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Biomedical subjects

J Buckman

Publications and source records attributed to J Buckman.

At least 19 recordsLinked to original sources
PubMed2000

Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein.

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) </= 50 nM) characterized by loss of NADPH fluorescence, subsequent rapid formation of a charge-transfer complex between NADPH and oxidized enzyme-bound flavin mononucleotide (FMN) cofactor, followed by rate-limiting reduction of the FMN, and finally dissociation of the oxidized pyridine nucleotide. The UV-visible absorbance behavior of this half-reaction is described by two apparent phases, with k(f)(obs) = 355 +/- 6 and k(s)(obs) = 3.30 +/- 0.03 s(-)(1), for the fast and slow phases, respectively. The reaction has also been evaluated in terms of the full, multi-equilibrium reaction scheme, and microscopic rate constants that lead to the observed behavior have been determined through convergent experimental techniques and computer simulation. Significant intrinsic kinetic isotope effects were noted on both the bond cleavage step and the preceding formation of the charge-transfer complex. The enzyme is reoxidized by transfer of hydride from the FMN to HXL in the second half-reaction that appears to consist of substrate binding to form a Michaelis-type complex and the subsequent chemical step. Characterization of the reaction in this simple manner allows determination of an apparent K(d) = 100 +/- 9 microM for the reduced enzyme.HXL complex.

Aldehydes↗
PubMed2000

Stabilization of a novel enzyme.substrate intermediate in the Y206F mutant of Candida albicans EBP1: evidence for acid catalysis.

EBP1-catalyzed reduction of alpha,beta-unsaturated ketones and aldehydes is proposed to proceed via transfer of hydride from the flavin to the beta-position of the olefinic bond, concomitant with or followed by uptake of a proton at the alpha-position. Structural analysis suggests that this proton is donated from Tyr206, and, hence, a protein was constructed in which it was replaced by phenylalanine. The mutation results in a slightly less stable protein than the wild type that nevertheless retains the fundamental flavin and phenol binding properties of EBP1 characterized previously. The pH profile for binding of phenol was characterized over the pH range 6.5-9.5 and was found to be simpler than that for the wild-type enzyme. Most importantly, a pK(a) of 8.7 that is perturbed to 9.4 upon binding of phenol to the wild-type enzyme is missing in the mutant, allowing assignment of this pK(a) to the Y206 hydroxyl group. Additionally, the pK(a) of phenol is further lowered from its value of 10.0 in solution to approximately 6.4 in the active site of the mutant, as compared to 7.1 in the wild type. Together, these perturbations lead to an increase of approximately 35-fold in the binding affinity of the mutant for phenol at high pH relative to the affinity of the wild-type enzyme. As expected, the mutation has little effect on the reductive half-reaction, in which a hydride equivalent is transferred from NADPH to the flavin. In contrast, the reduction of trans-2-hexenal by the reduced enzyme is significantly affected. The results indicate formation of a previously unobserved charge-transfer (CT) complex following formation of the Michaelis complex between substrate and reduced enzyme and preceding reduction of the substrate, which occurs at a greatly reduced rate (>/=440-fold) relative to wild type. Thus, while the oxidative half-reaction with wild-type enzyme is limited by the rate of formation of the CT complex, it is the chemical step that is rate-limiting in the reaction with EBP1:Y206F, consistent with the role of this residue as a general acid.

Base Sequence↗
PubMed2000

Viscoelastic properties of normal and atherosclerotic carotid arteries.

BACKGROUND: remodelling of the arterial wall occurs with ageing, even in the absence of atherosclerotic risk factors. With increasing age, arteries dilate, thicken, and get stiffer. The aim of this study was to correlate carotid artery stiffness with wall thickness and plaque presence between healthy individuals and patients with early and advanced atherosclerosis. METHODS: twenty healthy volunteers, 40 carotid segments and 90 patients, 174 carotid segments, with vascular disease were included in the study. The carotid artery was imaged longitudinally and measurements of the intimal-medial thickness (IMT) and plaque were obtained. Systolic and diastolic blood pressures were taken from each arm. The carotid artery stiffness (pressure-strain elastic modulus, Ep) was calculated in all sites from the changes in pressure and diameter. M-mode was used to detect the diameter change (systolic to diastolic) over five cardiac cycles. RESULTS: in the healthy volunteers there was no evidence of plaque or increased IMT. The mean IMT was significantly higher in the patients compared to control (0.83+/-0.27 mm vs. 0.54+/-0.08 mm, p <0.0001). The IMT had a poor correlation with Ep at lower thickness (r=0.24, p=0.08) but this association became stronger with increasing thickness (r=0.62, p<0.001). Arterial segments with an IMT 5 0.88 mm became significantly stiffer compared to the controls (p<0.001) and to patients with an IMT<0.88 mm (p <0.01). Carotid Ep was markedly greater in arterial segments with plaques than in those with increased IMT (p <0.001) and the controls (p<0.0001). CONCLUSIONS: carotid wall areas with small increase in IMT have a poor correlation with carotid artery stiffness. The carotid stiffness increases in areas with marked wall thickening and particularly in segments with plaque. The simultaneous study of vessel-wall elastic behaviour with IMT and plaque changes may increase our understanding of atherosclerotic progression and wall remodelling.

Adult↗
PubMed2000

The impact of isolated lesser saphenous vein system incompetence on clinical signs and symptoms of chronic venous disease.

PURPOSE: The purpose of this study was to determine the patterns of isolated lesser saphenous vein (LSV) system incompetence and correlate the distribution and extent of such reflux with symptoms and signs of chronic venous disease (CVD). METHODS: During a 3-year period, 2254 limbs in 1682 patients with signs and symptoms of CVD were evaluated with color flow duplex scanning. Extremities with isolated reflux in the LSV system were selected for this study. Limbs with perforating venous reflux connected to this system only were also included. Limbs that had marked reflux in the greater saphenous or deep vein, that had a documented history of deep venous thrombosis, and that previously underwent surgery or sclerotherapy were excluded. The clinical severity of the limbs was graded with the CEAP classification system. RESULTS: There were 226 limbs in 200 patients with reflux in the LSV system; 61% were female patients with a mean age of 49 years (range, 18-82 years). There were 174 patients (87%) with unilateral and 26 with bilateral disease, and 41% of the limbs belonged in CVD class 2, 26% in class 3, 12% in class 4, 3.5% in class 5, and 3% in class 6. Classes 0 and 1 were present in 14.5% of the limbs. Symptoms were present in 139 limbs (61.5%). Some degree of ache or burning sensation was the most frequent symptom (41%), followed by itching (32%), heaviness (29%), cramps (24%), and restless limbs (18%). Reflux in the main trunk of the LSV was the most prevalent (177 limbs [78%]), followed by the saphenopopliteal junction (146 limbs [64.6%]), the vein of Giacomini (39 limbs [17%]) and the gastrocnemial vein (23 limbs [10%]). Reflux involving both the saphenopopliteal junction and the LSV was seen in 50% of limbs, but almost any other combination of reflux was present, which indicated the complexity of this system. Perforator vein incompetence was detected in 56 limbs (25%). We found 83 perforator veins, resulting in a mean of 1.5 veins per limb. Both the number of incompetent perforator veins and the extent of superficial reflux correlated with clinical severity. Four main types of termination of the LSV were identified with at least nine variations. The LSV was duplicated for at least half of its length in five limbs (2.2%). Nonsaphenous reflux was detected in seven limbs (3.1%). Superficial vein thrombosis in the LSV system was found in eight limbs (3.5%), and in the gastrocnemial vein it was found in four (1.8%). CONCLUSIONS: Isolated LSV system incompetence can cause the entire range of signs and symptoms of CVD. Clinical deterioration is associated with a longer extent of reflux and perforator incompetence. Classes 2 to 4 are the most frequent clinical presentations, whereas classes 5 and 6 are uncommon. The complex anatomy of this system and the great variation in the patterns of reflux warrant the use of color flow duplex scanning before planning treatment.

Adolescent↗
PubMed1999

Patterns and distribution of isolated calf deep vein thrombosis.

PURPOSE: In the search for calf deep vein thrombosis (DVT) with color-flow duplex scanning (CFDS), most vascular laboratories investigate only the posterior tibial and peroneal veins. Few laboratories assess the soleal and gastrocnemial veins. This study was designed to determine the patterns and distribution of isolated calf DVT, including the soleal and gastrocnemial veins. METHODS: In the last 3 years, 5250 patients (mean age, 66 +/- 15 years; range, 22 to 93 years) were referred to the vascular laboratory for clinical suspicion of DVT and underwent examination with CFDS. All superficial and deep named veins, excluding the anterior tibial from groin to ankle, were imaged. Of the deep veins in the calf, the peroneal, the posterior tibial, the gastrocnemial, and the soleal veins were examined throughout their length. RESULTS: DVT was detected in 14% of the patients. Isolated calf DVT was detected in 282 limbs of 251 patients (4.8%). No significant difference was noted for the sex (114 men vs 137 women; P =.15) or the limb preference (145 left vs 137 right; P =.5). The peroneal veins were most frequently involved, with 115 limbs (41%) affected. The soleal veins were involved in 109 limbs (39%), followed by the posterior tibial in 105 limbs (37%) and the gastrocnemial in 79 limbs (29%). Thrombus in the soleal vein alone was found in 57 limbs (20%), in the gastrocnemial in 48 limbs (17%), in the peroneal in 41 limbs (15%), and in the posterior tibial vein in 35 limbs (12%). Thrombus confined to a single or paired vein was found in 181 limbs (64%). Thrombus involving two different veins (27%) was the second most frequent pattern, and thrombus in three (7%) or four (1.4%) different veins was less prevalent. Isolated thrombosis in veins not routinely investigated was found in 113 limbs (40%; soleal, n = 57; gastrocnemial, n = 48; soleal + gastrocnemial, n = 8). Multifocal origin of thrombosis, defined as thrombi in two different veins that do not anatomically communicate, was identified in 63 limbs (22%). CONCLUSION: Forty percent of the patients with acute isolated calf DVT would be judged to have normal CFDS examination results if the muscular veins in the calf were not imaged. Multifocal origin of thrombosis was found in 22% of the involved limbs. The prevalence of thrombosis in any calf vein either alone or in combination is comparable. Accordingly, the soleal and gastrocnemial veins should be examined routinely.

Adult↗
PubMed1999

Evaluation of a model of collaboration: academic and clinician partnership in the development and implementation of undergraduate teaching.

The difficulty nursing students experience in making the transition from the university to clinical context is attributed to the gap between theory and practice, and education and service. Collaboration between academics and clinicians in the provision of undergraduate education is considered to be a strategy for overcoming these problems. A project team consisting of four academics and six clinicians collaboratively developed and implemented an acute care topic, in the third year of a pre-registration nursing course at the School of Nursing, Flinders University. A review of the literature did not provide examples of collaborative models relevant to undergraduate teaching. The subsequent model, devised by the project team, focused on technical, cultural and interpersonal aspects of collaborative teaching. The model was evaluated by illuminative research methods. Participants in the project included academics (n = 4), clinicians (n = 6) and students (n = 104). Student questionnaires and academic and clinician interviews were used to inform evaluation procedures. The qualitative data was coded and analysed based upon the definitions and characteristics of collaboration described by Henneman et al (1995). Study findings demonstrated that the model was effective in facilitating collaborative relationships necessary for the successful development and implementation of reality-based learning for students.

Cooperative Behavior↗
PubMed1999

Primary superficial vein reflux with competent saphenous trunk.

OBJECTIVES: because reflux in superficial vein tributaries is most often collectively reported with the main saphenous veins, its importance remains largely unrecognised. This study was designed to identify the distribution and extent of non-truncal superficial venous reflux and its association with the signs and symptoms of chronic venous disease (CVD). PATIENTS AND METHODS: eighty-four limbs in 62 patients with signs and symptoms of CVD and evidence of reflux on continuous-wave Doppler were subsequently examined with colour-flow duplex imaging. Incompetent superficial vein tributaries were imaged throughout their extent and both ends were identified. Limbs with reflux in the main trunk of the saphenous veins or the deep, perforator or muscular veins, superficial or deep vein thrombosis, injection sclerotherapy, varicose-vein surgery, arterial disease and inflammation of non-venous origin were excluded from the study. The CEAP classification system was used for staging clinical severity of CVD. RESULTS: the prevalence of tributary reflux alone was 9.7% (84/860). Reflux was detected in 171 tributaries. The number of incompetent tributaries ranged from 1 to 5 per limb. Most prevalent were the tributaries to the greater saphenous (111, 65%<0. 0001), followed by those of lesser saphenous (33, 19%) or a combination of both (12, 7%). Incompetent non-saphenous tributaries were uncommon (15, 9%). Among the named tributaries in the lower limb the posterior arch vein was most often incompetent (46, 27%) followed by the anterolateral vein of the thigh (30, 18%), the medial accessory vein (16, 9%) and the anterior arch vein (14, 8%). Reflux in above-the-knee tributaries alone was found in 18 limbs (21%), in below the knee in 23 (28%) and in both sites in 43 (51%). The vast majority of the limbs (71%,p <0.0001) belonged to CVD class 2, 14% in class 3, 9% in class 1 and only 6% in class 4. Class 3 and 4 patients tended to have a longer duration of signs and symptoms, higher number of incompetent tributaries per limb and also a higher prevalence of combined above- and below-knee reflux. CONCLUSIONS: these data indicate that reflux confined to superficial tributaries is found throughout the lower limb. Because this reflux is present without greater and lesser saphenous trunk, perforator and deep-vein incompetence or proximal obstruction, it shows that reflux can develop in any vein without an apparent feeding source. Greater saphenous tributaries are affected significantly more often than those of lesser saphenous, while non-saphenous reflux is uncommon. Most limbs have signs and symptoms of CVD class 2 and 15% belong in classes 3 and 4.

Adolescent↗
PubMed1998

Binding and reactivity of Candida albicans estrogen binding protein with steroid and other substrates.

In this report recombinant estrogen binding protein (EBP1), isolated originally from Candida albicans as a result of its high affinity for 17beta-estradiol, has been purified extensively using a modified affinity purification scheme originally developed for a homolog of EBP1, old yellow enzyme (OYE). It is shown that like OYE, the protein binds a variety of compounds with a phenolic structure, including 17beta-estradiol, and compounds with an alpha, beta-unsaturated keto or aldehyde structure. In addition, EBP1 exhibits an NADPH oxidoreductase activity, transferring electrons from NADPH to all alpha,beta-unsaturated ketones and aldehydes tested via the tightly bound FMN cofactor. Analysis of the steady-state kinetics of these reactions indicate a tetra uni ping-pong mechanism. Inhibition of the steady-state reaction by 17beta-estradiol gives a Ki = 10 +/- 2 nM, and indicates exclusive binding of this steroid to the enzyme in its oxidized state. In contrast, 19-nortestosterone binds to both oxidized and reduced forms of the enzyme with dissociation constants of 600 +/- 100 and 650 +/- 90 nM, respectively. EBP1 also catalyzes a disproportionation reaction with certain compounds, in which two molecules of a cylic alpha,beta-unsaturated ketone, including the steroid 19-nortestosterone, are individually aromatized and reduced to the corresponding saturated ketone. Despite the extensive similarity in sequence and enzymic activity, notable differences between EBP1 and the OYE family of proteins exist with regard to the binding behavior and reactivity with the two steroids tested here, estradiol and 19-nortestosterone.

Binding, Competitive↗
PubMed1997

Prevalence and clinical significance of posterolateral thigh perforator vein incompetence.

PURPOSE: Posterolateral thigh perforator (PLTP) veins are part of the lateral thigh venous system, which in most people remains undeveloped. This study was designed to determine the prevalence and clinical significance of these veins. METHODS: Over the past 6 years, 2820 lower limbs with signs and symptoms of chronic venous disease (CVD) were evaluated for venous reflux using color flow duplex imaging. Superficial, perforating, and deep veins were examined in the standing, sitting, and reversed Trendelenburg positions. PLTP veins were best identified in the standing position with the patient facing away from the examiner. RESULTS: Twenty-six incompetent PLTP veins were found in 24 limbs (0.85%) of 21 patients (mean age, 43 +/- 16 years; range, 22 to 77 years). All PLTP veins pierced the fascia lata 12 to 25 cm (mean, 16 +/- 3 cm) above the popliteal skin crease in the lateral aspect of the thigh. At this level, the PLTP veins dove posteriorly 3 to 8 cm to join primarily tributaries of the deep femoral vein, superficial femoral vein, or both. Eight PLTP veins were duplicated at 1 to 2 cm below the fascia. Seven PLTP veins gave rise to superficial tributaries that were extended to the lower lateral and posterior thigh, whereas the remaining 19 PLTP veins gave rise to tributaries alongside the lesser saphenous vein and the anterior arch of the greater saphenous vein. On nine occasions, reflux was found in the PLTP veins and their associated tributaries alone. In all of these cases, reflux was adequately controlled with a tourniquet placed distal to the fascial defect. In the remaining 17 PLTP veins, reflux was also seen in the greater saphenous vein, the lesser saphenous vein, or both. None of the limbs that had PLTP vein reflux alone exceeded CVD class 3. When PLTP vein reflux was combined with saphenous reflux, there were five limbs classified as CVD class 4 and one limb each as CVD classes 5 and 6. Twenty limbs underwent ligation and stripping of the varicosities. Three of the earlier patients in the series underwent incomplete operations, which resulted in immediate residual varicosities from the PLTP tributaries. All three patients underwent reoperation successfully within a year. CONCLUSIONS: The prevalence of PLTP vein reflux is quite low. Reflux in the PLTP veins alone is associated with mild to moderate clinical presentation. However, when it is combined with saphenous reflux skin damage can be present. Failure to recognize PLTP veins may result in an incomplete or unnecessary operation, leaving the patients with residual varicose veins.

Adult↗
PubMed1992

Long-term preservation of explanted hearts perfused with L-aspartate-enriched cardioplegic solution. Improved function, metabolism, and ultrastructure.

The effects of supplementing oxygenated St. Thomas' Hospital cardioplegic solution No. 2 with L-aspartate and/or D-glucose for the long-term preservation of excised rat hearts were determined with isolated working heart preparations. Left ventricular function was assessed at 37 degrees C with a crystalloid perfusate, before cardioplegic arrest and after 20 hours of low-flow perfusion (1.5 ml/min) with continuing arrest at 4 degrees C, and after this period, again at 37 degrees C with a crystalloid perfusate. Four groups (n = 8/group) of hearts were studied with four cardioplegic solutions: St. Thomas' Hospital solution alone, St. Thomas' Hospital solution with aspartate 20 mmol/L, St. Thomas' Hospital solution with glucose 20 mmol/L, and St. Thomas' Hospital solution plus both aspartate and glucose (20 mmol/L each). The addition of glucose to St. Thomas' Hospital solution made no significant difference in the recovery of aortic flow rates (17.7% +/- 8.6% and 21.6% +/- 7.8% of prearrest values), but when aspartate or aspartate and glucose were present, hearts showed significant improvements (89.8% +/- 5.2% and 85.0% +/- 6.2%, respectively). These improvements were associated with a reduction in the decline of myocardial high-energy phosphates during reperfusion, a reduction in cellular uptake of Na+ and Ca++, and a reduction in ultrastructural damage. These results indicate that low-flow perfusion with St. Thomas' Hospital solution plus aspartate can considerably extend the duration of safe storage of explanted hearts.

Adenine Nucleotides↗
PubMed1990

Diagnostic results of psychiatric evaluations of state police officers.

Twenty-six state police officers referred by their department or self-referred were evaluated by a university department of psychiatry over a 28-month period. The most common primary diagnosis was adjustment disorder, followed by substance abuse and personality disorder. Sixteen of the officers were assessed to be psychiatrically capable of returning to duty; concurrent outpatient treatment was recommended for most of them. The most challenging cases for evaluation were those involving psychiatric symptoms after trauma on duty, behavioral manifestations of personality disorder, and substance abuse.

Adult↗
PubMed1977

Brainwashing, LSD, and CIA: historical and ethical perspective.

The history of various attempts at thought control and chemical warfare is briefly reviewed. Brainwashing, thought control, industrial and national espionage, and covert activities are becoming more sophisticated. These issues have been revived and accentuated by the Vietnam war, the Middle East Crisis, Watergate, the CIA investigations and the Patty Hearst trial. Historical perspective and the ethical implications of these activities are explored. It is suggested that there is a growing level of individual and international mistrust amounting to paranoia and complicating the issues of individual freedom, civil rights and human experimentation.

Ethics↗