Biomedical subjects

K A Williams

Publications and source records attributed to K A Williams.

At least 55 records · Page 3Linked to original sources

PubMed1996

Survival of rabbit limbal stem cell allografts.

Failure of a specialized population of corneal epithelial stem cells found in the peripheral cornea and limbus results in ocular surface disease, which may be amenable to treatment by transplantation of limbal tissue. This study was designed to investigate donor limbal stem cell allograft survival in rabbits with ocular surface disease. Rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal stem cell allotransplantation, by allotransplantation plus topical steroid, or by topical steroid only (n = 7 for each group). Donors and recipients were sex mismatched. Recipients were followed for up to 5 months. Outcome was assessed by daily slit-lamp examination, weekly impression cytology and photographic record, end-point sex chromatin and fluorescent cell tracer analyses, histology, and immunohistochemistry. In no case was a completely normal ocular surface regained, but some animals that received grafts plus corticosteroids fared best by all criteria used. In the absence of immunosuppression, graft hemorrhagia (believed to be a manifestation of graft rejection) occurred within the first month, the cornea became resurfaced with conjunctiva-derived cells, and no donor cells survived centrally in the long term. Topical corticosteroids reduced the number and severity of these episodes significantly, and were associated with survival of some donor-derived cells in the central cornea of some grafted animals. Thus, rabbit limbal stem cell allografts appeared to undergo rejection, which could be modified by immunosuppression, but useful regeneration of the ocular surface occurred only where rejection was circumvented.

Animals↗

PubMed1996

Biophysical characterization of wild-type and mutant bacteriophage IKe major coat protein in the virion and in detergent micelles.

Interactions between the filamentous bacteriophage major coat protein and its environment differ markedly between the membrane-bound assembly intermediate which spans the lipid bilayer and the phage coat protein which makes up the capsid of the virion. Nonetheless, both reflect successful strategies to sequester the hydrophobic regions of the coat protein away from the aqueous milieu. To characterize the roles of individual residues in the conformation, stability, and oligomerization of the coat protein in both the virion and in detergent micelles, wild-type IKe and M13 coat proteins, together with a library of over 40 IKe coat protein mutants, were studied using circular dichroism (CD), fluorescence, and solution nuclear magnetic resonance (NMR) spectroscopies. The largely helical conformations of coat protein in IKe wild-type and mutant virions were found to be very similar by CD, demonstrating that the overall organization of the phage can accommodate a diverse range of amino acid substitutions in the major coat protein. Intrinsic Trp fluorescence showed that the polarity of the Trp 29 environment in the virion was modulated by residues within one helical turn of this locus. Characterization of IKe phage growth and plaquing properties highlighted the importance of Pro 30 in maintaining viability. As well, the Pro 30 mutants were the only substitutions which rendered the detergent-solubilized coat protein less thermostable and additionally altered the polarity of the Trp 29 environment. The Pro 30 Gly mutant exhibited numerous 1H and 15N chemical shift changes between residues Ile 25 and Ala 38 in the 2D 1H-15N HSQC spectrum in myristoyllysophosphatidylglycerol (MPG) micelles, demonstrating that the effect of the substitution is propagated beyond adjacent residues. The overall results highlight the stabilizing effect of Pro in the first turn of a transmembrane helix and the importance of hydrophobicity in modulating the oligomerization and stability of coat protein both in the phage and in detergent micelles.

Amino Acid Sequence↗

PubMed1996

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Amino Acid Sequence↗

PubMed1996

Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR.

The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.

Amino Acid Sequence↗

PubMed1996

Comparison of technetium-99m sestamibi-gated tomographic perfusion imaging with echocardiography and electrocardiography for determination of left ventricular mass.

Left ventricular (LV) mass estimates obtained from post-stress gated single-photon emission computed tomographic (SPECT) perfusion images were compared with 2-dimensionally targeted M-mode echocardiograms and with resting electrocardiographic voltage in 32 patients with stress perfusion scans that were either normal or only mildly abnormal. Myocardial pixel volumes were obtained from SPECT transaxial slices at end-diastole, end-systole, and summed ("ungated") static reformatted SPECT images at 2 levels of background subtraction, 37.5% and 35% of peak myocardial activity. The S-wave amplitude in lead V1 and the R-wave amplitude in V5 were summed for an electrocardiographic index of voltage. Echocardiographic LV mass was calculated using the modified Penn convention formula. SPECT myocardial mass estimates were significantly greater at diastole when compared with systolic or summed images. There was a moderated, although highly significant, correlation between echocardiographic and SPECT indexes of LV mass with the lower (35%) background threshold (r = 0.59, 0.60, and 0.53 for diastole, summed, and systole, each p < 0.001). The diastolic SPECT estimate of LV mass correlation with electrocardiographic voltage (r = 0.56) was superior to the correlation between echocardiography and electrocardiography (r = 0.30). With use of published criteria for the presence of LV hypertrophy on echocardiography, diastolic and systolic gated SPECT predicted echocardiographic results with 78% accuracy.

Cardiac Volume↗

PubMed1996

Left ventricular function in patients with coronary artery disease assessed by gated tomographic myocardial perfusion images. Comparison with assessment by contrast ventriculography and first-pass radionuclide angiography.

OBJECTIVES: This study evaluated the use of gated single-photon emission computed tomographic (SPECT) myocardial perfusion images for determination of left ventricular ejection fraction. BACKGROUND: Gated SPECT has expanded the applications of myocardial perfusion imaging to include the evaluation of left ventricular size, regional wall motion and regional systolic thickening. Accurate automated or semi-automated methods for quantitation of left ventricular ejection fraction from tomographic perfusion images would provide additional valuable clinical information. METHODS: Rest gated SPECT was performed on the stress distribution of technetium-99m sestamibi, using eight frames per cardiac cycle. Mid-horizontal long-axis and vertical long-axis gated tomographic perfusion images were analyzed after digital matrix inversion, which enhances edge detection, for ejection fraction determination. These ejection fractions were compared with those determined by contrast ventriculography (n = 54, including 45 biplane and 9 single plane) and first-pass radionuclide angiography (n = 38) in patients with coronary artery disease. RESULTS: Myocardial perfusion SPECT image inversion-derived ejection fractions were slightly lower (2.7 ejection fraction units, p < 0.01), and first-pass ejection fractions were much lower (8.0 ejection fraction units, p < 0.001) than those obtained with contrast ventriculography. There was excellent correlation between SPECT and contrast ventriculographic ejection fractions (r = 0.93) over a wide range of ejection fractions (14% to 89%). Good correlation was also observed between first-pass radionuclide angiography and both contrast ventriculography (r = 0.83) and SPECT (r = 0.87). Reproducibility of SPECT image inversion ejection fractions was excellent (intraobserver r = 0.99, interobserver r = 0.93). CONCLUSIONS: Semiautomated ejection fractions can be obtained from gated SPECT technetium-99m sestamibi perfusion images using the image inversion technique. These results are reproducible and correlate well with results of first-pass radionuclide angiography but are closer in value to those obtained with contrast ventriculography.

Aged↗

PubMed1996

Factors affecting bovine corneal endothelial cell density in vitro.

AIMS: To examine factors influencing the density and contact inhibition of bovine corneal endothelial cells cultured in vitro. METHODS: Cell counts were performed on bovine corneal endothelial cells cultured for various times in the presence of 10% fetal calf serum, with or without varying concentrations of growth factors, 5% dextran T-500, or 2% chondroitin sulphate, at 32 degrees C or 37 degrees C, and after treatment with beta galactosidase. RESULTS: Both basic fibroblast growth factor (FGFb) and retinal crude extract (RCE), but neither epidermal growth factor (EGF) nor acidic fibroblast growth factor (FGFa), increased endothelial cell density in vitro (p < 0.05). Continuous exposure to RCE resulted in a higher cell density than did a 24 hour pulse (p < 0.01), and higher cell densities were achieved at 37 degrees C than at 32 degrees C (p < 0.0001). In the absence of RCE, dextran T-500 increased cell density modestly (p < 0.05); in the presence of RCE, the addition of dextran T-500 had no effect on final cell density, whereas chondroitin sulphate significantly decreased final cell density (p < 0.01). In the absence of exogenous growth factors, beta galactosidase treatment resulted in a 50% increase in final cell density compared with controls (p < 0.0001). CONCLUSIONS: Bovine corneal endothelial cell growth can be augmented under conditions different from those used in corneal preservation systems. The final cell density in a confluent monolayer can be increased by treatment with beta galactosidase, suggesting that corneal endothelial cells may be contact inhibited through a beta galactosidase sensitive receptor system.

Animals↗

PubMed1996

An intelligent learning environment for advanced cardiac life support.

Resuscitation from clinical cardiac arrest is complex and often takes several years to learn. This paper describes an intelligent simulation-based tutor for ACLS which increases students' opportunity to practice before, during and after the ACLS course, thus bridging the gap between studying theory and didactic textbook material and working with patients. Sophisticated reasoning about student performance, compared to an expert model, distinguishes this system from other computerized instruction systems. Intelligence in the tutor allows the system to make the simulation dynamically adaptive to focus on areas where the student's learning needs are greatest. A formative evaluation with two classes of fourth year medical students suggested that the tutor was helpful, realistic and effective. Positive reactions and strong student involvement with the simulation suggest that this simulation-based tutor may improve learning and retention while decreasing anxiety for most students.

Computer Simulation↗

PubMed1995

Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.

The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane.

Alkylation↗

PubMed1995

Experimental orthotopic corneal xenotransplantation in the rat. Mechanisms of graft rejection.

Orthotopic penetrating guinea pig to rat and chicken to rat corneal xenografts were performed to examine the nature of the host response. Guinea pig to rat xenografts failed at a median of day 3 after surgery. A similar but slightly accelerated pattern of failure was seen in guinea pig xenografts performed in prevascularized recipient rat corneas. Chicken to rat xenografts failed at a median of day 2 after grafting. Rat corneal isograft controls survived indefinitely. Corneal endothelial cells were visible by silver staining on the xenografts immediately after operation, which indicates that failure was not due to loss of these cells during surgery. Histopathology and immunoperoxidase staining indicated that xenograft failure in euthymic recipients was characterized by early corneal epithelial and endothelial cell damage, granulocytic infiltration, and hemorrhage from recipient corneal and iris capillaries, followed at 7-14 days by infiltration with T cells, macrophages, and eosinophils. An accelerated pattern of graft failure was also observed in guinea pig grafts into homozygous nude rat recipients, which suggests that preformed anti-donor antibody and complement were responsible for some of the early graft damage. Flow cytometry demonstrated the presence of pre-existing natural antibodies to guinea pig and chicken lymphocytes and erythrocytes, as expected from other studies. Immunohistochemistry showed the presence of rat IgG2a, IgG1, and IgM, but not IgD deposited on grafts in immunocompetent recipients on the first postoperative day. We conclude that orthotopic corneal xenografts undergo substantial accelerated damage mediated by pre-existing antibody, followed at 7-14 days by a cell-mediated response that causes further destruction.

Animals↗

PubMed1995

Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization.

Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8). This protein occurs as a membrane protein while stably inserted into its E. coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion. To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation. In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved. These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e. specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices.

Amino Acid Sequence↗

PubMed1995

Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft.

PURPOSE: The extent to which limbal epithelial stem cell allografts will repopulate the human corneal ocular surface, and the time frame over which such cells survive, are uncertain. We investigated the survival of donor-derived epithelial cells after limbal stem cell allotransplantation in a patient with bilateral limbal stem cell failure by using short tandem-repeat DNA polymorphisms to distinguish donor and recipient cells. METHODS: Epithelial cells were harvested by impression cytology from the grafted eye before and at various times after transplantation. DNA was extracted and amplified by the polymerase chain reaction at an informative locus, D8S264. RESULTS: Cells of donor genotype were present over the grafted areas at the time of surgery but were not detected in the central cornea until 12 weeks postoperatively, indicating that repopulation of the epithelial surface from transplanted limbal stem cells took considerable time. However, by the 20th postoperative week, only recipient-type cells were detected in the grafted eye, despite systemic immunosuppression of the recipient with azathioprine and cyclosporine. CONCLUSIONS: Discrimination between donor and recipient cells on the ocular surface after limbal allotransplantation was possible using genotypic variation at DNA polymorphic sites (microsatellites). Long-term survival of donor cells after limbal transplantation did not occur in this patient. Detection of DNA polymorphisms amplified by the polymerase chain reaction is a simple, rapid, and noninvasive method of following the course of transplanted cells at the ocular surface.

Adult↗

PubMed1995

How successful is corneal transplantation? A report from the Australian Corneal Graft Register.

Corneal graft outcome was assessed within a large, prospectively collected database of 4499 records. Penetrating corneal graft survival was 91% at 1 year, 72% at 5 years and 69% at 7 years. The three most common indications for graft were keratoconus (30%), bullous keratopathy (25%) and failed previous graft (18%); the three most common causes of graft failure were rejection (34%), infection (18%) and glaucoma (9%). The vast majority of grafts were performed for improved visual acuity. About four-fifths of recipients achieved at least one line of better acuity on the Snellen chart post-operatively; of the remainder with unchanged or worse acuity, only 21% had failed grafts. Overall, 43% of recipients achieved a best corrected Snellen acuity of 6/12 or better, 52% achieved 6/18 or better, and 20% had acuitities of less than 6/60. Reasons for poor post-operative acuity (recorded as less than 6/60) included graft failure (41%) and comorbidities in the grafted eye (43%). A number of risk factors for graft failure were examined: in most instances, there was little room for decision-making or expert intervention.

Astigmatism↗

PubMed1995

The host response in experimental corneal xenotransplantation.

In addressing the worldwide shortage of human donor cornea for transplantation, animal cornea may be a substitute if mechanisms of xenogeneic (cross-species) rejection can be identified and controlled. Xenotransplantation of solid organs is followed by hyperacute rejection with minutes due to humoral graft rejection. In an experimental model corneal xenografts in rats survived for 2-3 days, depending on the phylogenetic disparity of the donor animal. Endothelial injury was the specific cause of graft failure, probably mediated by humoral rejection mechanisms. A later cell-mediated rejection response was seen. The potent humoral response is the most important feature differentiating xenograft from allograft rejection.

Animals↗

PubMed1995

The effect of IL-4 on antigen expression: a comparison of fluorescence intensity and antigen density.

We have compared the membrane expression of MHC class II antigen and Ig on murine splenic B lymphocytes by two flow cytometric methods. The first method, commonly used, determined the mean immunofluorescence intensity; the second measured antigen density as a ratio of mean fluorescence intensity to cell volume. The data generated by the two methods allow for different interpretations of antigen density in comparison to antigen expression. By mean fluorescence intensity, activated B lymphocytes expressed more MHC class II antigen and membrane Ig than did resting B cells. However, resting B cells had the same density of MHC class II antigen and membrane Ig as activated B cells. Next we examined the effect of IL-4 on antigen expression by the two methods. We found that although IL-4 increased the mean fluorescence intensity and density of MHC class II and membrane Ig on each B cell population, the density of expression of membrane Ig tended to be greater on the resting B cells than on the activated, while the density of MHC class II antigen was essentially the same on each of the IL-4 treated B cell populations.

Animals↗

PubMed1995

Use of ultrasound to facilitate antibiotic diffusion through Pseudomonas aeruginosa alginate.

The effect of ultrasonic treatment of two pseudomonal alginate samples was studied by investigation of their rheological characteristics and interaction with tobramycin, piperacillin and ciprofloxacin. Whilst exposure to ultrasound at an amplitude of 20 microns for 8 min caused a significant decrease in viscosity of both alginates, molecular fragmentation, as demonstrated by PAGE and gel-permeation chromatography, was minimal. Antibiotic penetration was improved substantially however with sonication, leading to a 100% increase in tobramycin and piperacillin diffusion from the alginate compared with an untreated control polymer.

Alginates↗

PubMed1995

Interleukin 4 production by human amnion epithelial cells and regulation of its activity by glycosaminoglycan binding.

The pro-inflammatory molecules, tumor necrosis factor alpha (TNF alpha), interleukin 1 (IL-1), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), are postulated to have a role in human pregnancy and parturition. The ability of interleukin 4 (IL-4) to suppress the production of TNF alpha, IL-1, IL-6, and PGE2 by activated monocytes prompted us to investigate a possible regulatory role for IL-4 in human gestation. Immunohistochemical techniques were used to show that human amnion epithelium stained positively for IL-4. Tissue from both the first (n = 5) and third (n = 46) trimester expressed immunoreactive IL-4, which was detected by the use of four antihuman IL-4 monoclonal antibodies. Analysis of mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA extracts of amnion epithelial cells indicated that they were the source of IL-4. One of the anti-IL-4 antibodies used stained IL-4 protein associated with the basement membrane of the amnion epithelium. The mechanism of this association was investigated. IL-4 was shown to be a heparin-binding cytokine, which would enable it to bind to components of the extracellular matrix. Thus, this study identified a previously undescribed cellular source of IL-4, implicating a role for IL-4 in human gestation. Additionally, glycosaminoglycan binding may regulate IL-4 activity in vivo.

Amnion↗

PubMed1995

Surgical management of ocular surface disorders using conjunctival and stem cell allografts.

AIMS: The aim of this work was to investigate different surgical options for the repair of the ocular surface, using various extensions of the procedure of limbal stem cell allotransplantation. METHODS/RESULTS: Straightforward lamellar limbal transplantation was performed in one patient with contact lens induced limbal stem cell failure. A second patient with a neoplastic corneal lesion underwent limbal allotransplantation, followed later by a second procedure in which 360 degrees of limbus and the entire ocular surface was transplanted. A third patient who had suffered extensive chemical burns was treated by penetrating keratoplasty to restore central corneal clarity, followed later by a lamellar allograft comprising a 360 degrees annulus of peripheral cornea to repair the ocular surface. A fourth patient with long standing, chronic trachomatous eye disease underwent allotransplantation of the upper lid tarsal plate and conjunctiva, with reconstruction of the fornix. Finally, a child with Goldenhar's syndrome underwent reconstruction of the medial fornix with autologous buccal mucosa, followed by a lamellar corneal and conjunctival allograft. A stable ocular surface has been achieved in each case and there have been no obvious rejection episodes. CONCLUSION: Limbal allotransplantation can be extended to engraftment of the entire superficial cornea, limbus, conjunctiva, and tarsal plate in patients with a range of pathologies. We have described the surgical management of five cases which demonstrate the potential of the technique, but which raise questions which still need to be explored.

Adult↗