Biomedical subjects
Publications and source records attributed to M W Smith.
One of the most debated questions in the field of molecular evolution is the possible role of horizontal transfer in evolution. Of all the claims that have been made over the years, those reporting transfers between eukaryotes and prokaryotes are the most controversial. Here we present the cases for and against several such possible gene acquisitions.
"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.
We have employed the baculovirus expression system for the production of insect cell membranes having GABA/benzodiazepine binding sites. Three recombinant baculoviruses each harboring a different GABAA receptor cDNA were introduced into insect cells by simultaneous infection. Infected cells expressed GABA responsive Cl- channels and benzodiazepine binding sites with the same pharmacological specificity as animal cells expressing these receptor subunits.
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Quantitative methods of in situ hybridization and immunocytochemistry have been used to measure 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induction of calbindin mRNA and calbindin protein expressed in jejunal enterocytes at all points along the crypt-villus axis over a 24 h period. Small amounts of calbindin mRNA detected in vitamin D3 deficient (D-deficient) chick intestine increased rapidly to maximal values 8 h after hormone injection. The magnitude of this response was inversely related to age of enterocyte measured separately by injecting tritiated thymidine into D-deficient and 1,25(OH)2D3-injected birds. Enterocytes of all ages expressed small amounts of calbindin 3 h after hormone injection. This amount of calbindin then increased up to 24 h after hormone injection. Maximal calbindin expression took place in basal villus enterocytes. Later decrease in the ability of upper villus enterocytes to express calbindin was associated with a similar fall in calbindin mRNA expression. Previously it was suggested that inefficient translation to calbindin mRNA might take place in basal villus enterocytes 48 h after vitamin D injection. Present work using 1,25 (OH)2D3 shows that calbindin expression takes place at a constant rate during this early stage of enterocyte development. Secondary events limiting higher rates of calbindin synthesis in upper crypt and basal villus enterocytes remain to be identified.
Development of microvillus structure on the surface of enterocytes migrating over follicle-associated lymphoid tissue appears to take place in an identical way to that seen on villus enterocytes. M-cells, which are not found in crypts, possess microvilli of constant length in all parts of the follicle dome epithelial tissue. The surface area of M-cell microvilli is not however different from that determined on fully formed follicle-associated enterocytes. These results support the hypothesis that M-cell formation takes place throughout the dome epithelium by immune-induced transformation of already existing enterocytes.
Glucose uptake by rabbit intestine has been shown to be confined to the upper two-thirds of jejunal villi. Inhibition by phloridzin shows most of this transport to take place through the Na(+)-glucose linked transporter (SGLT1). Parallel measurements on the other hand show SGLT1 mRNA expression to increase rapidly as enterocytes reach the crypt-villus junction. Levels of SGLT1 mRNA then remain elevated in all villus enterocytes. These results provide direct evidence that SGLT1-mediated glucose transport is subject to post-transcriptional control.
Latex beads coated with secretory immunoglobulin (IgA) instilled into mouse intestinal loops are taken up by M cells present in Peyer's patch follicle-associated epithelial tissue. Bead adsorption and uptake is greater at the edge compared with the apex of follicle domes. Coating beads with bovine serum albumin (BSA) causes a fourfold reduction in adsorption and a twentyfold reduction in uptake. Results demonstrate selectivity between adsorption and uptake and between the ability of different proteins to facilitate uptake.
Cell injury was studied in cultured rabbit proximal tubular epithelial cells using digital-imaging fluorescent microscopy to relate changes in cytosolic Ca2+ ([Ca2+]i) to bleb formation and cell death. Fura-2-loaded cells were treated in normal (1.37 mM) and low (less than 5 microM) extracellular Ca2+ ([Ca2+]e) with 1) inhibitors of glycolysis (iodoacetate) and/or mitochondrial oxidation (KCN), 2) thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzene, and p-chloromercuribenzene sulfonate), and 3) Ca2+ ionophore (ionomycin). All three types of injury produced both [Ca2+]e-independent and [Ca2+]e-dependent increases in [Ca2+]i. KCN + iodoacetate +/- [Ca2+]e did not produce blebbing or death within 60-90 min. Thiol modifiers and ionomycin produced blebbing, which correlated with sustained threefold or greater elevations of [Ca2+]i and loss of viability only after [Ca2+]i had risen severalfold. Blebbing and cell death could be prevented or delayed by treatment in low [Ca2+]e. Trypsin (x0.5) caused a transient (less than 5 min) elevation in [Ca2+]i as well as increases in intracellular Ca2+ pools.
Validity of research in a medical environment is enhanced through incorporating the patient's perspective by using qualitative approaches. Our efforts to improve the validity of measures led us to examine the research process more broadly through mechanisms of patient participation: a protocol adviser, a participant advisory panel, and focus groups. In our research program of biopsychosocial HIV studies in the military population, feedback from patient participants provided the research staff with information important for refining standard measures, improving the research process, and interpreting the quantitative data analyses.
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The present work investigates the ability of galactose to affect enterocyte differentiation during normal development in vivo. Energy intake has also been varied to take account of the fact that galactose is poorly metabolized in mice. Brush-border lactase, alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N, alkaline phosphatase and microvillus length were measured as markers of enterocyte differentiation in mice fed diets containing galactose (G diet), corn oil (E diet) or galactose + corn oil (G + E diet). Maintaining mice on a G instead of E diet reduced brush-border lactase activity and enterocyte migration rates; alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N and microvillus length expression increased and alkaline phosphatase activity remained unchanged. Feeding the G + E diet restored enterocyte migration rates, lactase, aminopeptidase N and dipeptidylpeptidase-IV activities to values found in mice fed the E diet. Galactose stimulation of alpha-glucosidase and microvillus length expression was, however, fully maintained in mice fed the G + E diet. Present results show that enterocyte differentiation is affected independently by varying dietary galactose and energy levels; that galactose effects always increase and energy effects usually decrease expression of enterocyte components and that energy stimulation of lactase activity is exceptional.
Acute injury to renal proximal tubule cells has previously been shown to result in elevated cytosolic Ca2+ ([Ca2+]i), blebbing, and eventual cell death. In this study, digital imaging fluorescence microscopy was used to evaluate these changes in response to HgCl2 treatment of cultured rabbit proximal tubular cells. Monolayer cells loaded with fura-2 were treated with 10, 50, or 100 microM HgCl2 in both 1.37 mM CaCl2-containing and nominally Ca(2+)-free (less than 5 microM) Hanks' balanced salt solution. [Ca2+]i was estimated by measuring the ratio of fluorescent image pairs (collected at 340- and 380-nm excitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blue exclusion. After exposure of cells to 10 microM HgCl2, [Ca2+]i initially increased about 2-fold by 5 min; after 50 or 100 microM HgCl2, [Ca2+]i rapidly rose 2- to 3-fold, peaked at 1-3 min, and then generally decreased slightly. In nominally Ca(2+)-free (less than 5 microM) medium, [Ca2+]i stabilized, but in 1.37 mM Ca(2+)-containing medium, [Ca2+]i continued to slowly rise, often reaching levels of fura-2 saturation. The rate and extent of blebbing and the rate of cell death were increased in the presence of 1.37 mM Ca2+. These results show that sustained elevations of [Ca2+]i precede both cell blebbing and cell death and that when these elevations are limited by removing extracellular Ca2+ the amount of blebbing is reduced and cell viability is prolonged.
Human tracheo-bronchial epithelium obtained from autopsy, surgery, and organ donation will have areas of both viable and non-viable cells. It is important in the initial establishment of epithelial explant and cell cultures that injured, non-viable mucosal epithelium not be used for the cultures. Autopsy cases selected for culture should initially be chosen on the basis of a shorter post mortem interval and cause of death in order to increase the rate of successful culture. Staining the epithelium with the vital dye, trypan blue, in combination with phase contrast microscopy of the bronchial tissues will further identify those areas of the mucosa that are enriched for viable cells. The dead, non-viable areas are trypan blue positive, while the viable areas are clear and have foci of beating, motile cilia. Treatment of the mucosal tissue with mucolytic agents to remove cell debris, dead cells, and microbes trapped in the mucus material will further improve the chances for successful culture. Human tracheo-bronchial epithelium, although non-sterile and often injured at time zero for numerous reasons, can effectively be used in vitro pathophysiology studies.
1. The total organ weights and the constituent weights of mucosal and muscle tissues have been determined in the small intestines of three lines of domestic fowls subjected to different degrees of artificial genetic selection for rapid growth rates. 2. Effects of selection have been examined on the basis of wet and dry tissue weights in duodenum, jejunum and ileum from each line of birds. 3. Effects of selection have been compared on both absolute tissue weights and relative weights (i.e. expressed per kilogram body weight). 4. Selection for rapid growth rate is associated with increases in absolute weight and length of small intestine but marked reductions in relative weight and length. 5. In the fastest growing group of birds the major effect is a decrease in the relative mass of mucosa which is most pronounced in the more proximal regions of the small intestine. 6. These findings are discussed in relation to possible enhancement of intestinal digestive and absorptive efficiency in birds selected for improved growth rate and feed conversion. 7. The differences in tissue composition of the small intestine between lines and between regions of the intestine in the same line emphasise the difficulty in selecting an anatomical parameter upon which to base the in vitro and in vivo measurements of nutrient absorption in a number of established preparations.
1. Mice fed low carbohydrate and galactose-containing diets have been used to determine both positional and temporal aspects of microvillus development during enterocyte migration from intestinal crypts towards the tips of jejunal villi. 2. The positional dependence of microvillus growth was found to be similar in mice fed low carbohydrate (3.0 kcal/g), galactose-containing lipid substituted (2.9 kcal/g) and galactose-containing agar substituted (5.1 kcal/g) diets. The daily calorific intake by mice fed these diets was about 10.4 kcal/mouse. The maximal microvillus length reached by enterocytes fed galactose was nearly twice that measured in mice fed the low carbohydrate diet. 3. Enterocyte migration rate in mice fed the low carbohydrate and the high calorie galactose-containing diet was twice that measured in mice fed the low calorie galactose-containing diet. These changes were not associated with any noticeable alteration in the size of intestinal crypts. 4. Changes in maximal microvillus length (M) can be predicted from the equation M = 0.0016 CD + 0.073 CD/R, where CD and R refer to crypt depth and enterocyte migration rate respectively, Smith M. W. and Brown D. (1989). Dual control over microvillus elongation during enterocyte development. Comp. Biochem. Physiol. 93A, 623-628. Substituting measured values for CD and R in this equation revealed a specific capacity of galactose to potentiate microvillus development when presented in the form of a high calorie diet. 5. The possibility that galactose, which is poorly metabolized in mice, can increase microvillus expression by interfering specifically with some aspect of carbohydrate metabolism is discussed.
Further elaboration on the structure-activity relationships in our U-50,488 series has revealed that benzologation of this cyclohexane-1,2-diamine derivative provides compounds which either maintain the interaction with the kappa receptor (e.g. compounds 3a and 5a in the phenylacetamido series) or eliminate the mu receptor mediated analgesia (e.g. compounds 3-6 in the benzamido series). Naphthologation also caused the elimination of mu receptor mediated analgesia (e.g. compounds 17a and 17b).
Fifty-eight patients scheduled for elective thoracotomy were randomly allocated to receive fentanyl by either the thoracic or the lumbar epidural route for postoperative analgesia. The infusion rate was adjusted to optimise analgesia. Dose adjustment, pain assessment and the incidence of side effects were monitored by a blinded observer at set times over the 24 hour study period. Similar pain scores were obtained in both groups at all assessment times. In addition, there was no significant difference in dose requirements or incidence of side effects between the two groups. There appears little justification for the use of the generally less familiar, and potentially more dangerous, thoracic approach when fentanyl alone is infused into the epidural space following thoracotomy.