Determination of the critical size for non-healing defects in the mandibular bone of sheep. Part 2: Healing of 12 mm circular defects after 16 weeks.
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Animals↗
Biomedical subjects
J Marshall
Publications and source records attributed to J Marshall.
Localization of the site of major resistance to fluid transport in Bruch's membrane.
PURPOSE: To determine the site of highest resistance to the movement of water across Bruch's membrane in humans. METHODS: A hydraulic conductivity chamber was designed that enabled us to measure flow across Bruch's membrane while ablating its subepithelial aspect using an excimer laser (193 nm). When resistance was lost, samples were fixed and processed for electron microscopy. RESULTS: Changes in the hydraulic conductivity of Bruch's membrane in response to the excimer-mediated sequential removal of tissue layers was studied in four control eyes of donors 26, 46, 61, and 76 years of age and in one eye of an 83-year-old donor with age-related macular degeneration. The number of laser pulses required to abolish the resistance in Bruch's membrane was found to be dependent on the age of the donor. The ablation rate was approximately 0.11 micron per pulse. Loss of resistance correlated with removal of layers internal to the layer of elastin. CONCLUSIONS: This study indicates that the inner collage-nous layer imparts the major resistance to fluid movement between the retinal pigment epithelium and the choroid. Aging changes in the ultrastructure of these compartments could be responsible for the decrease in hydraulic conductivity observed in early life in previous studies.
Adult↗
Technique for efficient information retrieval in outpatient systems.
In the era of managed care, quality of medical care standards continue to materialize. Most of these standards have long, cumbersome, and complex rules. In light of such problematic rules, efficient ways of retrieving information for a computerized score card are needed. A technique for making such rules less difficult to use is to create Boolean expressions for each quality of care indicator. These Boolean expressions partition the indicators into key words and phrases so that information can be retrieved readily from a system. This study incorporates an outpatient clinical information system of a major university hospital. The technique used to retrieve information and related issues are discussed in the following text.
Ambulatory Care↗
An evolutionarily conserved binding site for serine proteinase inhibitors in large conductance calcium-activated potassium channels.
Complementary DNA coding for the channel-forming alpha-subunit of a large conductance Ca(2+)-activated K+ channel (maxi Kca channel) was cloned from bovine aortic smooth muscle cells. This cloned mammalian KCa channel (Bslo) and its homolog from Drosophila (Dslo) were expressed in the HEK293 human embryonic kidney cell line. Both Bslo and Dslo KCa channels were sensitive to inhibition by the internally applied serine proteinase inhibitors: bovine pancreatic trypsin inhibitor (BPTI, KD = 7.0 microM for Bslo and 2.6 microM for Dslo) and chicken ovoinhibitor (OI, KD = 1.5 microM for Bslo and 11.4 microM for Dslo). BPTI and OI are members of the Kunitz and Kazal families of proteinase inhibitors, respectively. The approximately 60-residue inhibitory domains of these proteins have a different tertiary structure except in the region of a loop formed by approximately 6 residues, in which the peptide backbone adopts a similar conformation complementary to the active site cleft of many serine proteinases. At the single-channel level, BPTI and OI were found to inhibit KCa channels by a similar mechanism involving the production of discrete low-conductance events. These two inhibitors also exhibited competitive behavior, suggesting that they bind to an overlapping site. Kinetic characterization revealed that the dissociation rate of BPTI from the bovine KCa channel is fast (k(off) = 0.41 s-1), whereas that from the Drosophila KCa channel is slow (k(off) = 9.0 x 10(-4) s-1) and indicative of a strong molecular interaction. The stable complex of BPTI and trypsin was inactive as a KCa channel inhibitor, further supporting the idea that the trypsin inhibitory loop of BPTI recognizes a specific site on the channel protein. These results lead to the conclusion that the alpha-subunit of maxi KCa channels contains a conserved proteinase inhibitor binding site. We hypothesize that this site corresponds to a C-terminal domain of the channel protein that structurally resembles serine proteinases.
Amino Acid Sequence↗
A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.
Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.
Administration, Intranasal↗
Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.
Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.
Adenoviruses, Human↗
Component characteristics of the vectorial transport system for taurine in isolated bovine retinal pigment epithelium.
1. A wide range of substrate concentrations (5-1600 microM) were used to screen for the presence of systems capable of transporting taurine into isolated and free-floating samples of bovine retinal pigment epithelium (RPE). Both high and low affinity systems displaying Michaelis-Menten saturation kinetics were identified. The high affinity system was characterized by a K(m) of 23 microM and a V(max) of 86.7 pmol (5 min)(-1) (4 mm disc of tissue sample)(-1). Similarly, the low affinity system was characterized by a K(m) of 507 microM and a V(max) of 344 pmol (5 min)(-1)(4 mm disc)(-1). 2. Ussing-type incubation chambers and double-label radiotracer techniques were used to assess the presence of specific taurine carriers on apical and basolateral surfaces of the RPE. High affinity carriers were shown to be present on both surfaces and the kinetic constants (K(m) and V(max)) for apical and basolateral systems were determined as 23.2 microM and 34.8 pmol (5 min)(-1) (4 mm disc)(-1) and 29 microM and 54.7 pmol (5 min)(-1)(4 mm disc)(-1), respectively. Both these high affinity systems were sodium dependent with a Hill coefficient of about 2.0 indicating that two sodium ions are required for the translocation of one molecule of taurine. The low affinity system was unevenly distributed over the two surfaces of the RPE, basolateral capacities being roughly twofold higher. The basolateral system was totally insensitive to sodium whereas the apical one with 50% sodium sensitivity suggested the presence of low affinity carrier heterogeneity. 3. A temperature-dependent mechanism for the release of pre-loaded taurine from bovine RPF was also demonstrated. 4. The effect of [K+]o trans-RPF gradients on the vectorial transport of taurine across the isolated preparation was also investigated. The results demonstrated that the direction and magnitude of taurine transport could be controlled by physiological variations in the extracellular concentration of potassium. 5. The determined kinetic parameters of the carriers were used to construct a working model of the vectorial pathway for the translocation of taurine across bovine RPE. This estimated the level of free intracellular taurine to be in the micromolar range. However, direct measurements of total RPE taurine by high performance liquid chromatography showed levels of 19.5 +/- 3.6 mM indicating that the major taurine pool in bovine RPE exists in the bound form. The model also showed that the magnitude and direction of net transport was a function of the transepithelial taurine gradient.
Animals↗
The role of the renin-angiotensin system in the renal response to moderate hypoxia in the rat.
1. In two groups of Saffan-anaesthetized rats, we studied the role of the renin-angiotensin system in mediating the antidiuresis and antinatriuresis induced by moderate systemic hypoxia. 2. In both groups, a first period of hypoxia (breathing 12% O2 for 20 min) induced a fall in arterial partial pressure of O2 (Pa,O2; to 42 mmHg), a fall in mean arterial pressure (MABP), no change in renal blood flow (RBF) due to an increase in renal vascular conductance (RVC = RBF/MABP) and falls in urine flow and absolute sodium excretion (UNaV). Concomitantly, plasma renin activity increased from 3.08 +/- 0.68 (mean +/- S.E.M.) to 8.36 +/- 1.8 ng ml-1 hr-1. 3. In group 1 (n = 11), Losartan (10 mg kg-1, I.V.), the angiotensin (AII) AT1 receptor antagonist, induced a fall in MABP (115 +/- 3 to 90 +/- 3 mmHg), an increase in RVC such that RBF was unchanged, and falls in glomerular filtration rate (GFR), urine flow and UNaV. However, hypoxia induced qualitatively similar changes to those seen before Losartan treatment. 4. In group 2 (n = 9), we occluded the aorta distal to the renal artery to prevent basal MABP and renal perfusion pressure (RPP) from falling after addition of Losartan and to keep the hypoxia-induced fall in MABP the same as before Losartan treatment. Nevertheless, Losartan induced an increase in basal RVC, RBF, urine flow and UNaV whilst hypoxia induced falls in urine flow and UNaV that were proportionately similar to those seen prior to addition of Losartan. 5. These results indicate that in the Saffan-anaesthetized rat, AII exerts tonic, renal vasoconstrictor and consequent antidiuretic and antinatriuretic influences in normoxia, but does not contribute to the hypoxia-induced antidiuresis and antinatriuresis. We propose that renin secretion is increased by the hypoxia-induced fall in RPP rather than by an increase in renal sympathetic activity. Thus, the AII generated cannot produce antidiuresis and antinatriuresis by its known facilitatory influence on the actions of an increase in sympathetic activity on the renal tubules and is insufficient to produce these effects by direct actions. Rather, these results support the view that the antidiuresis and antinatriuresis of moderate hypoxia is predominantly due to the fall in RPP.
Animals↗
Genetic testing for colon cancer susceptibility: Anticipated reactions of patients and challenges to providers.
The commercial availability of genetic tests for colon cancer susceptibility is creating new opportunities and challenges for both patients and providers. To provide information useful in the education and counseling of individuals considering genetic testing, we conducted structured interviews with 45 male and female first-degree relatives of colorectal cancer patients. Fifty-one percent of respondents indicated that they definitely would want to obtain a genetic test for colon cancer susceptibility when it is available and 31% said that they probably would want to be tested. Interest in genetic testing was significantly higher among persons with less formal education and those with a Catholic religious preference. Motivations for genetic testing included the following: to know if more screening tests are needed, to learn if one's children are at risk and to be reassured. Barriers to testing included concerns about insurance, test accuracy and how one's family would react emotionally. Most participants anticipated that they would become depressed and anxious if they tested positive for a mutation, while many would feel guilty and still worry if they tested negative. Of note, about one-half of respondents expected that they would decrease their use of screening tests and make fewer attempts to reduce dietary fat if they tested negative. These preliminary results underscore the importance of educating patients about the potential risks, benefits and limitations of genetic testing, with particular emphasis on the possibility of adverse psychological effects and implications for health insurance. The potential for false reassurance following a negative test result should be addressed by emphasizing the residual risks of cancer among non-carriers of predisposing mutations.
Adolescent↗
Hydrodynamics of ageing Bruch's membrane: implications for macular disease.
The hydrodynamic properties of isolated human Bruch's membrane and choroid were investigated as a function of age and retinal location. Macular and peripheral regions of the fundus showed an exponential decline of hydraulic conductivity with half-lives of 15 and 22 years respectively. Comparison of age profiles for hydraulic conductivity and lipid deposits suggests the involvement of two discrete processes for reduction in transport capability. The first appears to involve 'membrane remodelling' with a programmed decay rate leading to a major reduction in hydraulic conductivity by the fifth decade of life. The second commences in the fourth decade and is apparently dependent on the lipid content of Bruch's membrane.
Adolescent↗
Phase I trial of a novel matrix metalloproteinase inhibitor batimastat (BB-94) in patients with advanced cancer.
Degradation of basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be required for tumor invasion, tumor-induced angiogenesis and vascular invasion. A synthetic hydroxamate, batimastat (also known as BB-94), inhibits MMPs by binding the zinc ion in the active site of the MMP. Batimastat inhibits at least 50% of MMP activity at concentrations less than or equal to 10 ng/ml in vitro. Batimastat retarded ascites accumulation and increased survival in mice with human ovarian tumor xenografts. Acute and long-term toxicological studies revealed no major toxicity in animals. Batimastat is poorly soluble and was administered intraperitoneally (i.p.) as a suspension. Previous studies in patients with malignant ascites have shown no major toxicities at doses as high as 1350 mg/m2.
Adult↗
Biologic and environmental risk factors for regression after photorefractive keratectomy.
BACKGROUND/PURPOSE: After photorefractive keratectomy (PRK), the final refraction in the majority of patients is near the intended refraction. However, a significant proportion of patients show regression, which is commonly associated with corneal haze. This study aims to determine whether biologic and environmental factors influence the outcome of PRK. METHODS: One hundred patients prospectively underwent -3.00- or -6.00-diopter corrections. Three months after surgery, 100% of patients answered a questionnaire that recorded their biologic characteristics, medical and ocular history, and postoperative exposure to physical and chemical insults. Multivariable analysis identified those factors associated with regression. RESULTS: Multivariable analysis showed that the risk of regression was increased significantly in patients who underwent higher dioptric or smaller diameter treatments (P = 0.045) and in those who had had regression after treatment of the first eye (P = 0.019). There was no difference between the sexes, but regression was 13.5 times more likely in females who were taking oral contraceptives. Regression was increased in those exposed to solar radiation (odds ratio, 7.6; 95% confidence interval, 1.82-32.22) and sun beds (odds ratio, 2.4; 95% confidence interval, 0.64-9.39), and was significantly greater in patients with ocular-surface disorders (on univariable analysis only; P = 0.034). Regression was not associated with previous contact lens wear, swimming, cigarette smoking, or minor ocular trauma. CONCLUSION: The questionnaire identified biologic and environmental risk factors for regression. The biologic risk factors may be used preoperatively to counsel patients and, where necessary, advise them against surgery. The environmental risk factors are largely avoidable by modification of patient behavior.
Adult↗
An in vivo investigation of the structures responsible for corneal haze after photorefractive keratectomy and their effect on visual function.
PURPOSE: To make serial measurements of corneal haze and microscopic anatomy after photorefractive keratectomy (PRK) and compare the results with visual function measured at the same time points in the same single group of human subjects. METHODS: Ten patients underwent -6.00-diopter, 6-mm PRK. The patients were reviewed frequently for 12 months. Corneal haze was measured objectively in two ways: (1) an opacification index was determined from the variance in digitized retroillumination images; and (2) light reflected and scattered back from the cornea was assessed by gray-scale analysis of video slit images. In vivo confocal microscopy recorded the anatomic changes occurring in the cornea, and computer analysis of the images quantified the keratocytes and subepithelial deposit. Visual performance was assessed by Snellen visual acuity, contrast sensitivity, and glare-induced visual dysfunction. RESULTS: In the first week, epithelial irregularity resulted in a transient reduction in all aspects of visual function. In the first month, keratocyte disturbances reduced contrast sensitivity at high frequencies and produced glare. Over the next couple of months, the subepithelial deposit resulted in a more prolonged loss of contrast sensitivity at low frequencies and glare-induced visual dysfunction due to the scattering of light. In several patients, these visual defects persisted after 1 year. CONCLUSIONS: Epithelial and keratocyte disturbances only transiently affect visual function. The subepithelial deposit is more persistent and can have a lasting effect on visual performance. Therefore, attempts to improve the visual outcome of PRK must be aimed at controlling the synthesis of subepithelial material.
Adult↗
Postoperative inflammatory response to phacoemulsification and extracapsular cataract surgery: aqueous flare and cells.
PURPOSE: To compare the postoperative blood-aqueous barrier (BAB) breakdown induced by phacoemulsification with continuous curvilinear capsulorhexis (CCC) and by extracapsular cataract extraction (ECCE) with a linear capsulotomy. SETTING: Cataract and Refractive Surgery Research Unit, Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. METHODS: Anterior chamber flare and cells were measured preoperatively and 1 day, 1 week, and 1 and 3 months postoperatively in two parallel groups of 31 consecutive cataractous eyes. In Group 1, one surgeon performed ECCE with a linear capsulotomy; in Group 2, a second surgeon performed divide and conquer phacoemulsification with CCC. The preoperative, intraoperative, and postoperative medication regimen was the same in both groups. RESULTS: Group 2 eyes had significantly lower anterior chamber flare and cell measurements in the first postoperative month than Group 1 eyes (.01 < P < .00001). CONCLUSIONS: Phacoemulsification with CCC induced a less severe BAB breakdown than ECCE with a linear capsulotomy. Phacoemulsification with CCC may be preferable in high-risk eyes such as those with glaucoma, diabetes, or uveitis, which are prone to complications resulting from postoperative BAB breakdown.
Aged↗
Cellular reaction on the anterior surface of poly(methyl methacrylate) intraocular lenses.
PURPOSE: To assess the cellular reaction on the anterior surface of poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) implanted by phacoemulsification with continuous curvilinear capsulorhexis (CCC) or by extracapsular cataract extraction (ECCE) with a linear capsulotomy. SETTING: Cataract and Refractive Surgery Research Unit. Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. METHODS: To document morphology, topography, and severity of the cellular reaction, specular microscopy of the anterior IOL surface was performed at 1 day, 1 week, and 1 and 3 months postoperatively in two parallel groups of 31 consecutive cataractous eyes operated on by phacoemulsification with CCC or by ECCE with a linear capsulotomy. RESULTS: The local tissue response consisted of a nonspecific foreign-body reaction to the IOL and a lens epithelial cell reaction. The foreign-body reaction was significantly less severe in the phacoemulsification group than in the ECCE group, and the number of IOLs without inflammatory cells was significantly higher. CONCLUSION: The foreign-body reaction to PMMA IOLs is significantly reduced when the lens is implanted by phacoemulsification with CCC. This could be of clinical benefit in high-risk eyes prone to the inflammatory complications of cataract surgery.
Cataract Extraction↗
Hypothesis for a serine proteinase-like domain at the COOH terminus of Slowpoke calcium-activated potassium channels.
Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.
Amino Acid Sequence↗
Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.
In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.
Amino Acid Sequence↗
Identification of the major starch synthase in the soluble fraction of potato tubers.
The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. Immunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for approximately 80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.
Amino Acid Sequence↗