Biomedical subjects

D H Evans

Publications and source records attributed to D H Evans.

At least 19 recordsLinked to original sources

PubMed2006

Cerebral critical closing pressure estimation from Finapres and arterial blood pressure measurements in the aorta.

Estimates of cerebral critical closing pressure (CrCP) and resistance-area product (RAP) are often derived using noninvasive measurements of arterial blood pressure (ABP) in the finger, but the errors introduced by this approach, in relation to intra-vascular measurements of ABP, are not known. Continuous recordings of ABP (Finapres and solid-state catheter-tip transducer in the ascending aorta), cerebral blood flow velocity (CBFV, bilateral Doppler), ECG and transcutaneous CO(2) were performed following coronary catheterization. CrCP and RAP were calculated for each of 12,784 cardiac cycles from 27 subjects using the classical linear regression (LR) of the instantaneous CBFV-ABP relationship and also the first harmonic (H(1)) of the Fourier transform. There was a better agreement between LR and H(1) for the aortic measurements than for the Finapres (p < 0.000,01). For LR there were no significant differences for either CrCP or RAP due to the source of ABP measurement, but for H(1) the differences were highly significant (p < 0.000,03). The coherence functions between either CrCP or RAP values calculated with aortic pressure (input) or the Finapres (output) were significantly higher for H(1) than for LR for most harmonics below 0.2 Hz. When using the Finapres to estimate CrCP and RAP values, the LR method produces similar results to intra-arterial measurements of ABP for time-averaged values, but H(1) should be preferred in applications analysing beat-to-beat changes in these parameters.

Airway Obstruction↗

PubMed2005

Objective selection of signals for assessment of cerebral blood flow autoregulation in neonates.

A number of different system identification techniques have been proposed to assess dynamic cerebral autoregulation in critically ill patients. From these methods, the response to a standard stepwise change in blood pressure can be estimated. Responses lacking physiological consistency are a common occurrence and could be the consequence of particular system identification procedures or, alternatively, caused by measurements with a poor signal-to-noise ratio. A multi-observer approach was adopted in this paper to classify cerebral blood flow velocity (CBFV) step responses to spontaneous changes in arterial blood pressure in a group of 43 neonates with a mean gestational age of 33.7 weeks (range 24-42 weeks) and a mean birthweight of 1,980 g (range 570-3,910 g). Three experienced observers independently analysed the estimated step responses in 191 recordings each lasting 100 s; for an autoregressive (ARX) model, 124 (65%) of the step responses were accepted by at least two of the three observers. Two other system identification methods, transfer function analysis and the moving average Wiener-Laguerre model, gave 90 (45%) and 98 (51%) acceptable responses, respectively. Only 54 epochs (28%) were accepted with all three methods. With 88 (46%) responses rejected by at least two methods, it can be concluded that signal quality was the main reason for nonphysiological step responses. To avoid the need for subjective visual selection, an automatic procedure for classifying step responses was implemented leading to sensitivities and specificities in the range 85-90%, with respect to the agreement with subjective evaluations. Objective selection of CBFV step responses is thus feasible and could also be adapted for other physiological measurement techniques relying on system identification methods.

Blood Flow Velocity↗

PubMed2004

Real-time identification and archiving of micro-embolic Doppler signals using a knowledge-based DSP system.

Identification of micro-emboli in the cerebral circulation using transcranial Doppler ultrasound provides valuable clinical information, but, currently, embolic signal detection and analysis are significantly limited because they mainly rely on costly off-line analysis by human experts. In this study, a reliable, high-resolution, real-time automated system for the detection and archiving of embolic signals was designed and implemented using expert system theory and modern DSP technology. Preliminary tests were conducted to evaluate the functions and the performance of the system using data from ten carotid endarterectomy patients and two normal volunteers. Using the widely accepted 7 dB threshold for human reliability and a human expert, majority-decision gold standard, the real-time system reached sensitivity and specificity of 93.6% and 99.3%, respectively, which were close to the results obtained by three human experts under ideal laboratory conditions (90.1% and 99.8%, 98.4% and 99.9%, 98.9 and 99.9%). The new system has the potential to be used either as a bedside monitoring and signal acquisition device, or as a laboratory investigation tool.

Endarterectomy, Carotid↗

PubMed2004

Neural network modelling of dynamic cerebral autoregulation: assessment and comparison with established methods.

A time lagged recurrent neural network (TLRN) was implemented to model the dynamic relationship between arterial blood pressure (ABP) and cerebral blood flow velocity (CBFV) and its performance was compared to classical linear model such as transfer function analysis, Aaslid's dynamic autoregulation model, and the Wiener-Laguerre moving average filter. A simple linear regression was also tested as a naive estimator. In 16 normal subjects, CBFV was continuously recorded with Doppler ultrasound and ABP with the Finapres device during six repeated thigh cuff manoeuvres. Using mean beat-to-beat values of ABP as input and CBFV as output, the performance of each method was assessed by the model's predicted velocity correlation coefficient and normalized mean square error (MSE). Cross-validation was performed using three thigh cuff manoeuvres for the training data set and the other three for the validation set. The four methods studied performed significantly better than the zero-order naive estimator. The TLRN performed better than transfer function analysis, but was not significantly different from the time-domain techniques, despite showing the minimum predictive MSE. CBFV step responses could be extracted from the TLRN showing the presence of non-linear behaviour both in terms of amplitude and directionality.

Adaptation, Physiological↗

PubMed2004

Association between dynamic cerebral autoregulation and mortality in severe head injury.

The objective of the study was to test the hypothesis that dynamic cerebral pressure-autoregulation is associated with the outcome of patients with severe head injury and to derive optimal criteria for future studies on the predictive value of autoregulation indices. Repeated measurements were performed on 32 patients with severe head injury. Arterial blood pressure (ABP) was measured continuously with an intravascular catheter, intracranial pressure (ICP) was recorded with a subdural semiconductor transducer and cerebral blood flow velocity (CBFV) was measured with Doppler ultrasound in the middle cerebral artery. Transfer function analysis was performed on mean beat-to-beat values, using ABP or CBFV as input variables and CBFV or ICP as the output variables. A dynamic index of autoregulation (ARI) ranging between 0 and 9 was extracted from the CBFV step response for a change in ABP. No significant differences between survivors and non-survivors were found due to mean values of ICP, ABP, CPP, CBFV, pCO2, GCS, age or heart rate. The transfer functions between ABP-ICP and CBFV-ICP did not show any significant differences either. The median [lower, upper quartiles] ARI was significantly lower for non-survivors compared with survivors [4.8 (0.0, 5.9) v. 6.9 (5.9, 7.4), p= 0.004]. The correlation between ARI and GOS was also significant (r=0.464, p=0.011). Cohen's coefficient was optimal for a threshold of ARI= 5.86 (kappa 0.51, p=0.0036), leading to a sensitivity for death of 75%, specificity=76.5%, odds ratio =9.75 and overall precision = 75.8%. The difference in ARI values between survivors and non-survivors persisted when results were adjusted for GCS (p = 0.028). A similar analysis for the Marshall CT scale did not reach significance (p = 0.072). A logistic regression analysis confirmed that apart from the ARI, no other variables had a significant contribution to predict outcome. In this group of patients, death following severe head injury could not be explained by traditional indices of risk, but was strongly correlated to indices of dynamic cerebral pressure-autoregulation extracted by means of transfer function analysis. Future studies using a prospective design are needed to validate the predictive value of the ARI index, as estimated by transfer function analysis, in relation to death and other unfavourable outcomes.

Adolescent↗

PubMed2002

Assessment of dynamic cerebral autoregulation based on spontaneous fluctuations in arterial blood pressure and intracranial pressure.

Assessments of dynamic cerebral autoregulation usually measure the cerebral blood flow velocity (CBFV) response to changes in arterial blood pressure (ABP). We studied the effect of substituting ABP by cerebral perfusion pressure (CPP), expressed as the difference between ABP and intracranial pressure (ICP), in estimates of dynamic autoregulation obtained by transfer function analysis. CBFV, ABP and ICP were recorded during periods of physiological stability in 30 patients with severe head injury. Transfer function analysis was performed using the following combinations of input-output variables: ABP-CBFV, CPP-CBFV and CBFV-ICP. Frequency and time-domain (step response) functions were averaged for recordings with mean ICP < 20 mmHg (group A) and mean ICP > or = 20 mmHg (group B). The ABP-CBFV transfer function parameters and step response for group A were similar to previous studies in normal subjects, but group B showed deterioration of dynamic autoregulation. Radically different step responses were obtained from both groups for the CPP-CBFV transfer function and the coherence was not significantly improved. The CBFV-ICP transfer function had the highest values of coherence and indicates that changes in CBFV are the cause of spontaneous fluctuations in ICP. Furthermore, the ICP step response plateau was significantly higher for group B than for group A. An alternative calculation of the CBFV step response to changes in CPP resembled the corresponding responses for the ABP input. For spontaneous fluctuations in ABP, ICP and CBFV, it is not possible to calculate the CPP-CBFV transfer function directly due to the high positive correlation between ICP and CBFV, but an alternative estimate can be obtained by using the CBFV-ICP transfer function. The latter could also be useful as a method to assess intracranial compliance in head injury patients.

Adult↗

PubMed2002

Cerebral autoregulation under moderate hypothermia in patients with acute stroke.

BACKGROUND AND PURPOSE: We undertook this study to examine the integrity of cerebral autoregulation in patients with acute ischemic stroke treated with moderate hypothermia (33 degrees C). METHODS: Fourteen patients, aged 58+/-11 years, with an acute anterior circulation infarction and National Institutes of Health Stroke Scale score >15 were evaluated. Patients received catecholamines (norepinephrine) via continuous intravenous infusion and were mechanically ventilated. Alpha-stat was used for pH maintenance. Arterial pressure (AP) and intracranial pressure (ICP) were invasively monitored. Flow velocity in the middle cerebral artery (MCA) supplying the unaffected hemisphere was continuously monitored. Instantaneous maximum flow velocity (V(max) MCA), ICP, and AP were simultaneously recorded in real time. Mean values of V(max) MCA (V(mean) MCA) and AP (MAP) were calculated over 1 minute. Static cerebral autoregulation (sCA) was calculated as sCA=(%DeltaCVR/%DeltaMAP)x100% (where %DeltaCVR is an estimate of percent change in cerebrovascular resistance). An sCA value of 0% indicates absent autoregulation, and a value of 100% indicates perfect autoregulation. Autoregulation is considered impaired when sCA values are <40%. MAP changes were produced by increasing the rate of the norepinephrine infusion. Six patients were examined under both normothermic and hypothermic conditions, while 8 were examined only under hypothermia. RESULTS: The induced MAP increase was 22+/-7 mm Hg (minimum 13, maximum 40 mm Hg). Mean sCA was 64+/-16% (minimum 40%, maximum 100%). No effect of moderate hypothermia on sCA or V(mean) MCA was evident in any of the 6 serially examined patients. Normocapnia was observed in all cases. CONCLUSIONS: sCA appears intact under moderate hypothermia with the use of alpha-stat for pH maintenance.

Acute Disease↗

PubMed2002

Deterioration in carotid baroreflex during carotid endarterectomy.

OBJECTIVE: Blood pressure instability after carotid endarterectomy (CEA) has been associated with a disturbance of the baroreflex control mechanism caused by the surgery in the carotid sinus region. The purpose of this study was to determine if a deterioration in carotid baroreceptors occurs during the surgery. METHOD: Heart rate (HR) and blood pressure (BP) were recorded continuously in 60 patients undergoing CEA as well as preoperatively and postoperatively at 2 days and 6 weeks. The baroreflex sensitivity was determined by cross-spectral analysis of HR and systolic blood pressure (SBP). During the surgery, three tests were used to assess the baroreflex response. The first test simulated a sudden fall in systemic blood pressure by clamping the common carotid artery. The second test simulated a rise in systemic blood pressure by applying pressure by using a rubbing action on the luminal surface of the carotid sinus region. The rub test was performed twice, once with the atheromatous plaque in situ and once when the plaque had been removed. The third test is clamp removal and restoration of blood flow through the carotid sinus. RESULTS: Carotid cross-clamping increased mean +/- standard error of the mean SBP from 117 +/- 3 mm Hg before clamping to 125 +/- 3 mm Hg (P <.05) at 30 beats after clamping. The first rub test with the plaque in situ decreased SBP from 121 +/- 3 mm Hg to 117 +/- 3 mm Hg (P <.01) at 10 beats after the rub test, indicating a functioning baroreceptor reflex. The second rub test increased SBP from 126 +/- 3 mm Hg to 128 +/- 3 mm Hg (P <.05). SBP dropped (P <.01) when unclamping suggesting a selective alteration of the baroreflex sensitivity. The baroreflex sensitivity was significantly reduced 2 days postoperatively when compared to preoperative values (P <.05). CONCLUSIONS: These findings suggest that the act of plaque removal could be associated with a partial disruption of baroreceptor mechanism in the carotid artery. This could affect type I baroreceptors.

Aged↗

PubMed2001

Vasoactivity of the ventral aorta of the American eel (Anguilla rostrata), Atlantic hagfish ( Myxine glutinosa), and sea lamprey (Petromyzon marinus).

To determine if vascular smooth muscle from teleost and agnathan fishes expresses receptors for signaling agents that are important in vascular tension in other vertebrates, we exposed rings of aortic vascular smooth muscle from the eel (Anguilla rostrata), the hagfish (Myxine glutinosa), and the lamprey (Petromyzon marinus) to a suite of putative agonists, including: acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostanoids. Acetylcholine constricted aortic rings from the eel, but had no effect on the rings from lamprey. On the other hand, endothelin constricted rings from all three species. Use of receptor-specific ET agonists demonstrated that only ET(A) receptors are expressed in the eel and lamprey aorta. The nitric oxide donor sodium nitroprusside or nitric oxide itself dilated rings from the eel, but both agonists constricted rings from the hagfish and NO produced a biphasic response (constriction followed by dilation) in the lamprey. Two natriuretic peptides, eel ANP and porcine CNP, produced marginally significant dilation in the eel aorta, human ANP dilated the hagfish rings, and pCNP and eANP dilated the lamprey rings. The prostanoids PGE(1) and PGE(2) both dilated the eel aortic rings, and PGE(1) and carbaprostacyclin (stable PGI(2) agonist) dilated the hagfish and lamprey rings. Our results suggest that receptors for a variety of vasoactive signaling agents are expressed in the aortic smooth muscle of the earliest vertebrates (lamprey and hagfish), as well as the more advanced teleosts (eel).

Acetylcholine↗

PubMed2001

Interposition flaps in transabdominal vesicovaginal fistula repairs: are they really necessary?

Objectives. To evaluate the use of interposition flaps in repairing vesicovaginal fistulas (VVFs) of benign and malignant etiologies. Interposition flaps are not routinely used in the repair of VVFs when the surrounding tissues appear healthy and well-vascularized, such as in a benign etiology.Methods. We retrospectively reviewed the charts of 37 women (mean age 49.1 years) at our institution who underwent transabdominal repair of their VVF by urologic surgeons between August 1978 and June 1999. The preoperative and postoperative medical records were reviewed.Results. Of the 37 VVFs repaired transabdominally, 29 had a benign etiology (25 related to gynecologic procedures) and 8 a malignant etiology (all related to gynecologic neoplasia). Of the 29 benign VVFs, an interposition flap was used in 10 repairs with all 10 successful (100%). The remaining 19 benign VVF repairs were performed without using a flap, with 12 successful (63%). Of the 8 malignant fistulas, an interposition flap was used in 2 repairs with both successful (100%). The remaining 6 malignant VVF repairs were performed without a flap, with 4 successful (67%).Conclusions. The results of our study indicate a higher success rate for transabdominal VVF repairs performed with an interposition flap (100% success rate at our institution). This observation holds true regardless of the appearance of healthy surrounding tissue or, more importantly, a benign or malignant etiology. We recommend interposition flaps in transabdominal repairs of VVFs, even in the cases of benign fistulas with well-preserved surrounding tissue.

Female↗

PubMed2001

Automated embolus identification using a rule-based expert system.

Transcranial Doppler ultrasound (US) can be used to detect microemboli in the cerebral circulation, but is still limited because it usually relies on "human experts" (HEs) to identify signals corresponding to embolic events. The purpose of this study was to develop an automatic system that could replace the HE and, thus, make the technique more widely applicable and, potentially, more reliable. An expert system, based around a digital signal-processing board, analysed Doppler signal patterns in both the time domain and frequency domain. The system was trained and tested on Doppler signals recorded during the dissection and recovery phases of carotid endarterectomy. It was tested with 74 separate 2.5-min recordings that contained at least 575 artefacts in addition to 253 s of diathermy interference. The results were compared with the results obtained by three HEs. Using a "gold-standard" that classified any event detected by the majority of HEs as an embolus, the automatic system displayed a sensitivity of 94.7% and a specificity of 95.1% for 1151 candidate events 7 dB or more above the clutter (signal-to-clutter ratio, SCR, > or = 7 dB), and 89.6% and 95.3%, respectively, for 2098 candidate events with SCR > or = 5 dB. The system had a very similar performance to individual HEs for SCR > or = 7dB, and was only marginally worse for SCR > or = 5 dB.

Artifacts↗

PubMed2001

Compensatory fuzzy neural networks-based intelligent detection of abnormal neonatal cerebral Doppler ultrasound waveforms.

Compensatory fuzzy neural networks (CFNN) without normalization, which can be trained with a backpropagation learning algorithm, is proposed as a pattern recognition technique for intelligent detection of Doppler ultrasound waveforms of abnormal neonatal cerebral hemodynamics. Doppler ultrasound signals were recorded from the anterior cerebral arteries of 40 normal full-term babies and 14 mature babies with intracranial pathology. The features of normal and abnormal groups as inputs to pattern recognition algorithms were extracted from the maximum velocity waveforms by using principal component analysis. The proposed technique is compared with the CFNN with normalization and other pattern recognition techniques applied to Doppler ultrasound signals from various arteries. The results show that the proposed method is superior to the others, and can be a powerful technique to be used in analyzing Doppler ultrasound signals from various arteries.

Algorithms↗

PubMed2001

A parametric approach to measuring cerebral blood flow autoregulation from spontaneous variations in blood pressure.

Autoregulation maintains cerebral blood flow (CBF) almost constant in the face of changes in arterial blood pressure (ABP). Tests for impairment of this process using only spontaneous fluctuations in ABP, without provoking large variations, are of great clinical interest, and a range of different approaches have previously been applied. Extending earlier work based on linear filters, we propose a simple parametric method using a first order finite impulse response filter. We evaluate the method on ABP and CBF velocity [(CBFV), from trancranial Doppler ultrasound] signals collected in 60 patients with stenosis or occlusion of the carotid arteries. Data were collected during the inspiration of ambient air, a 5% CO2/air mixture, and finally the return to ambient air. Equivalent data were collected in 15 normal subjects. The filters estimated from the data segments with constant inspiratory pCO2 showed the expected high-pass characteristic, which was reduced during hypercapnia and also in patients. Highly significant correlation between the filter parameters and cerebrovascular reactivity (percent increase in CBFV per unit change in end-tidal pCO2) gives further evidence that the filters reflect autoregulation. The method allows simple parametrization of the dynamic autoregulatory responses in CBFV, and the analysis of short (1 min) data segments.

Aged↗

PubMed2001

Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR.

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding approximately 500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

Animals↗

PubMed2001

Effects of DNA structure and homology length on vaccinia virus recombination.

Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting approximately 4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3'-to-5' exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.

Animals↗

PubMed2001

Contractile properties of the elasmobranch rectal gland.

The importance of the rectal gland in elasmobranch osmoregulation is well established. The rate of secretion by the gland is under the control of a variety of secretagogues and inhibitors. Early morphological work suggested that a band of smooth muscle cells surrounds the periphery of the shark rectal gland between the secretory tubules and the connective tissue capsule. To confirm the presence of the muscle ring, we examined histological sections from two species of shark, Squalus acanthias and Carcharodon carcharius, and from the stingray Dasyatis sabina and stained sections from S. acanthias with the actin-specific ligand phalloidin. In all three species, a distinct band of what appeared to be smooth muscle cells was evident, and the putative muscle ring in S. acanthias stained specifically with phalloidin. Moreover, isolated rings of rectal gland tissue from S. acanthias constricted when acetylcholine or endothelin was applied and responded to nitric oxide with an initial dilation, followed by a more substantial constriction. Subsequent addition of porcine C-type natriuretic peptide dilated the rings, but two prostanoids (carbaprostacyclin and prostaglandin E(1)) did not change ring tension significantly. The rings did not respond to the endothelin-B-specific agonist sarafotoxoin S6c, suggesting that the response to endothelin was mediated via endothelin-A-type receptors. Our data confirm the presence of a smooth muscle ring in the periphery of the elasmobranch rectal gland and demonstrate that the gland responds to a suite of smooth muscle agonists, suggesting that changes in the dimensions of the whole rectal gland may play a role in its secretory function.

Acetylcholine↗

PubMed2001

Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na(+)/K(+)-ATPase.

In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H(+)-ATPase) is found on the apical membrane of pavement and chloride (Na(+)/K(+)-ATPase-rich) cells, and is an important transporter for energizing Na(+) uptake and H(+) excretion. In the gills of elasmobranch fishes, the V-H(+)-ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H(+)-ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina) and determine if V-H(+)-ATPase and Na(+)/K(+)-ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H(+)-ATPase and greatest number of V-H(+)-ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H(+)-ATPase abundance and the number of V-H(+)-ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H(+)-ATPase and least number of V-H(+)-ATPase-rich cells. In contrast to teleost fishes, localization of V-H(+)-ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H(+)-ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H(+)-ATPase and Na(+)/K(+)-ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid-base regulation where the V-H(+)-ATPase-rich cells and Na(+)/K(+)-ATPase-rich cells are the sites of Cl(-) uptake/HCO(3)(-) excretion and Na(+) uptake/H(+) excretion, respectively.

Animals↗

PubMed2000

In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase.

During poxvirus infection, both viral genomes and transfected DNAs are converted into high-molecular-weight concatemers by the replicative machinery. However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unknown. Here we show that vaccinia virus DNA polymerase can catalyze single-stranded annealing reactions in vitro, converting linear duplex substrates into linear or circular concatemers, in a manner directed by sequences located at the DNA ends. The reaction required > or =12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpI3L). Varying the structures at the cleaved ends of the molecules had no effect on efficiency. These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3'-to-5' proofreading exonuclease, as judged by the fact that only a 5'-(32)P-end label is retained in the joint molecules and the reaction is inhibited by dNTPs. The resulting concatemers are joined only through noncovalent bonds, but can be processed into stable molecules in E. coli, if the homologies permit formation of circular molecules. This reaction provides a starting point for investigating the mechanism of viral concatemer formation and can be used to clone PCR-amplified DNA.

Animals↗