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Biomedical subjects

R L Williams

Publications and source records attributed to R L Williams.

At least 73 records · Page 4Linked to original sources
PubMed1999

Mammalian phosphoinositide-specific phospholipase C.

Mammalian phosphoinositide-specific phospholipases C (PI-PLCs) are involved in most receptor-mediated signal transduction pathways. The mammalian isozymes employ a modular arrangement of domains to achieve a regulated production of two key second messengers. The roles of the PH, EF hand, C2, SH2 and SH3 modules in regulation of these enzymes and in interactions with membranes and other proteins is becoming apparent from recent structural and functional studies.

Animals↗
PubMed1999

Structural insights into phosphoinositide 3-kinase catalysis and signalling.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Amino Acid Sequence↗
PubMed1999

The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.

C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.

Amino Acid Sequence↗
PubMed1999

Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.

Biological Transport↗
PubMed1999

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding.

Amino Acid Sequence↗
PubMed1999

Bioequivalence of immediate-release theophylline capsules.

A three-way crossover study in 18 healthy male volunteers was conducted to evaluate the bioequivalence of three different 200 mg anhydrous theophylline immediate-release (IR) capsules. The products had not been rated as therapeutically equivalent by the US Food and Drug Administration (FDA) owing to a lack of bioequivalence data. Serum samples were obtained from 0 to 34 h after dosing. Mean time of maximum serum concentration (T(max)) ranged from 1.3 to 1.4 h. Mean values for the maximum serum concentration (C(max)) and the area under the serum concentration-time curves (AUC) differed by <5% for the three products. The confidence limits for Ln-transformed C(max) and AUC ranged from >/=89 to </=113%. It was concluded that the three products were bioequivalent. In addition, the rapid in vitro dissolution of these formulations, as well as the reported high solubility and high permeability of theophylline, was predictive of the lack of any bioavailability differences among the three products.

Adult↗
PubMed1999

Three-dimensional reconstruction of pulmonary arteries in plexiform pulmonary hypertension using cell-specific markers. Evidence for a dynamic and heterogeneous process of pulmonary endothelial cell growth.

The plexiform lesions of severe pulmonary hypertension (PH) are complex vascular structures composed primarily of endothelial cells. In this study, we use immunohistochemical markers to identify the various cell layers of pulmonary vessels and to identify different endothelial cell phenotypes in pulmonary arteries affected by severe PH. Our computerized three-dimensional reconstructions of nine vessels in five patients with severe PH demonstrate that plexiform (n = 14) and concentric-obliterative (n = 6) lesions occur distal to branch points of small pulmonary arteries. And, whereas plexiform lesions occur as solitary lesions, concentric-obliterative lesions appear to be only associated with, and proximal to, plexiform structures. The endothelial cells of plexiform lesions express intensely and uniformly the vascular endothelial growth factor (VEGF) receptor KDR and segregate phenotypically into cyclin-kinase inhibitor p27/kip1-negative cells in the central core of the plexiform lesion and p27/kip1-positive cells in peripheral areas adjacent to incipient blood vessel formation. Using immunohistochemistry and three-dimensional reconstruction techniques, we show that plexiform lesions are dynamic vascular structures characterized by at least two endothelial cell phenotypes. Plexiform arteriopathy is not merely an end stage or postthrombotic change--it may represent one stage in an ongoing, angiogenic endothelial cell growth process.

Actins↗
PubMed1999

The corrosion behaviour of Ti-6Al-4V, Ti-6Al-7Nb and Ti-13Nb-13Zr in protein solutions.

Ti alloys are used in orthopaedic applications owing to their appropriate mechanical properties and their excellent corrosion resistance. The release of titanium and the other alloying elements into the surrounding tissue has been reported due either to passive corrosion or accelerating processes such as wear. Since the passive layer can be broken down in certain circumstances by wear it is important to study the ability of these alloys to repassivate in biological environments, in particular in the presence of proteins, and evaluate how the repassivated surface may vary from the original surface. In this study we investigated the ability of Ti-6Al-4V, Ti-6Al-7Nb and Ti-13Nb-13Zr to repassivate in phosphate buffered saline (PBS), bovine albumin solutions in PBS and 10% foetal calf serum in PBS at different pH values and at different albumin concentrations. It was found that an increase in pH had a greater effect on the corrosion behaviour of Ti-6Al-4V and Ti-6Al-7Nb than on Ti-13Nb-13Zr in PBS and that the addition of protein to the PBS reduced the influence of pH on the corrosion behaviour of all the alloys. The effect of the corrosion and repassivation was investigated by measuring changes in the surface hardness of the alloys and it was found that corrosion reduced the hardness of the surface oxides of all the alloys. In PBS the reduction was smallest for Ti-6Al-4V and largest for Ti-13Nb- 3Zr and that corrosion in protein solutions further reduced the hardness of the surface oxides. This effect was greater for Ti-6Al-4V and Ti-6Al-7Nb than for Ti-13Nb-13Zr. In conclusion, proteins in the environment appear to interact with the repassivation process at the surface of these alloys and influence the resulting surface properties.

Alloys↗
PubMed1999

Conjoint corrosion and wear in titanium alloys.

When considering titanium alloys for orthopaedic applications it is important to examine the conjoint action of corrosion and wear. In this study we investigate the corrosion and wear behaviour of Ti-6Al-4V, Ti-6Al-7Nb and Ti-13Nb-13Zr in phosphate buffered saline (PBS), bovine albumin solutions in PBS and 10% foetal calf serum solutions in PBS. The tests were performed under four different conditions to evaluate the influence of wear on the corrosion and corrosion on the wear behaviour as follows: corrosion without wear, wear-accelerated corrosion, wear in a non-corrosive environment and wear in a corrosive environment. The corrosion behaviour was investigated using cyclic polarisation studies to measure the ability of the surface to repassivate following breakdown of the passive layer. The properties of the repassivated layer were evaluated by measuring changes in the surface hardness of the alloys. The amount of wear that had occurred was assessed from weight changes and measurement of the depth of the wear scar. It was found that in the presence of wear without corrosion the wear behaviour of Ti-13Nb-13Zr was greater than that of Ti-6Al-7Nb or Ti-6Al-4V and that in the presence of proteins the wear of all three alloys is reduced. In the presence of corrosion without wear Ti-13Nb-13Zr was more corrosion resistant than Ti-6Al-7Nb which was more corrosion resistant than Ti-6Al-4V without proteins whereas in the presence of protein the corrosion resistance of Ti-13Nb-13Zr and Ti-6Al-7Nb was reduced and that of Ti-6Al-4V increased. In the presence of corrosion and wear the corrosion resistance of Ti-13Nb-13Zr is higher than that of Ti-6Al-7Nb or Ti-6Al-4V in PBS but in the presence of proteins the corrosion resistance of Ti-13Nb-13Zr and Ti-6Al-7Nb are very similar but higher than that of Ti-6Al-4V. The wear of Ti-13Nb-13Zr is lower than that of Ti-6Al-7Nb and Ti-6Al-4V with or without the presence of proteins in a corrosive environment. Therefore the overall degradation when both corrosion and wear processes are occurring is lowest for Ti-13Nb-13Zr and highest for Ti-6Al-4V and the presence of proteins reduces the degradation of all three alloys.

Alloys↗
PubMed1999

trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets.

1. The phytoestrogenic compound trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. 3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 microM. trans-Resveratrol at 0.1, 1.0 and 10.0 microM produced 20+/-6, 37+/-6 and 57+/-4% inhibition respectively of the effect of thrombin (0.01 u ml(-1)) to increase [Ca2+]i. 4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 microM trans-resveratrol producing 10+/-5, 30+/-5 and 50+/-7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. 5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. 6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. 7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.

Adult↗
PubMed1999

Practical skills and valued community outcomes: the next step in community-based education.

OBJECTIVES: Community-based medical education (CBE) has clear value. However, there are aspects of CBE where improvement is possible. First, communities do not generally receive valued outcomes in exchange for participation in the CBE process. Secondly, students are usually not trained to influence health in the community using methods that are realistic in busy clinical practice. DESIGN: A CBE rotation was designed to address these problems. Rotation activities were structured to facilitate development of a health programme desired by the community while giving students practical skills for later use. Working with community residents and health staff, sequential groups of students carried out, in turn, problem analysis, resource identification, planning and implementation activities aimed at establishing a community tuberculosis (TB) control programme. SETTING: The University of Natal in Durban, South Africa. SUBJECTS: Final-year medical students. RESULTS: At the end of the academic year, the TB control programme was approximately 60% in place, and 90% of TB patients cared for by the students were completing treatment. Overall, students rated the experience good for learning about health care in community settings and about methods for community health programme development. Student ratings were significantly higher for those groups whose activities brought them into greater contact with community residents. The 'real-time' nature of planning the sequential student groups' work created logistical problems and, as an isolated activity, the rotation had little impact on student attitudes toward community-based careers. CONCLUSIONS: Expanding the goals for CBE is both feasible and important. Further work should focus on refining designs for this next step in CBE.

Community Health Planning↗
PubMed1999

Development of in vivo bioequivalence methodology for dermatologic corticosteroids based on pharmacodynamic modeling.

BACKGROUND: Dermatologic corticosteroid products produce skin blanching that is related to clinical potency and dose. (For application of the vasoconstrictor assay to bioavailability and bioequivalence assessment, dose is defined in terms of duration of treatment exposure [dose duration], so the terms dose and dose duration have been used interchangeably). The vasoconstrictor assay is the method of choice to assess dermatologic corticosteroid products bioequivalence if dose-response is validated. This article examines dose-response validation to meet objectives of US Food and Drug Administration (FDA) bioequivalence guidance for dermatologic corticosteroid products. METHODS: An exploratory dose-response study was conducted to determine applicability of the empirical maximum effect (Emax) model to the individual subject and population dose-response relationships of six dermatologic corticosteroid product creams that varied from the most to the least potent classes. Products were applied to the skin of 10 healthy subjects in each of two dosing periods for dose durations of 0.5, 1, 2, and 6 hours. Skin blanching was measured by reflectance colorimeter through 24 hours after application. Area under the effect curve (AUEC) was determined for each dose duration. An Emax model was fitted to the AUEC versus dose duration data. A similar analysis was conducted for a bioequivalence study on two formulations of a dermatologic corticosteroid product in 40 healthy subjects. RESULTS: In the exploratory study, the number of individual subject data sets for which the Emax model provided an acceptable fit generally increased with the potency of the dermatologic corticosteroid product. On the basis of population modeling, dose-response data of all products, except the lowest potency cream, were adequately described by the Emax model. Values for population ED50 (the dose duration required to achieve 50% of the fitted AUECmax value) decreased with increase in dermatologic corticosteroid product potency. CONCLUSIONS: Acceptable model fits to all individual subject dose-response data were not achieved for any dermatologic corticosteroid product. However, population dose-responses were adequately described by the Emax model. On the basis of these data, the optimal dose duration used for comparison of multisource dermatologic corticosteroid products is recommended to be equal to the ED50 based on population modeling of pilot dose-response study data.

Adrenal Cortex Hormones↗
PubMed1999

Evaluation of the test system used for in vitro release of drugs for topical dermatological drug products.

The purpose of this research was to evaluate different parameters that can influence in vitro drug release from topical dermatological drug products such as creams, gels, and ointments. In vitro release from topical dermatological drug products was carried out by using a static diffusion cell, a synthetic membrane, and an appropriate receptor medium. The receptor medium was mixed by means of a magnetic bar rotated at 400 rpm. Experiments were designed to evaluate the influence of (i) receptor media, (ii) different lots of synthetic membranes, and (iii) agitation on drug release. Release experiments were also carried out to study inter- and intralot variability. The in vitro release test was used to evaluate the marketed glucocorticoid products. Among the parameters studied, the receptor medium was found to be the most important and critical variable that influenced drug release. The release rate ranged between 0.61 and 2.68 micrograms/cm2/min0.5 for betamethasone dipropionate, depending on the percentage of ethanol in the receptor medium. The drug release was not influenced by agitation or by different lots of synthetic membranes. Very small inter- and intralot variability was observed. These experiments establish the ruggedness of the in vitro diffusion cell test system used for the drug release measurements.

Administration, Topical↗
PubMed1999

MR imaging of intraventricular silicone: case report.

A 42-year-old man with human immunodeficiency viral infection developed cytomegaloviral retinitis that was complicated by retinal detachment and was treated with an intravitreous injection of silicone. Fifteen months later, magnetic resonance imaging revealed intraocular and intraventricular silicone. Signal intensity characteristics and chemical shifts of silicone in the two locations were identical.

AIDS-Related Opportunistic Infections↗
PubMed1999

Assessment of the quality and quantity of drug-drug interaction studies in recent NDA submissions: study design and data analysis issues.

This report investigates the quality and quantity of drug-drug interaction studies in recent new drug applications (NDAs). Eighty-nine studies contained in 14 NDAs submitted between December 1995 and November 1996 to the U.S. Food and Drug Administration (FDA) were reviewed. The results indicated that the median number of clinical drug-drug interaction studies per NDA was 6, almost double that of a 1994-1995 survey. In vitro metabolism data were present in 70% of the submissions. More than 50% of the submissions contained interaction studies using a battery of drugs (cimetidine, digoxin, or warfarin) without optimal use of the in vitro metabolism or in vivo mass balance data. Various study designs using a median number of 12 subjects were employed in the evaluation of drug-drug interactions. Some of the important study design factors such as dose size, dosing regimen, dosing duration, and timing of coadministration were considered, although not consistently, by the sponsors in their study design. Seventy-five percent of the studies used normal, healthy male subjects, and 25% used patients for whom the new molecular entities were intended. In 33% of the studies, female subjects were also recruited. Although the majority (80%) of the submissions still used p-values to determine the significance of drug interactions, 30% used a more relevant equivalence approach with 90% confidence intervals for key pharmacokinetic and/or pharmacodynamic parameter ratios to assess the extent of drug interactions. Overall, 82% of the studies concluded no interaction. Although population pharmacokinetic analysis can be a useful tool in studying drug-drug interactions, only 21% of the submissions used this approach. In summary, this assessment reveals that the quantity and quality of drug-drug interaction studies in NDAs have improved over the years. These improvements, as well as others that can be implemented, should result in more informative labeling and better patient care. FDA guidance for industry dealing with the design, analysis, and labeling language of in vivo metabolic drug-drug interactions has been developed to assist sponsors and FDA reviewers with these issues.

Clinical Trials as Topic↗
PubMed1999

Fungal spinal osteomyelitis in the immunocompromised patient: MR findings in three cases.

The MR imaging findings of fungal spinal osteomyelitis in three recipients of organ transplants showed hypointensity of the vertebral bodies on T1-weighted sequences in all cases. Signal changes and enhancement extended into the posterior elements in two cases. Multiple-level disease was present in two cases (with a total of five intervertebral disks involved in three cases). All cases lacked hyperintensity within the disks on T2-weighted images. In addition, the intranuclear cleft was preserved in four of five affected disks at initial MR imaging. MR features in Candida and Aspergillus spondylitis that are distinct from pyogenic osteomyelitis include absence of disk hyperintensity and preservation of the intranuclear cleft on T2-weighted images. Prompt recognition of these findings may avoid delay in establishing a diagnosis and instituting treatment of opportunistic osteomyelitis in the immunocompromised patient.

Aspergillosis↗
PubMed1999

Acute spontaneous spinal epidural hematomas.

BACKGROUND AND PURPOSE: Although previous reports have characterized MR imaging features of spinal epidural hematomas (EDH), few cases have been reported during the acute or hyperacute phase within the first 48 hours. Our goal in this investigation was to correlate the MR imaging features of acute (< or =48 hours) spontaneous EDH with clinical management and outcome. METHODS: Eight patients with acute spontaneous EDH (five men and three women; age range, 31-81 years) underwent MR imaging at 1.5 T (T1-weighted, n = 8; T1-weighted after the administration of 0.1 mmol/kg contrast material, n = 6; T2-weighted, n = 8; and T2-weighted, n = 4). The interval from symptom onset to hospital admission ranged from immediate to 5 days. Two neuroradiologists reviewed the MR images for signal characteristics, contrast enhancement, and cord compression. Treatment and clinical outcome were correlated with the imaging findings. RESULTS: The EDH were located in the cervical (n = 3), cervicothoracic (n = 2), thoracolumbar (n = 2), and lumbar (n = 1) regions. On T1-weighted images, the signal intensity of the EDH was isointense to spinal cord in five cases, hyperintense in two cases, and hypointense in one case and did not correlate with time to imaging. Isointensity on T1-weighted images persisted for 5 days in one case. On T2-weighted images, all EDHs were hyperintense with focal, heterogeneous hypointensity. Cord compression was severe in six patients, moderate in one patient, and minimal in one patient. Four cases were treated conservatively with complete resolution or improvement of symptoms within 1 to 3 weeks. CONCLUSION: MR imaging findings were useful in establishing the diagnosis of EDH but did not influence management or predict outcome in this series. Heterogeneous hyperintensity to cord with focal hypointensity on T2-weighted images should suggest the diagnosis of acute spinal EDH. Severity of neurologic impairment had the greatest impact on management and outcome. Nonoperative treatment may be successful in cases with minimal neurologic deficits, despite cord compression revealed by MR imaging.

Acute Disease↗