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Biomedical subjects

R M Thomas

Publications and source records attributed to R M Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1996

Antioxidant enzymes in intramural nerves of the opossum esophagus.

Superoxide radical (O2-.) combines with nitric oxide (NO) to form peroxynitrite, thereby nullifying the biological activity of NO. Superoxide dismutase (SOD) prevents this reaction by converting O2-. to H2O2. We tested the hypotheses that the antioxidant enzymes catalase (CAT), Mn SOD, and Cu/Zn SOD are present in enteric neurons of the opossum esophagus, and that O2-. alters esophageal motor function. Immunostaining demonstrated CAT, Mn SOD, and Cu/Zn SOD immunoreactivity in interganglionic nerve bundles and ganglia of the myenteric and submucosal plexuses. Western blot analysis confirmed the presence of these enzymes in homogenates of esophageal muscularis propria, and enzyme assays demonstrated Cu/Zn SOD and Mn SOD activities of 262 and 73 U/mg protein, respectively. Both diethyldithiocarbamic acid, an inhibitor of Cu/Zn SOD, and xanthine (X) with xanthine oxidase (XO), which generate O2-., shortened the latency of the nerve-mediated contraction of circular esophageal muscle, the off response, by 20.2 and 23.4%, respectively. SOD alone did not affect the latency, but it inhibited the effect of X with XO on the latency. Antioxidant enzymes found in intramural esophageal nerves may play a role in regulating NO-mediated neuromuscular communication in the esophagus.

Animals↗
PubMed1996

The role of ultraviolet light in the induction of cellular DNA damage by a spark-gap lithotripter in vitro.

PURPOSE: The purpose of this study was to investigate the mechanisms for DNA damage induced by a spark-gap lithotripter. MATERIALS AND METHODS: Cultured Chinese hamster ovary cells suspended in phosphate buffered saline were exposed in small chambers placed at the focus of a lithotripter in a 37C water bath. Viability was checked by trypan blue exclusion, and DNA strand breaks were evaluated with the comet assay after exposure. RESULTS: About 50% cell lysis and significant DNA damage in surviving cells were found after 500 discharges. The strand break effect, but not the lysis, could be eliminated by blocking the light emitted by the discharge. The exposure chamber material influenced the results, and use of nutrient medium with serum reduced the observed effects. The DNA damage was eliminated by added novobiocin and enhanced by added aphidicolin, as expected for strand breaks associated with repair of ultraviolet light damage. The DNA strand-break effect of 500 spark-gap discharges approximated that obtained from a positive control treatment with 38 J/m2. of 254 nm. ultraviolet light. CONCLUSIONS: Thus, the observed DNA damage appears to result from exposure of cells to the ultraviolet light emissions of the spark-gap discharge, rather than to the shockwaves or shockwave induced cavitation, which causes the cell lysis. Exposure of the skin to this ultraviolet light during lithotripsy might have some clinical implications for prolonged or repeated treatment.

Animals↗
PubMed1995

Kinetics of folding of leucine zipper domains.

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Gastrointestinal cancer.

BACKGROUND: There are great differences in incidence and prognosis of cancer among the component sites of the digestive tract. Furthermore, a number of the histologic types of tumors at these sites differ in their incidence and prognosis. METHODS: The SEER data from 1973 to 1987 on frequency, incidence, staging, and survival for the various histologic types of 194,452 cancers of the esophagus, stomach, and intestines were studied. RESULTS: There was an increased incidence of adenocarcinomas of esophagus, gastric cardia, small intestine, and colon; incidence rates for rectal adenocarcinoma and esophageal squamous cell carcinoma remained stable; and rates for gastric adenocarcinoma decreased. Blacks had higher incidence rates than whites for gastric adenocarcinoma; squamous cell carcinoma of the esophagus; carcinoids of the small intestine, colon, and rectum; and gastric sarcomas. Whites, especially males, had higher incidence rates for adenocarcinomas of the esophagus and gastric cardia than did blacks. Blacks had lower rates for rectal adenocarcinomas than did whites, but slightly higher rates than whites for rectal mucinous carcinomas and colonic adenocarcinomas. Data on race, sex, survival, and time trends indicate that mucinous colorectal carcinomas are biologically different from other adenocarcinomas. Males had poorer overall survival than did females for most histologic types of digestive tract tumors. Appendiceal mucinous adenocarcinomas with distant metastases were associated with a high (50%) 5-year survival. The poor prognosis of cases diagnosed as colonic carcinoid raises the possibility that some of these actually may be carcinomas. CONCLUSION: Studies of digestive tract cancer require analysis by histologic type for adequate assessment.

Adolescent↗
PubMed1995

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.

Amino Acid Sequence↗
PubMed1995

Comet assay reveals DNA strand breaks induced by ultrasonic cavitation in vitro.

The induction of DNA strand breaks in cultured Chinese hamster ovary (CHO) cells was investigated in suspensions directly exposed to 2.17-MHz ultrasound. Production of hydrogen peroxide, a DNA-damaging sonochemical, by inertial cavitation was enhanced by the use of argon-and-oxygen-bubbled media and cell survival was improved by establishing standing waves and minimizing tube rotation. Viable cells were separated from the suspension after exposure and kept on ice for evaluation with the single-cell gel electrophoresis (comet) assay. With this assay, DNA damage from as little as 2-mumol/L hydrogen peroxide treatment for 30 min could be detected, and cell survival as low as 2-5% after ultrasound exposure was adequate for assay. An ultrasound dose-response trend was noted for increasing pressure amplitude up to 0.82 MPa (free field) and increasing exposure duration up to 4 min. The cells were able to repair some of the strand breaks when warmed to 28 degrees C for 30 min. The effect was not eliminated by addition of catalase, which indicates that the DNA damage was not due to the action of residual H2O2 alone. The results confirm the hypothesis of DNA damage in cells surviving inertial cavitation.

Animals↗
PubMed1995

Ultrasound contrast agents nucleate inertial cavitation in vitro.

Some ultrasound contrast agents contain stable bodies of gas, and this study was undertaken to determine if these agents could provide nuclei for inertial cavitation. Inertial cavitation was detected and assessed by the measurement of the sonochemical hydrogen peroxide after exposure to 2.17-, 2.95- or 3.8-MHz ultrasound. A noncavitating system was obtained by removing cavitation nuclei from the rotating tube exposure chambers by vacuum degassing, and from the phosphate-buffered saline medium by filtering. Albunex added at 10-2, 10-3 or 10-4 dilutions, or Levovist added at 2 mg mL-1, 0.2 mg mL-1 or 0.02 mg mL-1 all initiated significant H2O2 production for 2.17-MHz ultrasound at 0.41 MPa or higher spatial peak pressure amplitude for 5 min exposure gated at 0.25 s on and off with 60-rpm rotation. Not rotating the tube virtually eliminated H2O2 production. For 2.5-min continuous exposure, both agents initiated significant H2O2 production for 2.95-MHz exposure at 0.58 MPa or higher, but not for 3.8-MHz exposure up to 1.16 MPa. Bubble-based ultrasound contrast agents therefore appear to be able to provide nuclei for inertial cavitation in the rotating tube exposure system.

Albumins↗
PubMed1995

Induction of base damage in DNA solutions by ultrasonic cavitation.

Ultrasound can damage macromolecules by the mechanical (shearing) and sonochemical (free radical generating) action of ultrasonic cavitation. Attributing macromolecular damage to either direct mechanical stress or to indirect mechanisms involving free radicals or other sonochemicals is a challenging problem. DNA damage induced by ultrasound was evaluated by measuring the formation of purine and pyrimidine products using combined gas chromatography-mass spectrometry with selected ion monitoring. Samples of DNA were prepared in 10 mmol dm-3 phosphate buffered saline (pH 7.4) and saturated with a mixture of argon:oxygen (3:1). Continuous 2.17 MHz ultrasound exposures at 0.82 mPa spatial peak negative pressure amplitude were performed in a 60 rpm rotating tube exposure system. Hydrogen peroxide yields were measured after each exposure to quantify the cavitation activity and ranged up to 350 mumol dm-3 for 1-h exposures. Purine and pyrimidine products identified were those typically observed following exposure of DNA to hydroxyl radical-generating systems, such as ionizing radiation, hypoxanthine/xanthine oxidase, or hydrogen peroxide in the presence of transition metal ions. The yields of these products were directly correlated with cavitation activity as measured by residual hydrogen peroxide concentrations. The yields of DNA products increased in the following order: thymine glycol approximately cytosine glycol > 8-oxoAde > FAPyAde approximately 5-HMU approximately 5,6-diOHCyt > FAPyGua. Unexpectedly, 8-oxoguanine did not exhibit a dose-dependent increase above background levels, and this observation is inconsistent with processes involving metal ion-dependent formation of hydroxyl radicals from hydrogen peroxide. In addition, the product yields were far too large to result from the residual hydrogen peroxide. Thus, ultrasonic cavitation appears to have a mode of action distinct from either ionizing radiation or formation of hydroxyl radicals via Fenton-like reaction with transition metals.

Adenine↗
PubMed1995

Thresholds for hemorrhages in mouse skin and intestine induced by lithotripter shock waves.

In vivo biological effects of ultrasound should be characterized as thermal or cavitational to understand their etiology and significance. A spark-gap shock-wave lithotripter was built and used to compare cavitation-induced hemorrhages to the heat-induced petechial hemorrhages caused by continuous-wave ultrasound in mouse intestine. Intestinal hemorrhages induced in anesthetized hairless mice by the lithotripter pulses involved tissue destruction with bleeding into the lumen of the intestine, and were associated with intestinal gas bubbles. Skin hemorrhages were also observed, which appeared to be contusions, with no actual breakage of the skin. Administration of 100 shock waves with peak positive amplitude of 18.5 MPa produced an average of 7.6 (standard error [SE] 3.1, n = 6) intestinal hemorrhages and 45 (SE 11) skin hemorrhages. The counts and severity of hemorrhages increased with increasing numbers (3 to 300) of shock waves. Absorbers of varying thickness were used to reduce the pressure amplitude of the shock waves, which were thereby modified into low frequency ultrasound pulses. For 100 pulse exposures, apparent thresholds for effects occurred between 1.6 and 4.0 MPa for the intestinal hemorrhages and between 0.6 and 1.6 MPa for the skin hemorrhages. The low 1-Hz pulse repetition frequency precluded significant heating, and so these effects were the result of cavitation, which probably occurred inside the intestines or in the surrounding water. Compared to the previously observed thermal petechia, the cavitation-induced hemorrhages could be distinguished on the basis of their appearance upon histological examination, and also by the relative values of the thermal and mechanical exposure indices associated with the two different exposure modes.

Animals↗
PubMed1995

Co-localization of manganese superoxide dismutase and NADH diaphorase.

Manganese superoxide dismutase (Mn SOD), mitochondrial enzyme, defends against the toxic effects of superoxide radical (O2.-) in pathological processes by catalyzing the conversion of O2.- to hydrogen peroxide (H2O2). The activity of another enzyme, NADH diaphorase, forms the basis for a histochemical method used commonly to demonstrate nerve cell bodies in the enteric plexuses. We found identical patterns of localization of Mn SOD immunoreactivity and NADH diaphorase activity in brain, esophagus, stomach, colon, liver, and kidney. NADH diaphorase enzymatic activity co-migrated with complexes of Mn SOD on a non-denaturing gel. This suggests that the NADH diaphorase may in some way be related to Mn SOD.

Animals↗
PubMed1994

Gastric carcinoids. An immunohistochemical and clinicopathologic study of 104 patients.

BACKGROUND: Gastric carcinoids are uncommon, and are unlike carcinoids at other gastrointestinal sites, clinically and pathologically. METHODS: The authors studied specimens from 104 patients with gastric carcinoid, with study emphasis being placed on pathologic features, immunohistochemistry, clinical associations, and prognostic factors. RESULTS: The average age of the 47 male patients and 57 female patients was 61 years. Twenty-seven patients had chronic atrophic gastritis, 12 had pernicious anemia, and 6 had hypergastrinemia; no patient had carcinoid syndrome. Most of the tumors were confined to the mucosa and submucosa. Lymph node metastases were present in only one patient. The tumors were argyrophilic in 84% and argentaffin in 14%. Chromogranin tested positive in all patients; serotonin was detected in one-third; other hormones were much less common. Gastrin-positive tumors were antral. Of the 62 patients with follow-up, 44 were alive without disease, 4 were alive with disease, and 14 were dead (4 died of carcinoid-related disease). None of the deceased had pernicious anemia or hypergastrinemia. The tumors in patients with a fatal outcome were 2 cm or larger. CONCLUSION: Gastric carcinoids generally are indolent tumors, particularly when associated with pernicious anemia or hypergastrinemia or when smaller than 2 cm. Chromogranin is the most sensitive marker.

Adult↗
PubMed1994

Cavitation dosimetry: estimates for single bubbles in a rotating-tube exposure system.

Cell lysis and hydrogen peroxide production from cavitation in a 60 rpm rotating-tube exposure system were observed for 2.17 MHz ultrasound at 0.8 MPa peak negative pressure amplitude. Synchronized 10 ms burst mode exposure was utilized to emphasize the phenomenon of bubble cycling each half rotation. Low cell numbers and inhibition of H2O2-consuming enzymes allowed measurement of the residual hydrogen peroxide in exposed cell suspensions. Canine red blood cells (RBCs) or Chinese hamster ovary cells (CHO) suspended at 1 x 10(6) mL-1 in phosphate buffered saline were lysed exponentially with the number of bursts. The CHO cells were lysed faster than RBCs. The H2O2 increased approximately in proportion to the number of bursts. Longer bursts (100 ms) or continuous exposure produced similar trends, but were less effective per unit of total on-time. The number of bubbles per 10 ms burst was estimated from a simple model to be about 7100. The faster lysis of the CHO cells could be explained mostly by the larger size of these cells, which makes them more likely to meet a bubble. The H2O2 production gave concentrations of about 93-155 fM per bubble per burst. Similar calculations gave estimates of 178 fM per bubble for 100 ms bursts and 150 fM per bubble for continuous exposure. The rate of H2O2 production was roughly 500 fmole s-1 while a bubble crossed the tube. This sonochemical yield could be biologically significant under favorable circumstances.

Animals↗
PubMed1994

Heating as a mechanism for ultrasonically-induced petechial hemorrhages in mouse intestine.

Anesthetized hairless mice were exposed to 1.035 MHz unfocused ultrasound in a temperature-controlled water bath. Visible petechial hemorrhages and hyperemia were produced in the intestines. Histologically, the petechiae appeared to be localized mostly to the lamina propria of the mucosa. For 2 min exposure, the number of petechiae and length of hyperemic tissue both increased with increasing spatial peak pressure amplitude above a threshold of about 0.4-0.57 MPa. The magnitude of these effects increased, and the threshold decreased, with increasing exposure duration from 1 min to 4 min. The effects also increased for increasing bath temperature from 32 degrees C to 42 degrees C. The effects decreased markedly for 1 ms burst-mode exposure with 2 ms and 4 ms repetition periods with constant pressure amplitude, but were essentially constant for constant temporal-average intensity. Both effects were also produced by heating from radio-frequency diathermy at 10.35 MHz. The effects were associated with abdominal temperatures above about 42-43 degrees C, which were estimated with a thermocouple at the end of the exposure. The hyperemia is a clearly thermal effect, while the petechiae have been previously associated with the cavitation mechanism. However, the ultrasonically-induced petechiae observed in this study do not represent an unequivocal marker for cavitation, but rather appear to be attributable to heating. These findings help to reduce the expectation of ultrasonic cavitation during medical therapy treatments.

Animals↗
PubMed1994

The effects of gestation on circulating progenitor cells.

The frequency of BFU-E in second-trimester fetal blood (484 +/- 104/10(5)) falls progressively during gestation to a value of 69 +/- 41/10(5) in cord bloods of 36 weeks gestation and beyond, but this is still significantly greater than adult blood values of 14 +/- 8 (P < 0.01). BFU-E obtained from unfractionated peripheral blood mononuclear cells from fetuses/neonates less than 36 weeks gestation were more sensitive to erythropoietin than adult BFU-E, but the sensitivity of highly purified BFU-E obtained from second-trimester fetal liver was similar to that in adult cells. Almost maximal growth of BFU-E from purified fetal progenitor cells could be achieved with erythropoietin alone, whereas adult cells required the presence of other factors with 'burst-promoting activity'.

Cell Division↗
PubMed1993

A comparison of hemolytic and sonochemical activity of ultrasonic cavitation in a rotating tube.

Biological effects of in vitro ultrasonic exposure may result from mechanical and from sonochemical mechanisms related to ultrasonic cavitation. Mechanical cell lysis by 1.61 MHz ultrasonic cavitation was assessed in a rotating tube exposure system by hemolysis measurements. Free radical generation was assessed by the terephthalic acid dosimeter, calibrated by gamma-ray dosage. Sonochemical production was assessed by measuring residual hydrogen peroxide using the sensitive isoluminol method. Exposure conditions were similar for all tests, except longer durations were needed for the free radical and hydrogen peroxide tests. The sonochemical mechanisms were relatively more important for increasing intensity, and increasing temperature. Increasing rotation speed or bubbling with argon before exposure enhanced all cavitation activity. Stopping the rotation reduced all cavitation activity. Burst-mode ultrasound (10.5 microseconds bursts, 1:1 or 1:3 duty cycle) reduced cavitation activity, but gave relatively greater sonochemical activity under some conditions. These results indicate that the mechanical and sonochemical mechanisms can be separately favored to some extent by varying exposure conditions. The observed trends should be helpful for selecting exposure conditions favorable for studying bioeffects of the sonochemical mechanism.

Erythrocytes↗
PubMed1993

Ultrasonic gas body activation in Elodea leaves and the mechanical index.

Membrane damage resulting from ultrasonic gas body activation was investigated in leaves of the aquatic plant Elodea using pulse-mode exposures from 0.745-15 MHz. The frequency response was similar to that previously observed for continuous exposures. Cell death thresholds were higher for the pulse modes; for example, at 6 MHz the threshold was 166 W/cm2 spatial-peak, pulse-average (SPPA) intensity for 1 microsecond pulses and 1 kHz pulse repetition frequency (PRF) for 60 s exposures, compared to 12 W/cm2 for 60 ms continuous exposure. Increasing the PRF for 2.5 MHz, 3 microseconds pulses from 10 Hz to 5 kHz resulted in a gradual decrease in the threshold. Increasing the pulse duration from 1 microsecond to 30 microseconds and PRF from 33 Hz to 1 kHz only weakly influenced the threshold, for the constant 60 ms total on-time. Decreasing the pulse duration for single 6 MHz pulses increased the threshold up to 1,540 W/cm2 at 30 microseconds. The gas body activity was modeled with linear theory for oscillation of the gas channel walls, and intracellular shear stress, which causes lethal damage to the plasma membranes, was modeled with theory for acoustic microstreaming. Theory and observation indicate that the minimum shear stress threshold as a function of resonance frequency has essentially the same form as the Mechanical Index (MI) in the 0.5 to 20 MHz range. Specifically, for 3 microseconds pulses with 0.2-2 kHz PRF and 60 ms total on-time, the pressure-amplitude threshold divided by the square root of the frequency was approximately constant at 0.95 MPa/MHz1/2.

Cell Death↗
PubMed1993

Frequency dependence of cavitation activity in a rotating tube exposure system compared to the mechanical index.

Cavitation was produced by focused and unfocused ultrasonic beams in a 60-rpm rotating-tube exposure system. Hemolysis, free radical generation, and hydrogen peroxide were assayed after exposure to determine the dependence of cavitation activity on pressure-amplitude p and frequency f. For unfocused exposure, hemolysis rose rapidly from an apparent threshold pressure amplitude, to a maximum value and then declined gradually at higher amplitudes. Cavitation activity decreased with increasing ultrasonic frequency for a fixed pressure amplitude. Apparent thresholds for unfocused exposure were similar for hemolysis after 15 s and H2O2 production after 15 min. These thresholds increased roughly in proportion to increasing ultrasonic frequency. Cavitation activity was lower and thresholds were higher for focused exposures than for unfocused exposures. The approximate pf-1 = 0.20 MPa/MHz dependence of the experimental threshold of H2O2 production from 0.745 to 2.95 MHz differs from the pf-1/2 = 0.24 MPa/MHz1/2 dependence of the theoretical "mechanical index" threshold for inertial cavitation.

Animals↗