Transversus abdominis plane block reduces morphine consumption in the early postoperative period following microsurgical abdominal tissue breast reconstruction: a double-blind, placebo-controlled, randomized trial.

BACKGROUND: The analgesic efficacy of the transversus abdominis plane peripheral nerve block following abdominal tissue breast reconstruction has not been studied in a randomized controlled trial. METHODS: The authors conducted a double-blind, placebo-controlled, 1:1 allocation, two-arm parallel group, superiority design, randomized controlled trial in patients undergoing microsurgical abdominally based breast reconstruction. Intraoperatively, epidural catheters were inserted under direct vision through the triangle of Petit on both sides of the abdomen into the transversus abdominis plane just before rectus fascial closure. Patients received either bupivacaine (study group) or saline (placebo group) through the catheters for 2 postoperative days. All patients received hydromorphone by means of a patient-controlled analgesic pump. The primary outcome was the difference in the parenteral opioid consumption on each postoperative day between the groups. The secondary outcome measures included the following: total in-hospital opioid; antinausea medication; pain, nausea, and sedation scores; Quality of Recovery Score; time to ambulation; and hospital stay duration. RESULTS: Between September of 2011 and June of 2013, 93 patients were enrolled: 49 received bupivacaine and 44 received saline. There were 11 postoperative complications (13 percent); none were related to the catheter. Primary outcomes were completed by 85 of 93 patients (91.3 percent); the mean parenteral morphine consumption was significantly reduced on postoperative day 1 in the bupivacaine group (20.7±20.1 mg) compared with 30.0±19.1 mg in the control group (p=0.02). There were no significant differences in secondary outcomes. CONCLUSION: Following abdominally based breast reconstruction, transversus abdominis plane peripheral nerve block is safe and significantly reduces morphine consumption in the early postoperative period. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

Baseline survey on functioning of abortion services in government approved CAC centers in three pilot districts of Nepal.

BACKGROUND: Abortion has been legalized in Nepal since September 2002 and under this law, Comprehensive Abortion Care (CAC) service is being provided through listed service providers and listed health facilities from 2004. Nepal Government has prioritized the national safe abortion program and is working with many government and non government partners for providing this service. Till date medical abortion services are not made available at any of the health facility. Government is now preparing to introduce this service in six selected pilot districts. OBJECTIVE: This survey was carried out to assess the functioning of existing abortion services in 12 Government approved CAC sites of three districts. MATERIALS AND METHODS: Direct observation of the functioning of these centers, assessment of physical facilities and service provider's skill was done. At the same time service provider's attitude and knowledge on CAC service and other abortion services were also assessed through semi structured interviews. Quality of record keeping and the feasibility of initiating the medical abortion service in these sites were also studied. RESULT: Number of listed centers in six pilot districts was twenty nine. Study districts have 16 listed centers. Visited sites were twelve; four managed by Government and eight by non government organizations. Thirty three thousand nine hundred and twenty women have availed this service so far: only 4.76% of them received service from Government facilities. Marie Stopes International (MSI) topped the list in providing service to the maximum number of clients (75.64%) and Family planning association of Nepal (FPAN) was the second. MSI centre was also first to initiate the service. Government facilities provide 24 hours service unlike private facilities which are open only up to 5.00 pm. Cost for the service varies from rupees 900/- to rupees 1365/- and is cheaper at Government facilities. Private sectors have separate setups and Government have allocated some space within their already existing infrastructure for CAC service. Private sectors were better in providing the information to public about the availability of service. There were total 20 trained service providers for first trimester abortion service. They are more at Government facilities. They seem to be positive to CAC service and had good knowledge and skill of service delivery. Complications were not recorded at most of the sites. Pain management and infection prevention practice needs improvement at the Government sites. All the sites had identified their referral sites and had one or the other arrangement for referral. CONCLUSION: CAC service has become accessible and affordable to Nepalese women even at peripheral level. CAC sites are functioning well. Initiation of medical abortion and second trimester abortion services at these sites are feasible and would expand the option and choices available.

Test of isospin symmetry via low-energy 1H(pi{-},pi{0})n charge exchange.

We report measurements of the pi;{-}p-->pi;{0}n differential cross sections at six momenta (104-143 MeV/c) and four angles (0 degrees -40 degrees ) by detection of gamma-ray pairs from pi;{0}-->gammagamma decays using a photon-pair spectrometer at TRIUMF. This kinematic region exhibits a vanishing zero-degree cross section from destructive interference between s and p waves, thus yielding special sensitivity to pion-nucleon dynamics. Our data and previous data do not agree, with important implications for earlier claims of large isospin-violating effects.

Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa.

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

Distribution of intimal and medial thickening in the human right coronary artery: a study of 17 RCAs.

OBJECTIVE: To quantify the distribution of intimal and medial thickening in human right coronary arteries (RCAs) obtained at autopsy. BACKGROUND: The shear and tensile stresses created by arterial bifurcation are believed to result in eccentric fibromuscular intimal thickening that leads to atherosclerosis. Vascular curvature has been cited as a cause of atherosclerosis; however, details of the location and extent of intimal and medial thickness in the largely curved human RCA are not adequately documented. METHODS: The right coronary arteries were obtained from 40 postmortem hearts and cut into 20-30 segments, each being 3-4 mm in length. Microscopic sections from the proximal, acute margin, and distal regions of the RCA were digitized around the circumference of the vessel. Seventeen arteries showed insignificant stenosis (<50%) and were selected for detailed examination. RESULTS: Seventy-one percent (12/17) of proximal sections displayed eccentric intimal thickening. Normalized ensemble averaging revealed a preferential thickening on the myocardial side of the artery. At the acute margin region where curvature is most pronounced and at the distal region, 51% (8/17) of the samples showed eccentric thickening, but the ensemble average thickening in these regions showed no preferential location. In these mildly diseased arteries, the thickened intima comprised of mainly smooth muscle cells with an extracellular matrix of collagen and some elastin. A relatively uniform medial smooth muscle layer was seen at all three locations. CONCLUSIONS: The proximal region of the RCA appears to be a site of intrinsic eccentric intimal thickening with maximum thickness on the myocardial side of the artery. Eccentric thickening does occur in the acute margin and distal regions; however, no distinct pattern or location was evident.

In vitro hemodynamic evaluation of a Simon nitinol vena cava filter: possible explanation of IVC occlusion.

PURPOSE: To evaluate the local hemodynamics in the region of the Simon nitinol filter (SNF), used to prevent pulmonary emboli by capturing clot and promoting lysis. MATERIALS AND METHODS: The hemodynamics of the Simon nitinol inferior vena cava (IVC) filter were evaluated under steady flow (Re = 600) in a 20-mm-diameter IVC model. The photochromic dye tracer technique was used to estimate the velocity and wall shear stress. These flow features were determined for the unoccluded and partially occluded (clot volume = 1,500 mm(3)) states of the SNF along its center plane. RESULTS: A region of low velocities developed around the central axis of the filter extending from the leading edge of the central strut to the filter tip. This phenomenon was created by the strong redirection of flow toward the periphery of the filter. With the presence of the clot, these effects were enhanced, causing flow separation and recirculation. In addition, the shear stress on the hip of the clot was about 30 times that of the upstream value, and turbulence developed in the near-downstream region. CONCLUSIONS: The extended region of almost-stagnant flow near the midsection of the umbrella region could lead to organization of thrombus and fibrin mesh network development. The presence of a simulated clot led to a significant increase in the size of the stagnant, thrombus-prone region as well as turbulence, which, overall, may contribute to caval occlusion.

Factors influencing blood flow patterns in the human right coronary artery.

Evidence suggests that atherogenesis is linked to local hemodynamic factors such as wall shear stress. We investigated the velocity and wall shear stress patterns within a human right coronary artery (RCA), an important site of atherosclerotic lesion development. Emphasis was placed on evaluating the effect of flow waveform and inlet flow velocity profile on the hemodynamics in the proximal, medial, and distal arterial regions. Using the finite-element method, velocity and wall shear stress patterns in a rigid, anatomically realistic model of a human RCA were computed. Steady flow simulations (ReD=500) were performed with three different inlet velocity profiles; pulsatile flow simulations utilized two different flow waveforms (both with Womersley parameter=1.82, mean ReD=233), as well as two of the three inlet profiles. Velocity profiles showed Dean-like secondary flow features that were remarkably sensitive to the local curvature of the RCA model. Particularly noteworthy was the "rotation" of these Dean-like profiles, which produced large local variations in wall shear stress along the sidewalls of the RCA model. Changes in the inlet velocity profiles did not produce significant changes in the arterial velocity and wall shear stress patterns. Pulsatile flow simulations exhibited remarkably similar cycle-average wall shear stress distributions regardless of waveform and inlet velocity profile. The oscillatory shear index was very small and was attributed to flow reversal in the waveform, rather than separation. Cumulatively, these results illustrate that geometric effects (particularly local three-dimensional curvature) dominate RCA hemodynamics, implying that studies attempting to link hemodynamics with atherogenesis should replicate the patient-specific RCA geometry.

Histology and morphology of 59 internal thoracic artery grafts and their distal anastomoses.

BACKGROUND: The left internal thoracic artery (LITA) is accepted as a superior graft for the left coronary system because of its better long-term patency rate than saphenous grafts. The postsurgical histomorphometric changes at the distal anastomosis of LITA grafts are not well documented. METHODS: The cellular changes within the intima of 59 LITA grafts were analyzed by light microscopy. RESULTS: Grafts implanted 1 week or less (n = 34) showed no postsurgical tissue proliferation. Of the 7 grafts implanted 1 to 8 weeks, only the suture sites exhibited intimal thickening (6 of 7 grafts, 0.08 +/- 0.07 mm). The remaining grafts (n = 18), aged 2 months to 10 years, showed significant intimal thickening at the suture sites (0.39 +/- 0.17 mm) and on the hood (0.29 +/- 0.25 mm), with variable thickening on the floor (10 of 18 left anterior descending coronary arteries, 0.11 +/- 0.12 mm). The graft body showed insignificant intimal changes (10 of 18, 0.03 +/- 0.04 mm), with mild focal atherosclerotic lesions in 2 of 18 late LITA grafts. CONCLUSIONS: Left internal thoracic artery grafts develop fibromuscular intimal hyperplasia primarily around the anastomosis. The response on the hood appears to be a hemodynamic response, secondary to that of the suture sites.

An in vitro comparison of the hemodynamics of two inferior vena cava filters.

PURPOSE: The effectiveness of an inferior vena cava (IVC) filter in preventing pulmonary embolism while preserving caval flow is significantly affected by its hemodynamic characteristics. Flow fields surrounding two types of IVC filters were compared to assess how the design of a filter may influence performance. METHODS: The 12F Titanium Greenfield and VenaTech LGM inferior vena cava filters were studied in vitro with a noninvasive flow visualization technique, the photochromic flow visualization and measurement technique. Axial velocity profiles and wall shear stress distributions were measured. These results were compared with analytical data corresponding to the flow field in the absence of a filter to determine the relative extent of the flow disturbances. RESULTS: The reductions in near-wall axial velocity and wall shear stress caused by the VenaTech filter were more extensive and severe than those caused by the Greenfield filter. These changes were the consequence of differences in the geometry and dimensions of the struts of the two filters. The measurements showed the flow fields to be laminar, with no evidence of turbulence in both cases. CONCLUSION: Two factors that have been linked to thrombogenesis, near-wall velocity and wall-shear stress, were significantly affected by the larger frontal profile area of the VenaTech filter. Although a larger area may increase clot-trapping efficiency, as shown by previous studies, the reduced near-wall velocities and wall shear stresses may increase the potential for thrombogenesis and, thus, caval occlusion. In contrast to other in vitro flow visualization studies, no turbulence was observed with either filter.

Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.

Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).

Velocity and wall shear stress patterns in the human right coronary artery.

Blood flow dynamics in the human right coronary artery have not been adequately quantified despite the clinical significance of coronary atherosclerosis. In this study, a technique was developed to construct a rigid flow model from a cast of a human right coronary artery. A laser photochromic method was used to characterize the velocity and wall shear stress patterns. The flow conditions include steady flow at Reynolds numbers of 500 and 1000 as well as unsteady flow with Womersley parameter and peak Reynolds number of 1.82 and 750, respectively. Characterization of the three-dimensional geometry of the artery revealed that the largest spatial variation in curvature occurred within the almost branch-free proximal region, with the greatest curvature existing along the acute margin of the heart. In the proximal segment, high shear stresses were observed on the outer wall and lower, but not negative, stresses along the inner wall. Low shear stress on the inner wall may be related to the preferential localization of atherosclerosis in the proximal segment of the right coronary artery. However, it is possible that the large difference between the outer and inner wall shear stresses may also be involved.

Flow waveform effects on end-to-side anastomotic flow patterns.

PURPOSE: Restenosis due to distal anastomotic intimal hyperplasia, a leading cause of arterial bypass graft failure, is thought to be promoted by hemodynamic effects, specifically 'abnormal' wall shear stress patterns. The purpose of this study was to quantify the effects of flow waveform on peri-anastomotic flow and wall shear stress patterns. METHODS: Blood flow and wall shear stress patterns were numerically computed in a representative three-dimensional anastomosis using femoral, iliac and coronary flow waveforms suitable for humans at rest. Numerical results were validated against experimental data. RESULTS: Peri-anastomotic wall shear stress patterns were influenced by a complex interplay between secondary flow effects and unsteadiness. Peripheral flow waveforms (iliac, femoral) produced large temporal and spatial wall shear stress gradients on the host artery bed. In comparison, the coronary flow waveform produced normalized bed wall shear stress gradients that were a factor of 2-3 less than for the peripheral waveforms, even though average bed wall shear stress magnitudes were similar for the two waveforms. CONCLUSIONS: If anastomotic intimal hyperplasia is promoted by large spatial and/or temporal gradients of wall shear stress, as has been proposed, this study predicts that there will be markedly less intimal hyperplasia on the host artery bed of coronary bypass grafts than for peripheral bypass grafts. This information, in conjunction with a comparative histopathologic study of intimal hyperplasia distribution, could help determine specific wall shear stress factors promoting intimal hyperplasia.

Histological and morphometric analyses of early and late aortocoronary vein grafts and distal anastomoses.

BACKGROUND: Aortocoronary vein grafts develop fibromuscular intimal hyperplasia within the first year of implantation. Tissue remodeling may promote development of graft atherosclerosis and thrombosis. Angiographic studies show that human aortocoronary vein grafts in situ for one or more years become stenosed, preferentially at the distal anastomosis versus the body or trunk of the graft or at the proximal anastomosis. Previous studies have not reported morphological data on the nature and distribution of intimal lesions around the distal graft/artery anastomoses. OBJECTIVE: To examine and quantify histological and morphometric changes within the intima of 27 aortocoronary vein grafts and their distal anastomoses. METHODS: Seventy-two hearts obtained at autopsy and one at heart transplantation were examined, photographed and fixed in 10% buffered formaldehyde solution. Three to seven 3 mm long segments of grafts and their distal anastomoses were sectioned, stained and examined by light microscopy. RESULTS: Eleven early grafts were implanted for six weeks or less, and they showed significant cellular hyperplasia mainly at the suture line. In 16 late grafts in situ 1.5 to 15 years, the degree of fibromuscular intimal thickening was greatest on the hood and at the suture line, whereas on the floor of the native artery and in the graft body the degree of thickening was approximately one-third and two-thirds, respectively, that seen on the hood. CONCLUSIONS: Stenosis of aortocoronary vein grafts at their distal anastomosis is likely related to the preferential development of intimal thickening on the hood of the graft and at the suture line. Because fibromuscular intimal hyperplasia has been reported to play a role in the development of atherosclerosis and thrombosis in the body of vein grafts, this focal hyperplasia at the distal anastomosis may also play a role in vein graft failure.

Compliance mismatch may promote graft-artery intimal hyperplasia by altering suture-line stresses.

The role of graft-artery compliance mismatch in the development of distal anastomotic intimal hyperplasia (DAIH) is not yet resolved. Although DAIH develops at all surgically created anastomoses, increased compliance mismatch does not lead to greater hyperplasia formation in end-to-end anastomoses, but in end-to-side anastomoses, it leads to a profound increase in hyperplasia. The current study was undertaken to determine whether suture-induced anastomotic stresses could explain these findings. A large strain finite element analysis of vascular wall mechanics was performed to compare the influence of compliance mismatch on intramural stresses in end-to-end versus end-to-side anastomoses. A novel modelling approach was implemented which includes suture-induced stress concentrations. End-to-end and end-to-side graft-artery simulations were executed using (1) artery (compliance = C = 0.44% kPa(-1)), (2) vein (C = 0.33% kPa(-1)), and (3) Dacron (C = 0.14% kPa(-1)) grafts. Residual stresses due to axial tension were included and the anastomoses were statically inflated to 13.3 kPa (100 mmHg). Elevated intramural stresses were found to exist at both the end-to-end and end-to-side graft-artery junctions; however, in the end-to-end anastomosis, the maximum anastomotic stress was not a function of the graft compliance, whereas in the end-to-side anastomosis, the maximum stress was a strong function of graft compliance. For the 45 degree end-to-side geometry considered in this study, the maximum anastomotic stress concentration obtained using a stiff Dacron graft was more than 40% greater than that obtained using a compliant artery graft. In the end-to-end anastomosis, the Dacron graft led to a less than 5% increase in maximum stress over the artery graft. Therefore, increased compliance mismatch increases stresses and promotes DAIH in end-to-side junctions, but, it has little influence on either stresses or DAIH in end-to-end junctions. Thus, the proliferative influence of increased compliance mismatch on suture-line hyperplasia in end-to-side anastomoses can be explained by the resulting increase in intramural stresses. In addition, since high stresses were found in both geometries, elevated suture-line intramural stresses may be an important proliferative stimulus for intimal hyperplasia formation in all vascular reconstructions.

In vitro assessment of the hemodynamic effects of a partial occlusion in a vena cava filter.

PURPOSE: Flow fields surrounding a vena cava filter were studied with a noninvasive method of flow visualization to examine the underlying hemodynamic factors that contribute to its function and patency. METHODS: The photochromic technique was used to measure axial velocity profiles and wall shear stress distributions in a 12F titanium Greenfield filter partially occluded with a simulated volume of entrapped thrombi. These results were compared with similar measurements obtained for an unoccluded filter and with a theoretic prediction of the velocity profile and wall shear stress without a vena cava filter. Shear stress distributions were obtained along the vessel wall and for the partially occluded filter along the surface of the simulated clot. RESULTS: The unoccluded filter was observed to have little effect on the flow field. In the case of the partially occluded filter, the results of these measurements show that caval blood flow is preserved by the creation of an annular region of increased flow around the periphery of the clot. Within this region high shear stresses that develop as a result of the increased flow are observed along the vessel wall and along the surface of the simulated clot. No vortices or turbulence were observed with either the unoccluded or the partially occluded filter. CONCLUSIONS: The elevated levels of shear stress may be a factor in the lysis of trapped clots observed in vivo. Although increased shear stress is reported to cause thrombogenesis in an in vivo study in canines, particularly under turbulent flow conditions, the levels of shear measured in this study around the simulated clot were well below such values.

Relation of the flow field distal to a moderate stenosis to the Doppler power.

An experimental investigation was undertaken to establish how different flow regimes affect the Doppler signal. A rigid tube model consisting of a 70% asymmetric area stenosis was used with steady and pulsatile flow conditions. The characteristics of the flow field at various sites was determined using a photochromic flow visualization method. Continuous-wave Doppler measurements were made using a 41% suspension of human red blood cells (RBCs) in saline as well as a dilute suspension of 4% fixed RBCs. For steady flow, the photochromic results indicated that for Reynolds numbers (Re) of 545 and 1410, turbulence was generated and the length of the turbulent region was found to increase with increasing Re. Under pulsatile flow conditions, turbulence was triggered around peak systole and began to dissipate in late deceleration, and by the end of diastole the flow field almost relaminarized. During the turbulent phase of the flow cycle, the poststenotic flow field was seen to consist of four distinct flow regimes similar to those observed for steady flow. For higher Womersley parameters and Reynolds numbers the turbulent zone was found to be larger and to occupy a greater fraction of the flow cycle. These flow visualization results were compared with the Doppler power measurements made at the same locations and under similar flow conditions. At physiological hematocrits (41%) the onset of turbulence for both steady and pulsatile flow increased the backscattered Doppler power. The location of the peak Doppler power coincided with the region of maximum turbulence observed using the photochromic technique.

Blood flow dynamics, atherosclerosis and bypass graft failure.

Atherosclerosis occurs at reproducible sites in the arterial tree and intimal proliferation that leads to bypass graft occlusion also show a well-defined focal distribution. These observations have led to the hypothesis that local blood flow conditions, especially low or fluctuating shear stresses, are important in the development of both disorders. Basic research using both cell culture and animal models has revealed that endothelial cell biology is very sensitive to local shear stresses and rapid progress is being made in characterizing how endothelial cells transduce shear stress. Endothelial sensitivity to shear stress affects control of hemostasis, leukocyte adherence and transmigration, growth factor production, vasomotor responses, endothelial repair and arterial wall remodeling, all of which can be expected to influence development of vascular pathologies. Also, substantial progress has been made in characterizing complex local hemodynamics at relevant arterial sites; however, further progress is needed in this area, as well as in the extrapolation of advances in basic vascular biology to human vascular disease. (Trends Cardiovasc Med 1997;7:111-118). © 1997, Elsevier Science Inc.

Defining the limitations of measurements from Doppler spectral recordings.

PURPOSE: The purpose of this study was to determine whether Doppler measurements of peak velocity and four other quantitative measures of spectral shape are affected significantly by the site of the Doppler recording in relation to the location of the maximum stenosis. METHOD: Continuous-wave and pulsed Doppler recordings were made distal to a 70% (area reduction or 45% diameter reduction) asymmetric stenosis in an in vitro flow model under steady and pulsatile flow conditions. Recordings were taken at six different locations proximal and distal to the stenosis. A photochromic dye technique was used to visualize the actual flow field in the model. RESULTS: Distal to the stenosis, the flow visualization results demonstrated a strong radial and axial variation of the velocity field and thus explained why the Doppler measurements of peak frequency and spectral broadening were strongly dependent on the recording site. The peak frequency was maximum within the throat of the stenosis and returned to the prestenotic value five tube diameters distal to the stenosis. Other measurements of spectral broadening and spectral shape varied greatly depending on the location of the recording site in the poststenotic region. Higher order spectral moments such as the coefficient of kurtosis were found to exhibit large temporal variability, which makes them inappropriate as diagnostic indicators. CONCLUSIONS: Because of the complex nature of the poststenotic flow field, these results clearly demonstrate that no single Doppler measurement can accurately quantify the severity of a stenosis. Of the Doppler measurements only peak velocity is related to the severity of stenosis. Reproducible peak velocity measurements are obtained only if the Doppler sample volume is positioned at or very near the throat of the stenosis and at an appropriate radial site that may not necessarily be at the center of the vessel.

Purification, properties and developmental regulation of a Ca(2+)-independent serine-cysteine protease from Allomyces arbuscula.

Reproductive differentiation in Allomyces takes place against the background of substrate limitation, a sharp increase in intracellular proteolysine and the induction of at least one specific protease. The aim of this report is to describe the purification, properties and developmental regulation of this enzyme. The enzyme has been partially purified by a combination of ion exchange chromatography, ultrogel filtration and affinity chromatography. The purified enzyme in SDS-PAGE appeared as a doublet of M(r) 40-43 kDa. Two bands corresponding to a relative molecular mass of 40-43 kDa were also apparent in activity gels. The protein has an apparent molecular mass in the region of 43 kDa as estimated by gel filtration. The enzyme therefore, seems to be a monomer of 43 kDa. The second band in SDS-PAGE and activity gels is probably the proteolyzed form of the enzyme. The protease recognized alanine and to a lesser extent phenylalanine in the P1 position when assayed with a range of synthetic peptides. The active site of the enzyme contains a reactive serine residue, as shown by its inhibition with PMSF and soya bean trypsin inhibitor. There is probably a reactive cysteine residue as well since the enzyme activity is strongly inhibited by HgCl2, a thiol group binding reagent. The enzyme is present in zoospores but disappears progressively during germination and hyphal growth. It reappears when actively growing cultures are transferred to dilute salt solution. In conclusion, we have purified a serine-cysteine protease of M(r) 43 kDa. This enzyme has a very restricted substrate specificity and appears to be developmentally regulated.