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Biomedical subjects

N Shepherd

Publications and source records attributed to N Shepherd.

At least 19 recordsLinked to original sources
PubMed2001

Cytoplasmic RNA Polymerase in Escherichia coli.

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase beta and beta' subunits in DNA-free minicells from the minicell-producing Escherichia coli strain chi925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase beta and beta' subunits in minicells produced by this strain corresponded to about 17% (or 2.5 microM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

Bacteriophage T7↗
PubMed2000

Defective hMSH2/hMLH1 protein expression is seen infrequently in ulcerative colitis associated colorectal cancers.

BACKGROUND: Ulcerative colitis is associated with an increased risk of colorectal cancer above that of the normal population. The relative risk correlates with the extent and duration of the disease but the genetic basis of ulcerative colitis associated cancer risk is not known. AIMS: To assess the prevalence of microsatellite instability and mismatch repair gene abnormalities in ulcerative colitis associated colorectal cancer. PATIENTS: Forty six patients with colorectal cancer, with a previous histological diagnosis of ulcerative colitis. METHODS: The frequency of microsatellite instability and/or immunohistochemical expression of hMSH2 and hMLH1 was assessed. Thirty three cases were investigated using both approaches. RESULTS: Although 6/41 (14.6%) cases showed microsatellite instability at one or more markers, only one case (2. 4%) exhibited high level instability (at least two markers affected). Of 38 cases which were assessed using antibodies against hMSH2 and hMLH1, only one case (2.6%) showed loss of expression. This case, which showed loss of hMSH2 expression, was the same case which exhibited high level microsatellite instability. The 33 cases which were investigated using both approaches showed that loss of expression of either hMSH2 or hMLH1 was not seen in any case which exhibited microsatellite instability in no more than one marker. CONCLUSIONS: This study suggests that both high level microsatellite instability and loss of expression of hMSH2/hMLH1 are infrequent events in ulcerative colitis associated colorectal cancers. Low level microsatellite instability was not associated with loss of expression of either hMSH2 or hMLH1.

Biomarkers↗
PubMed1998

Ionic diffusion in transverse tubules of cardiac ventricular myocytes.

We have estimated the rate of diffusion of calcium ions in the transverse tubules of isolated cardiocytes by recording changes in peak calcium current (ICa) caused by rapid changes of the extracellular calcium concentration ([Ca]o) at various intervals just preceding activation of ICa. Isolated ventricular cells of guinea pig heart and atrial cells from rabbit heart were voltage-clamped (whole cell patch), superfused at a high flow rate, and stimulated continuously with depolarizing pulses (0.5 Hz, 200- or 20-ms pulses from a holding potential of -45 or -75 mV to 0 mV). In ventricular cells, the change in peak ICa following a sudden change of [Ca]o increased rapidly as the delay between the solution change and depolarization was increased, up to a delay of approximately 75 ms [time constant (tau) approximately 20 ms, 30-40% of total current change), and then increased more slowly (tau approximately 200 ms, 60-70% of total current change); 400-500 ms were needed to achieve 90% of the total current increase. In atrial cells, a clear separation into two phases was not possible and 90% of the current change occurred within 85 ms. The slow phase of current change, which was unique to the ventricular cells, presumably reflects the slow equilibration of ions between the bulk perfusate and the lumina of the transverse tubules. If the lengths of the transverse tubules were equal to the cell thickness, the slow rate of change of current would be consistent with an apparent diffusion coefficient for calcium ions of 0.95 x 10(-6) cm2/s, considerably smaller than the value in bulk solution (7.9 x 10(-6) cm2/s). Most likely, this discrepancy is due to a high degree of tortuosity in the transverse tubular system in guinea pig ventricular cells or possibly to ion binding sites within the tubular membranes and glycocalyx.

Animals↗
PubMed1998

Altered cadherin and catenin complexes in the Barrett's esophagus-dysplasia-adenocarcinoma sequence: correlation with disease progression and dedifferentiation.

The maintenance of adult tissue architecture is largely dependent on the function of cadherins. E-cadherin is expressed in most epithelia, although it may be co-expressed with P-cadherin in basal layers of stratified epithelia. Adhesive function of cadherins relies on interactions with catenins. Many reports have characterized reduced expression of cadherins and catenins in tumors, including those of the gastrointestinal tract. This study aimed to characterize expression of E- and P-cadherins, and the catenins, in the progression of Barrett's esophagus to adenocarcinoma. Immunohistochemical analysis and Western blotting were performed on paraffin-embedded and fresh-frozen tissue using antisera to the selected cadherins and catenins. The results of this study have shown inappropriate expression of cadherins and catenins in neoplastic Barrett's mucosa. There was a significant reduction of E-cadherin expression as the Barrett's metaplasia-dysplasia-adenocarcinoma sequence progressed (P < 0.01). In contrast, P-cadherin, expressed in basal layers of squamous esophagus, was usually absent from Barrett's and dysplasia but was expressed in 17 of 24 carcinomas, especially at the advancing tumor edge. Reduced expression of catenins was also seen, but in some specimens, immunoreactivity was observed in neoplastic nuclei, suggesting mediation of a nuclear function such as transcriptional regulation.

Adenocarcinoma↗
PubMed1997

Restitution of contractility in single ventricular myocytes of guinea pig heart.

OBJECTIVE: Our aim was to assess the extent to which changes in intracellular Ca2+ stores contribute to mechanical restitution in heart muscle. METHODS: Single, isolated guinea pig ventricular cells were voltage clamped at -45 mV and stimulated continuously at 0.5 or 2 Hz with 200 ms depolarizing pulses (35 degrees C). The recoveries of the peak of contraction force (Fp) and the calcium current (ICa) between beats were measured in contractions interpolated at various intervals (td) after a conditioning twitch. Recovery of SR Ca2+ load was inferred from the peak magnitude (Cp) of similarly interpolated contractures, induced by rapid application of 5 mM caffeine. RESULTS: For a conditioning stimulus rate of 0.5 Hz, both Fp and ICa were very small for small td and recovered along similar time courses with a t1/2 of about 50 ms. Cp was maximal at as early a time after a previous contraction as could be measured, at which time Fp was 56% of maximal. Cp declined throughout the stimulus interval to about 50% of its maximal value. Similar results were obtained for a conditioning stimulus rate of 2 Hz, at which rate both Fp and Cp were increased by a factor of 2. CONCLUSIONS: The time course of mechanical restitution is coincident with the recovery of ICa from inactivation. Caffeine-releasable intracellular calcium stores are fully recovered soon after a contraction and well before mechanical restitution is complete.

Animals↗
PubMed1997

Cadherin and catenin biology represent a global mechanism for epithelial cancer progression.

The cell undergoes a diverse range of stimulations including growth factor activation and signal transduction from adhesion receptors, such as cadherins. In the absence of a mitogenic signal from outside the cell, beta catenin is sequestered in complexes with the product of the adenomatous polyposis coli (APC) gene and a serine threonine glycogen kinase (GSK 3 beta) enabling degradation of free beta catenin. Residual catenins hold cells together by binding to cadherins both at adherens junctions and the actin cytoskeleton. When a mitotic signal is delivered by the wnt pathway, GSK 3 beta is antagonised so that beta catenin can no longer be degraded. Cytosolic concentrations rise and binding to other newly synthesised proteins occurs, especially transcription factors that are transported to the nucleus, such as lymphocyte enhancing factor and T cell factor. This article discusses the signalling between mitogenic and adhesion pathways and suggests that it is a global mechanism for development, differentiation, and disease. These changes in catenin and APC biology may not be sufficient alone to transform cells fully but they appear to be a necessary final common pathway for several cancers of the mucous secreting crypts (including Barrett's oesophageal lesions and colorectal cancer) or stratified secreting epithelium (melanoma) before invasion.

Cadherins↗
PubMed1994

Interobserver variation in the reporting of the histopathological grading of anal intraepithelial neoplasia.

AIM: To assess the consistency in the histological reporting of anal intraepithelial neoplasia (AIN) among experienced histopathologists. METHOD: One hundred anal biopsy specimens were retrieved from archival material at St Mark's Hospital, London and graded by five histopathologists according to criteria outlined by Fenger (six point scale, ranging from normal to invasive carcinoma). RESULTS: There was only moderate agreement among the pathologists, with unweighted k scores ranging from 0.09 to 0.48, and weighted k scores of 0.17 to 0.60. CONCLUSIONS: There is considerable interobserver variation in the reporting of AIN. A simplified system of grading may help to abolish this.

Anus Neoplasms↗
PubMed1994

Tension-voltage relations of single myocytes reflect Ca release triggered by Na/Ca exchange at 35 degrees C but not 23 degrees C.

Contractile tension in response to 200-ms voltage-clamp pulses was measured in isolated guinea pig ventricular cells conditioned to constant Ca load. At 23 degrees C, the tension-voltage relation was bell shaped, decaying from a maximum at +20 mV to zero at +100 mV, but at 35 degrees C it was sigmoidal, with similar twitch tensions at +20 and +100 mV. Tension at 35 degrees C and +100 mV was reduced by ryanodine or caffeine and abolished by removal of Ca just before the test pulse. At 35 degrees C and +100 mV, twitch tension increased markedly as the Na concentration in the patch pipette ([Na]p) was varied between 0 and 20 mM. Cd (300 microM) blocked tension at all potentials at 23 degrees C, but tension remained in the presence of Cd at 35 degrees C (29% of control at +2 mV and 100% of control at +100 mV). Cd-resistant tension began to relax during the clamp pulse at all potentials (80 +/- 10 ms at +2 mV and 140 +/- 12 ms at +100 mV). Ni (3.6 mM) both reduced and slowed tension transients at all potentials. The results suggest that fast contractions due to sarcoplasmic reticulum Ca release can be triggered by Ca influx through either Ca current (ICa) or Na/Ca exchange and that those triggered through exchange are much more temperature sensitive than those triggered by ICa.

Animals↗
PubMed1992

Assessment of the accuracy of cytology in women referred for colposcopy and biopsy: the results of a 1 year audit.

The results of weekly colposcopy review meetings have been audited for 1 year and cases where there was a discrepancy between the referral cervical smear and the initial colposcopy biopsy have been analysed. New referrals (n = 476) for colposcopy were studied. In the final outcome 80% of 326 women referred for moderate or severe dyskaryosis were found to have cervical intraepithelial neoplasia (CIN) grade II or III or invasive carcinoma. Three women found to have invasive carcinoma had been referred for severely dyskaryotic smears. Twenty women were referred for smears with cell changes suggesting glandular neoplasia: five were found to have adenocarcinoma in situ, whereas eight had CIN and seven had negative biopsies. The results justify the referral policy and demonstrate the need for further investigation when initial colposcopic biopsies are negative.

Adenocarcinoma↗
PubMed1991

Cadmium block of isometric contractions of isolated bullfrog atrial cells.

We studied the effect of cadmium, verapamil, and quinacrine on the force of contraction (Fp) of isolated, single, field-stimulated bullfrog atrial cells. All agents were applied or removed rapidly (t1/2 approximately 15 ms) to minimize intracellular concentration changes other than intracellular calcium concentration. Two components of twitch force were observed, one blocked by micromolar Cd2+ and the other by millimolar Cd2+. The two contributed about equally to the activation of the twitch. The "cadmium-sensitive" portion of force (that affected by [Cd] less than or equal to 100 microM) had a K1/2 approximately 1 microM, was identical in magnitude to, and not additive with, a "verapamil-sensitive" (10 microM) component of force, was most strongly affected by 50-ms pulses of Cd2+ when they were applied in the mechanical latent period, and was potentiated by catecholamines. The cadmium-insensitive portion of force was abolished by the removal of extracellular calcium and was greatly potentiated by quinacrine (3 or 10 microM), a blocker of Na-Ca exchange. The results are consistent with the idea that activating calcium enters the cell via both an inactivating cadmium-sensitive L-type channel and a noninactivating cadmium-insensitive mechanism that is not Na-Ca exchange and leaves the cell via Na-Ca exchange.

Animals↗
PubMed1990

Combined force and voltage measurement in rapidly superfused guinea pig heart cells.

We describe the construction and use of a setup that allows the rapid exchange of the solution surrounding an isolated guinea pig heart cell while simultaneously measuring the isometric force and membrane potential (Em). Cells were stably attached, by means of poly-L-lysine, to a force transducer which was adapted from one previously used for a study of frog atrial cells [N. Shepherd and F. Kavaler.Am. J. Physiol. 251 (Cell Physiol. 20): C653-C661, 1986]. The modified transducer is simple to construct and use and can be readily added to existing patch-clamp setups. The strength of attachment of a cell to the transducer exceeded the strength of the gigaseal in all of the experiments. The membrane potential was measured by means of patch electrodes and a high-impedance voltage follower. Rapidly changing extracellular K concentration [( K]o) from 5.4 to 10.8 mM caused a positive change of Em by 16.5 +/- 1.4 mV with a half-time (t1/2) of 27 +/- 4 ms. Replacing calcium in the perfusate by magnesium instantly abolished the contraction and shortened the action potential. Twitch tension returned stepwise to the control value on return of calcium to the perfusate. Our initial observations show that the patch electrode can be used successfully in conjunction with the isometric force transducer and rapid extracellular solution changes for studies of excitation and contraction coupling in isolated mammalian heart cells.

Action Potentials↗
PubMed1990

Force measurements from voltage-clamped guinea pig ventricular myocytes.

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Administration, Topical↗
PubMed1990

Infant hearing screening 1984 to 1989: the Henry Ford Hospital experience.

From 1984 to 1989 the Infant Hearing Screening (IHS) program at Henry Ford Hospital identified 1,300 infants as being "at risk" for hearing loss. The prevalence of significant sensorineural hearing loss in this sample was 1.4%. Additionally, 80 infants who passed the IHS program and reached 3 years of age were found to have normal hearing sensitivity by conventional audiometric techniques (ie, no false-negative predictions). There were three false-positive predictions. It was discovered that infants of low birthweight (ie, less than 1,500 g) were three times more likely to fail IHS than those whose weight exceeded 1,500 g. A higher return rate was found for infants failing an initial hearing screening conducted in the neonatal intensive care unit in comparison to those screened as outpatients one week postdischarge. The sensitivity and specificity of behavioral observation audiometry were 43% and 92%, respectively, when brainstem auditory-evoked potentials was used as the criterion validity measure.

Audiology↗
PubMed1988

The occurrence of pancreastatin in tumours of the diffuse neuroendocrine system.

We have reported previously the localization of the 49 amino acid peptide pancreastatin to all identifiable endocrine cells of porcine gut, pancreas and adrenal, thyroid and pituitary glands. In this study, we have investigated the occurrence of pancreastatin in a series of human neuroendocrine tumours using an antibody to whole synthetic porcine pancreastatin. The most consistent immunostaining for pancreastatin was found in carcinoid tumours of ileum (four out of six), rectum (four out of six), ovary (two out of two) and lung (nine out of 10). Radioimmunoassay of tumour extracts showed that the concentrations of pancreastatin in ileal carcinoids were very high (mean 71.6, range 31.0-184.0 pmol g-1). The high rate of positivity in lung carcinoids contrasted sharply with the results of 10 pulmonary small cell carcinomas which displayed no immunoreactivity and contained minimal concentrations of pancreastatin (mean 2.0, range 0-6.0 pmol g-1). Extra-adrenal paragangliomas also contained pancreastatin (seven out of 10), but although radioimmunoassay detected peptide in phaeochromocytomas (mean 29.8, range 8.0-69.0 pmol g-1), immunocytochemistry did not. Porcine pancreastatin shows structural homology with bovine chromogranin A, an observation which has led to suggestions that chromogranin is a precursor for the peptide. More recently, a sequence homologous to porcine pancreastatin has been identified in the human chromogranin A molecule. In this study, immunostaining with an antiserum to human chromogranin gave positive results in most cases of each tumour type except the small cell carcinomas. The lack of consistent relationships between chromogranin and pancreastatin immunoreactivities may reflect the fact that the antiserum to pancreastatin was raised against the porcine peptide. When antibodies to human pancreastatin become available, the peptide may prove to be a more consistent marker for neuroendocrine tumours.

Adenocarcinoma↗
PubMed1987

Carcinoid tumours of the rectum and anus.

Carcinoid tumours of the anorectum comprise only 0.1 per cent of all rectal tumours. Their behaviour and treatment are still debatable. We identified 35 cases from the records of St Mark's Hospital; 31 were benign and 4 were malignant. Benign tumours were of three distinct histopathological patterns. All benign tumours were cured by local treatment. Malignant tumours can be identified clinically; the only consistent microscopic feature was invasion of the muscularis propria. All patients with malignant tumours died, despite radical surgical treatment; lymph node metastases were invariably present in the operation specimens. Because radical treatment for malignant carcinoid tumours of the anorectum is largely unsuccessful, a flexible surgical approach is advocated.

Adult↗
PubMed1986

Submaximal sodium-lack contractures in rapidly perfused frog ventricular strips.

Rapid perfusion of strips of frog ventricular muscle (width less than or equal to 8 mm) by sodium-free Ringer yielded contractures with very fast rates of rise (half time approximately equal to 2 s) and flat plateaus. Tension in lithium Ringer was 0.64 (0.38-0.77) of the maximal tension the muscle could produce [i.e., for an extracellular potassium concentration ([K]o) of 160 mM, and an [Na]o of 0 mM at less than 15 degrees C], showing that intracellular calcium concentration ([Ca]i) in zero [Na]o and normal [K]o was insufficient to saturate the myofilaments. Tension was related to [Ca]o in an S-shaped manner (apparent Km = 1.3 microM), was rapidly increased by electrical depolarization, high potassium, or low temperature, but was unaffected by up to 10(-5) M dihydroouabain. From these data was derived the working hypothesis that the value of [Ca]i during the contracture plateau is a steady-state value due to influx through a sodium-dependent mechanism and calcium uptake or efflux via a sodium-independent mechanism. Also, 1 microM dihydroouabain increased peak twitch tension by 60% but did not potentiate tension during the plateaus of contractures obtained by partial replacement of Nao. This result supports the working hypothesis and suggests that the potentiating effect of raising Nai may depend on the membrane potential (D. A. Eisner, W. J. Ledener, and R. D. Vaughan-Jones. J. Physiol. Lond. 335: 723-743, 1983).

Animals↗
PubMed1986

Direct control of contraction force of single frog atrial cells by extracellular ions.

We describe a method by which the ionic surround of an isolated frog heart cell can be changed within a small fraction of a contraction cycle while continuously measuring contraction force. With this method, we have investigated the effect on force development of changing the extracellular concentrations of Ca [( Ca]o) and Na [( Na]o) in the period between electrically driven contractions and during the rising phase of a contraction. Raising or lowering either [Ca]o or [Na]o more than 300 ms prior to a stimulus caused peak force of the next contraction to be changed 100% of the way to the steady-state value characteristic of the new ionic concentrations. Similar maneuvers at later times relative to the stimulus caused progressively smaller changes. Lowering [Ca]o from 2 to 1 mM or raising [Na]o from 78 to 110 mM 100 ms after stimulation brought twitch force 35 and 67% of the way to the new steady states, respectively. We conclude that extracellular Ca is the source of activator Ca in these cells and that extracellular Na plays a role in regulation of the intracellular Ca concentration early in the contraction cycle.

Animals↗