Biomedical subjects

D Dennis

Publications and source records attributed to D Dennis.

At least 19 recordsLinked to original sources

PubMed1992

Polyhydroxyalkanoate production in recombinant Escherichia coli.

The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-beta-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-3HV).

Escherichia coli↗

PubMed1992

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.

Alcaligenes↗

PubMed1992

Evidence of spare A1-adenosine receptors in guinea pig atrioventricular node.

In normoxic, isolated perfused guinea pig hearts instrumented for measurement of atrioventricular nodal conduction time (AVCT), an analysis utilizing the irreversible A1-adenosine (Ado) antagonist, meta-1,3-phenylene diisothiocyanate xanthine amine cogener (m-DITC-XAC), a novel isothiocyanate derivative of 1,3-dialkylxanthine, was used to investigate whether spare A1-Ado receptors exist in the guinea pig atrioventricular (AV) node and the degree of amplification (reserve) between A1-Ado receptor occupancy and dromotropic response (e.g., AVCT slowing). The potency, dose dependency, and kinetic profile (time dependence of washout and washin) of m-DITC-XAC was determined and compared with those of known competitive (reversible) A1-Ado receptor antagonists. In the presence of m-DITC-XAC, Ado and N6-cyclopentyladenosine (CPA) produced submaximal dromotropic responses. In a series of 19 hearts, m-DITC-XAC caused 100% apparent antagonism of the effect of Ado on AVCT even after 60 min of washout. In contrast, greater than 90% of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and XAC-induced antagonism of the effect of Ado on AVCT dissipated within 35 min. Unlike XAC, which caused maximal attenuation of Ado's AVCT effect within 5 min and remained constant thereafter, m-DITC-XAC showed marked time- and concentration-dependent behavior. It was found that 5 min of 0.5 microM m-DITC-XAC pretreatment irreversibly inactivated 72% of the A1-Ado receptors mediating the dromotropic effect, and the estimated agonist equilibrium dissociation constant for CPA was 84 +/- 4 nM. The percent of spare A1-Ado receptors at the EC50 and extrapolated maximal S-H interval prolongation levels was 20 and 54%, respectively, and the reserve (coupling amplification) varied from 1 to 2.3 within the 0-50% maximal response range. In summary, m-DITC-XAC appears to specifically and irreversibly antagonize the negative dromotropic effect of Ado and CPA, and guinea pig AV nodal tissue possesses spare A1-Ado receptors.

Adenosine↗

PubMed1991

Open versus laparoscopic cholecystectomy: an initial analysis.

Laparoscopic cholecystectomy is a new procedure in the armamentarium of the general surgeon. Its utility was investigated by comparison to open cholecystectomy in terms of procedure time, complications, hospital stay, and total hospital cost. Procedure time was approximately 200% longer with a higher incidence of intraoperative stone and bile spillage (17%) in the laparoscopic group. Hospital stay was reduced by 60% using the laparoscopic technique. No difference in total hospital cost existed between the two groups. The learning curve had an affect on hospital costs, which will decrease as more experience is gained with this procedure. Although laparoscopic cholecystectomy, at least initially, has no cost advantage over open cholecystectomy, laparoscopic cholecystectomy may be preferred by patients seeking shorter hospital stays and presumably shorter total recovery time.

Adult↗

PubMed1990

Yeast RNA polymerase I. Derivatization of the 190 and 135 subunits by 4-thiouridine monophosphate positioned uniquely at the 3' terminus of an enzyme-bound 32P-containing transcript initiated by a triribonucleotide primer on synthetic single-stranded DNA.

Specific transcription complexes were formed with yeast RNA polymerase I using a cognate oligoribotri-nucleotide primer (GCG) to initiate transcription on short synthetic single-stranded DNA templates. The templates were designed to limit the incorporation of a photoprobe, 4-thiouridine triphosphate, to a single unique position at the 3' terminus of the product RNA (position 12, 13, 14, or 15). The resulting transcription complexes were photolyzed to cross-link the bound transcript (radiolabeled with [alpha-32P]CTP) to the protein with the probe located at the catalytic site. Separation of the protein subunit components by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by autoradiography and silver staining revealed that the two largest subunits (A190 and A135) were radiolabeled. The ratio of subunit labeling (A190/A135) decreased as the RNA transcript increased from 12 to 15 nucleotides in length. This decrease in ratio resulted from a progressive reduction of A190 subunit labeling while the A135 subunit derivatization remained essentially constant. It was also observed that the DNA template was radiolabeled.

Affinity Labels↗

PubMed1990

The morbidity, mortality, and economic costs of cigarette smoking in New Hampshire.

The Surgeon General of the United States Public Health Service has identified cigarette smoking as the single most important source of preventable morbidity and premature mortality. An analysis was conducted in the state of New Hampshire to determine the consequences of smoking: morbidity, mortality, and economic costs to the population. Data were collected on smoking prevalence, smoking attributed deaths, years of potential life lost, hospital days attributed to smoking diagnoses, direct medical costs, and per capita incomes. Smoking attributable fractions were applied to these data. In 1983, 16% of total statewide deaths were attributable to cigarette smoking. These deaths included 15% of the cardiovascular deaths, 20% of cancer deaths, 42% of respiratory disease deaths, 3% of digestive disease deaths, and 5% of infant deaths, in a population of less than 1 million. These deaths represented almost 3100 years of potential life lost. Smoking attributable hospital days totaled almost 70,000, for 8% of male and 4% of female hospital days. Direct medical care costs attributable to cigarette smoking were over $76 million, 7% of the total statewide medical costs. Indirect costs (present value of lost earnings due to premature mortality and morbidity attributable to smoking) were almost $118 million. These economic costs totaled almost $200 million. The results of this study were used extensively by the New Hampshire media and volunteer agencies. This methodology can be a model for other local area analyses.

Cause of Death↗

PubMed1990

Problems in diagnosis and management of desmoid tumors.

Thirty-two primary desmoid tumors occurred in 29 patients. The median patient age was 32 years, and 55% of the patients were females. An antecedent history of trauma at the tumor site was elicited from 28% of patients. Thirty-one of 32 primary tumors were completely excised at initial presentation. Five tumors were treated with adjuvant radiotherapy. The overall recurrence rate for primary and recurrent lesions was 60%. The recurrence rate in children (88%) was more than twice that found in adults (38%). A single recurrence did not significantly increase the likelihood of a subsequent recurrence. Greater than 90% of all recurrences took place within 3 years of treatment. The rate of recurrence was not clearly influenced by the status of histologic margins, although this was examined in less than half the tumors. Desmoid tumors are aggressive neoplasms that exhibit a strong propensity for local recurrence. They should be treated as low-grade malignancies with documentation of histologic margins and close clinical follow-up within the framework of a tumor registry.

Abdominal Muscles↗

PubMed1988

Formation and characterization of precise eucaryotic transcription complexes using a semisynthetic DNA template and specific oligoribonucleotide primers.

An artificial template of defined sequence which supports specific in vitro initiation and elongation by yeast RNA polymerase II has been constructed. This template is a pBR322 derivative which contains a synthetic oligonucleotide inserted into the BamHI cloning site. The sequence of this oligonucleotide is such that when the plasmid is restricted with SacI the two ends obtained are identical. The addition of an oligodeoxycytidylate chain to the 3' hydroxy termini produces a DNA template, (poly dC-p+22), with the sequence: 3'(C)nTCGA-GAGTCTCCTA. . . . The underlined position denotes the beginning of the duplex region. When initiation is primed with the diribonucleotide GpC the predicted sequence of the transcript obtained is: 5'GCUCUCAGAGGAU. . . . Kinetic and product analyses indicate that a ternary complex containing a precise length of transcript can be produced which is subsequently resistant to heparin inactivation. Initiation can also be directed to a specific position dictated by a tri or tetraribonucleotide primer.

Base Sequence↗

PubMed1988

The mortality, morbidity, and economic costs of alcohol abuse in New Hampshire.

Since alcoholism and alcohol abuse are the number one health problem in the United States, community-based estimates of mortality, morbidity, and economic costs associated with alcohol abuse are needed to convey their impact in local areas. In the state of New Hampshire, data were collected on alcohol consumption patterns, alcohol-associated mortality, years of potential life lost, hospital days associated with alcohol-related diagnoses, direct medical care costs, employment levels, and per capita incomes. Alcohol-attributable mortality and morbidity percentages were applied to these data to estimate the effects of alcohol abuse. In 1983, alcohol was associated with 4% of total statewide deaths. These included 37% of the deaths due to injury, 26% of the deaths due to digestive disease, and 3% of the deaths due to cancer. These deaths represented over 6,000 years of potential life lost. Between 4 and 7% of hospital days were attributable to alcohol-related diagnoses. Direct medical care costs attributable to alcohol were over $101 million; 10% of the direct medical costs in the state. Indirect costs (present value of lost earnings due to premature mortality and morbidity associated with alcohol) represented over $142 million. Property damage and insurance costs associated with alcohol were almost $13 million, and alcohol-related arrests added another $17 million. Excess absenteeism due to alcohol abuse cost another $33 million and lost productivity at work cost over $278 million. These economic costs totaled almost $600 million, or 5% of the gross state product. The methodology used to obtain these results is easily applied and is shown in the Appendix.

Alcoholism↗

PubMed1987

RNA polymerase. Limit cognate primer for initiation and stable ternary complex formation.

Various lengths of oligoribonucleotides corresponding to regions flanking the initiation site of the A1 promoter of the T7 delta D111 template were examined in order to determine their ability to function as primers of transcription for the DNA-dependent RNA polymerase from Escherichia coli. The oligoribonucleotides which functioned as primers were also examined as precursors for the formation of a stable ternary complex (enzyme X DNA template X oligoribonucleotide product) by systematically extending each primer with one or more specific cognate substrate nucleotide triphosphates. A stable ternary complex (resistant to a salt jump challenge) was formed whenever the primer oligoribonucleotide and augmenting nucleotide triphosphate(s) allowed the formation of the normal third phosphodiester bond of the transcript to occur on the enzyme surface. An oligoribonucleotide (a cognate having the correct base-pairing substituents) containing the preformed third phosphodiester bond does not function as a primer. For example the cognate oligoribonucleotide corresponding to the region flanking the A1 promoter of the T7 delta D111 is: formula; see text The limit primer is the oligoribonucleotide trimer AUC (+1...+3). Other acceptable primers are constructed by adding to this limit primer, cognate bases in the negative registry. The oligonucleotide containing one base added to this limit primer in the positive registry (e.g. AUCG) is completely inactive as a primer. We have also demonstrated these phenomena for the A2 and the A3 promoter of the T7 template.

Chromatography, High Pressure Liquid↗

PubMed1985

RNA polymerase. Direct evidence for a unique topographical site for initiation.

The compound 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl)adenine 5'-monophosphate is an inhibitor (Ki = 330 microM) of the initiation binding site of the DNA-dependent RNA polymerase derived from Escherichia coli. The alpha-32P derivative of this photo-labile compound is used to derivatize a site on the sigma subunit of the holoenzyme (E sigma) using either T7 delta D111 or poly[d(A-T)] as a DNA template. The incorporation of the 32P label into the sigma subunit could be prevented by the addition of either 5'-AMP or 5'-ATP. The results are suggested to support the existence of a unique initiation binding site, topographically distinct from the sites employed during the elongation phase.

Binding Sites↗

PubMed1985

RNA polymerase. Direct evidence for two active sites involved in transcription.

The [alpha-32P]3'-azido-3'-deoxyxylofuranosyl analogues of 5'-ATP and 5'-GTP were used as photoaffinity probes to derivatize two distinctly different active sites of the DNA-dependent RNA polymerase obtained from Escherichia coli. The sites derivatized by these probes are both located on the beta subunit. Radioactive 32P containing cyanogen bromide peptides produced from the derivatized beta subunits were observed to give either one of two distinct patterns by high pressure liquid chromatography or TLC analysis. These sites are used alternately for the synthesis of successive phosphodiester bonds. These results support our rotational translocation model for transcription.

Affinity Labels↗

PubMed1985

Amplitude of ventricular fibrillation waveform and outcome after cardiac arrest.

The amplitude of ventricular fibrillation found initially in 394 patients was compared to clinical and logistical findings at the time of cardiac arrest. Peak-to-peak amplitude averaged 0.55 +/- 0.25 mV; a very low amplitude (0.2 mV or less) or "fine" fibrillation was present in 66 patients (17%). The amplitude was not found to be related to clinical histories, but depended on the length of the period from collapse until start of basic life support (p = 0.004) and the delay until assessment by paramedics (p = 0.001). Survival rates were strongly associated with amplitude: only 4 patients (6%) with fine ventricular fibrillation survived, compared to 117 or 328 patients (36%) in whom the initial amplitude was higher (p less than 0.001). Patient outcome related to amplitude even after adjusting for clinical history and logistical delays (p less than 0.005). We conclude that fine ventricular fibrillation is in part the result of delay in initiation of treatment, and that fibrillation amplitude is a powerful indicator of outcome after cardiac arrest.

Aged↗

PubMed1985

RNA polymerase: direct evidence for two active sites involved in transcription.

Photoaffinity reagents (3'-azido analogues of 5' ATP and 5' GTP) were used to label two different sites at the catalytic surface of the beta subunit of the E. coli DNA dependent RNA polymerase. These sites are used alternately and successively during consecutive phosphodiester bond formation. This result is consistent with our model of rotational translocation for transcription.

Affinity Labels↗

PubMed1984

RNA polymerase. Synthesis and kinetic inhibition by 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl) derivatives of 5'-ATP and 5'-GTP.

The 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl) nucleoside 5'-triphosphate derivatives of adenine (3'-azido, x-dATP) and guanine (3'-azido, x-dGTP) were prepared by chemical phosphorylation of the corresponding nucleosides. The compounds were characterized by 31P and 1H NMR, high performance liquid chromatography, IR, and TLC. The compounds were examined kinetically and observed to be linear mixed inhibitors for the DNA-dependent RNA polymerase of Escherichia coli (EC 2.7.7.6); Ki values for the 3'-azido, x-dATP and 3'-azido, x-dGTP compounds are 33 and 0.95 microM, respectively. Neither compound functions as an alternate substrate or as a chain terminator during the normal kinetic time course. The 3'-azido, x-dGTP does exhibit a slow time-dependent irreversible inhibition and may therefore function as an alternate substrate and chain terminator with prolonged incubation. Both compounds (3'-azido, x-dATP and 3'-azido, x-dGTP) are photolabile and will derivatize lysine in a coupled photolytic reaction.

Chromatography, Thin Layer↗

PubMed1984

Transactivation of a late herpes simplex virus promoter.

We have asked whether the promoter for the gene encoding the major capsid protein (VP5) of herpes simplex virus functions in uninfected mouse cells. Our experimental strategy was to first fuse the VP5 promoter to the herpes simplex virus thymidine kinase (TK) structural sequence and then to use the resulting hybrid gene to transform TK- cells to TK+. The recombinant gene transferred TK at an extremely low frequency by comparison with the wild-type TK gene, and the TK transcripts present within the resulting rare transformants initiated within the TK structural gene, rather than in the vicinity of the VP5 promoter. However, after infection with herpes simplex virus, large amounts of RNA driven from the VP5 promoter accumulated. We conclude that the VP5 promoter does not function in uninfected cells but is efficiently activated by virally coded factors, most likely one or more immediate-early proteins.

Animals↗

PubMed1982

RNA polymerase: correlation between transcript length, abortive product synthesis, and formation of a stable ternary complex.

In order to investigate the relationship between the stability of the ternary complex RNA polymerase-T7 D111 DNA-RNA product and the length of the bound RNA product, we have developed a protocol for the production of stable ternary complexes of known length and composition. The assembly of the ternary complex is achieved by utilizing a dinucleotide tetraphosphate (pppApU) as a selective primer, which is augmented by one or more appropriate nucleotides. The labeled products were characterized by autoradiography of gel electrophoresis patterns, which were then quantified. The criterion for stability is the protection from perturbations (a salt-jump or a rifampicin challenge), which effectively inhibit initiation. The formation of a bound ribotetranucleotide ternary complex confers stability and terminates abortive product synthesis.

Chromatography, Thin Layer↗

PubMed1981

Otoplasty--a contemporary survey.

This report examines the various common congenital auricular deformities categorized under the broad heading of the "outstanding ear" and reviews briefly the major contributions to otoplasty surgery.

Child↗