157:H7.

Amino Acid Sequence↗
PubMed1993

The mitochondrial genome of Anopheles quadrimaculatus species A: complete nucleotide sequence and gene organization.

The complete sequence (15,455 bp) of the mitochondrial DNA of the mosquito Anopheles quadrimaculatus species A is reported. This genome is compact and very A+T rich (77.4% A+T). It contains genes for 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement is the same as in Drosophila yakuba, except that the positions of two contiguous tRNAs are reversed and a third tRNA is transcribed from the complementary strand. Protein-coding genes, rRNAs, and most tRNAs were similar to D. yakuba. Two tRNAs had nonstandard secondary structures comparable with those of nematode mitochondrial tRNAs. The very small putative control region (625 bp) contains no sequence motifs similar to those used in vertebrates and other insects for initiation of transcription and replication.

Amino Acid Sequence↗
PubMed1993

Multiple-technique identification of sibling species of the Anopheles quadrimaculatus complex.

In the past, most researchers used a single technique for identification of cryptic taxa, population structures, biosystematics, and phylogenetic studies. Our experience with the Anopheles quadrimaculatus complex shows the importance of using several methods on individual mosquitoes. This approach consists of analysis of the polytene chromosomes in ovarian nurse cells, gas chromatographic profiles of cuticular hydrocarbons, isozyme electrophoresis, and restriction site analysis of mitochondrial or genomic DNA. We recommend use of this multiple-technique approach when analyzing feral populations for the first time, or for correlating information obtained by investigators using different techniques.

Animals↗
PubMed1992

Segmental renal artery embolization for treatment of pediatric renovascular hypertension.

Selective intrarenal arterial embolization was used to treat three children with documented renovascular hypertension. Embolization resulted in complete cure (ie, elimination of all antihypertensive medicines) in all three patients and caused only minimal loss of renal parenchyma. Renal vein renin sampling, including sampling after furosemide administration, correlated well with the location of identified vascular lesions and helped direct selective angiography when lesions were not found initially. Intrarenal arterial embolization is a safe, effective alternative to surgical resection in the treatment of renovascular hypertension in children who have identifiable intrarenal arterial lesions not amenable to balloon angioplasty.

Child↗