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Biomedical subjects

B L Smith

Publications and source records attributed to B L Smith.

At least 127 records · Page 7Linked to original sources
PubMed1995

Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney.

The localization of aquaporin-1 water channels (AQP-1) in nephron and vascular structures in rat kidney were characterized, because vascular bundles are known to play a key role in urinary concentration. Immunohistochemistry and immunoelectron microscopy were applied on thin cryosections or ultrathin Lowicryl sections, using an optimized freeze-substitution method. Within the vascular bundles, AQP-1 is localized in descending thin limbs (DTL) of short nephrons in apical and basolateral membranes. The expression in DTL of short nephrons is considerably lower compared with the expression in long nephrons, consistent with the known lower osmotic water permeability of this segment. Furthermore, DTL of short nephrons expressing AQP-1 continue abruptly into a thin limb segment without AQP-1. This suggests the existence of a novel thin limb epithelium in the outer medulla. Extensive expression of AQP-1 is observed in apical and basolateral membranes of DTL of long nephrons, which are localized in the periphery of the vascular bundles. The expression decreases along the axis of long nephron DTLs in correlation with the known water permeability characteristics of thin limb segments. DTLs of both short and long nephrons continue abruptly into thin limb segments without AQP-1 expression, revealing an abrupt cell-to-cell transition. In vasa recta, AQP-1 is selectively localized in the nonfenestrated endothelium of descending vasa recta, whereas the fenestrated endothelium of ascending vesa recta and peritubular capillaries do not express AQP-1. AQP-1 is localized in both apical and basolateral plasma membranes, which is logical for transendothelial water transport. Isolated perfused descending vasa recta display high water permeability, and, unlike sodium permeability, diffusional water permeability is partly inhibited by mercurials, thus substantiating the presence of mercurial-sensitive water channels in descending vasa recta. Thus AQP-1 is localized in DTL and descending vasa recta within vascular bundles, and AQP-1 expression in DTL segments is in exact concordance with the known water permeability characteristics, strongly supporting that AQP-1 is the major constitutive water channel of the nephron.

Animals↗
PubMed1995

Variability of lip and jaw movements in the speech of children and adults.

One purpose of the present study was to examine displacement variability of lower lip and jaw movements to determine whether sounds that are generally learned earlier and should, therefore, have been practiced more (e. g., stops and nasals) would be less variable than sounds that tend to develop later (e. g., fricatives). It was also of interest to determine whether individual articulators such as the lower lip and the jaw show any differences in displacement variability, given that lower lip gestures may need to be more precise than jaw movements. Repetitions of several labial-initial, CVC stimuli embedded in short phrases produced by three groups of children (5-, 8-, and 11-year-olds) and a group of adults were examined to determine the variability of articulatory gestures. No evidence was found to suggest that fricatives were more variable than stops or nasals for any of the groups. For the children but not the adults, lower lip gestures tended to be more variable than jaw movements.

Adult↗
PubMed1995

Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (mean +/- SE, fmol/mm): S-1 proximal, 10.8 +/- 2.1; S-2, 10.0 +/- 2.3; S-3, 21.3 +/- 3.1; type 1 thin descending limb (DTL), 12.9 +/- 4.6; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.

Animals↗
PubMed1995

Point-of-care testing.

Point-of-care testing refers to testing outside of the central laboratory at or near the patient's bedside. The practice greatly decreases turnaround time for testing and has improved outcome and decreased length of stay in some patient groups. Advances in technology have made analyzers increasingly portable with expanded testing capacities while maintaining standards for accuracy required by regulatory agencies. It is possible for clinicians to perform testing that historically was performed only in the central laboratory by trained laboratory technicians. Determination of all appropriate bedside testing for different clinical areas and patient groups will require further investigation and debate.

Blood Chemical Analysis↗
PubMed1994

DNA damage induced by bleomycin, neocarzinostatin, and melphalan in a precisely positioned nucleosome. Asymmetry in protection at the periphery of nucleosome-bound DNA.

The antitumor drugs bleomycin, neocarzinostatin, and melphalan all damage DNA by mechanisms which involve binding in the minor groove. In order to examine at high resolution the modulating effects of chromatin structure on the action of these drugs, an end-labeled DNA fragment from the Xenopus laevis 5 S rRNA gene was reconstituted with histone octamers to form a precisely positioned nucleosome. For each drug, DNA damage at specific sequence positions in the fragment was then compared for nucleosome-bound versus naked DNA. Reconstitution into nucleosomes resulted in a marked inhibition of the DNA cleavage induced by bleomycin (5-fold) and neocarzinostatin (2.4-fold) in the central region of nucleosomal DNA. However, at the periphery of nucleosome-bound DNA, a distinct asymmetry was apparent, with marked inhibition of cleavage toward the upstream side, but little if any inhibition toward the downstream side, which overlaps the binding site of the transcription factor TFIIIA. In the case of melphalan, alkylation at adenine N-3 was inhibited by nearly 2-fold throughout the nucleosome, whereas alkylation at guanine N-7 was either slightly inhibited or slightly enhanced, depending on sequence position. None of the drugs showed the 10-base pair periodicity characteristic of hydroxyl radical-induced cleavage of nucleosomal DNA. The results are consistent with a model in which minor groove sites in nucleosome-bound DNA remain relatively accessible to small molecules, even where the minor groove faces the histone core, and in which drug-induced DNA damage is inhibited by conformational constraints imposed on DNA by nucleosome structure. Furthermore, the degree of such constraints appears to be sequence-dependent, at least near the periphery of nucleosome-bound DNA.

Animals↗
PubMed1994

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Animals↗
PubMed1994

The three-dimensional structure of human erythrocyte aquaporin CHIP.

Water-permeable membranes of several plant and mammalian tissues contain specific water channel proteins, the 'aquaporins'. The best characterized aquaporin is CHIP, a 28 kDa red blood cell channel-forming integral protein. Isolated CHIP and Escherichia coli lipids may be assembled into 2-D crystals for structural analyses. Here we present (i) a structural characterization of the solubilized CHIP oligomers, (ii) projections of CHIP arrays after negative staining or metal-shadowing, and (iii) the 3-D structure at 1.6 nm resolution. Negatively stained CHIP oligomers exhibited a side length of 6.9 nm with four-fold symmetry, and a mass of 202 +/- 3 kDa determined by scanning transmission electron microscopy. Reconstituted into lipid bilayers, CHIP formed 2-D square lattices with unit cell dimensions a = b = 9.6 nm and a p422(1) symmetry. The 3-D map revealed that CHIP tetramers contain central stain-filled depressions about the fourfold axis. These cavities extend from both sides into the transbilayer domain of the molecule leaving only a thin barrier to be penetrated by the water pores. Although CHIP monomers behave as independent pores, we propose that their particular structure requires tetramerization for stable integration into the bilayer.

Aquaporin 1↗
PubMed1994

Molecular structure of the water channel through aquaporin CHIP. The hourglass model.

Aquaporin channel-forming integral protein (CHIP) is the first characterized water channel protein (genome symbol AQP1), but the molecular structure of the aqueous pathway through CHIP remains undefined. The two halves of CHIP are sequence-related, and each has three bilayer-spanning domains with the motif asparagine-proline-alanine (NPA) at residues 76-78 (in cytoplasmic loop B) and 192-194 (in extracellular loop E). The NPA motifs are oriented 180 degrees to each other, and the second NPA is near cysteine 189, the known site where mercurials inhibit osmotic water permeability (Pf). When expressed in Xenopus oocytes, the double mutant A73C/C189S exhibited high, mercurial-sensitive Pf similar to wild-type CHIP. Conservative substitutions of slightly greater mass in or near NPA motifs in loop B or loop E in CHIP caused reduced Pf and failure of the protein to localize at the plasma membrane. Certain nonfunctional loop E mutants complemented the truncation mutant D237Z. Formation of mixed oligomers was demonstrated by velocity sedimentation, immunoprecipitation, and analysis of dimeric-CHIP polypeptides. Cellular distributions of individual mutants or complementing pairs of mutants were verified by plasma membrane isolation and confocal microscopy. An hourglass structural model is proposed in which a cytoplasmic chamber (loop B) connects within the membrane to an extracellular chamber (loop E) forming a single, narrow aqueous pathway through each of the CHIP subunits; subunit oligomerization may provide the vertical symmetry necessary for residence within the lipid bilayer.

Amino Acid Sequence↗
PubMed1994

Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes.

Reconstitution of highly purified aquaporin CHIP (channel-forming integral protein) into proteoliposomes was previously shown to confer high osmotic water permeability (Pf) to the membranes [Zeidel et al. (1992) Biochemistry 31, 7436-7440]. Here we report detailed ultrastructural, pharmacologic, and transport studies of human red cell CHIP in proteoliposomes. Freeze-fracture and transmission electron microscopy revealed a uniform distribution of CHIP which was incorporated into the membranes in both native and inverse orientations. Morphometric analysis of membranes reconstituted at three different concentrations of CHIP revealed that the intramembrane particles correspond to tetramers or possible higher order oligomers, and the Pf increased in direct proportion to the CHIP density. Proteolytic removal of the 4-kDa C-terminal cytoplasmic domain of CHIP did not alter the Pf or oligomerization in red cell membranes. CHIP exhibited a similar conductance for water when reconstituted into membranes of varied lipid compositions. The sensitivities of CHIP-mediated Pf to specific sulfhydryl reagents were identical to known sensitivities of red cell Pf, including a delayed response to p-(chloromercuri)benzenesulfonate. CHIP did not increase the permeability of the proteoliposome membranes to H+/OH- or NH3. These studies demonstrate that CHIP proteoliposomes exhibit all known characteristics of water channels in native red cells and therefore provide a defined system for biophysical analysis of transmembrane water movements.

Aquaporin 1↗
PubMed1994

Biologically active two-dimensional crystals of aquaporin CHIP.

Plasma membranes of several mammalian tissues are highly permeable to water due to the presence of CHIP, the 28-kDa channel-forming integral protein which is the archetypal member of the aquaporin family of water channel proteins. To define its native structure, purified red cell CHIP protein was reconstituted into lipid bilayers at a high protein-to-lipid ratio, and the resulting 3-microns diameter membrane vesicles were examined by high resolution electron microscopy. The reconstituted membranes contained highly ordered two-dimensional crystalline lattices of p422(1) symmetry in which each CHIP tetramer contained a central depression extending from the outer and inner surfaces of the membrane into the transbilayer domain of the molecule. The reconstituted membranes also exhibited extremely high osmotic water permeability, Pf = 0.472 cm/s, corresponding to the sum of activities of all incorporated CHIP molecules. These studies report the first two-dimensional crystallization of a biologically active water channel and provide direct evidence of the structure responsible for its pore-like behavior.

Aquaporin 1↗
PubMed1994

Comparative toxicity of alkyl-1,4-naphthoquinones in rats: relationship to free radical production in vitro.

2-Methyl-1,4-naphthoquinone causes haemolysis in vivo. This toxic effect is believed to result from oxidative damage to erythrocytes by "active oxygen" species formed via one-electron reduction of the naphthoquinone by oxyhaemoglobin. In the present investigation, seven 2-alkyl-1,4-naphtoquinones have been studied with regard to their haemolytic activity in rats, their ability to cause oxidative damage in erythrocytes in vitro, and their reactivity toward oxyhaemoglobin. A close correlation was observed between the in vivo and in vitro parameters, suggesting that the proposed mechanism of toxicity of 2-methyl-1,4-naphthoquinone is correct and is also applicable to other alkylnaphthoquinones.

Animals↗
PubMed1994

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells.

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

Animals↗
PubMed1994

Endoscopic anatomy and map of the equine bronchial tree.

To develop a bronchoscopic map of the equine respiratory tree, the major airways of the lungs of 6 healthy Thoroughbred horses were systematically explored with a flexible fibreoptic endoscope through a tracheostomy while the horses were sedated in stocks. With the carina as the reference point, measurements were made of distances to the branches of the major airways using markers on the shaft of the endoscope. All branches were explored until the narrowing of their diameters prevented further advancement of the endoscope. Positions of origins of branches from the parent bronchus were recorded in relation to a 12 h clock. Branching patterns of the right and left lungs were similar. Seventeen branches of the principal and caudal lobar bronchi of the left lung, and 18 branches of the principal and caudal lobar bronchi of the right lung were identified. Mean explorable distances from the carina to the ends of the right and left caudal lobar bronchi were 34.0 +/- 3.5 (sd) and 34.5 +/- 3.0 cm, respectively. Generally, smaller horses had shorter explorable bronchial lengths. Branching patterns of the parent bronchi were fairly consistent among horses, particularly the branches closest to the carina. After endoscopy and euthanasia, the lungs were removed, and dried with pressurised air flowing through them for 7-10 days. Attempts to explore the airways of the dried lungs endoscopically were relatively unsuccessful, because airways were much smaller in the dried lungs, and many of the branches were distorted when compared with their antemortem appearances. However, having a dried lung specimen as a reference during the bronchoscopic procedure was useful for maintaining orientation in the lungs. Radiographs were used to estimate the location of the origin and destination of each airway branch in relation to the nearest intercostal space. This makes the airway map useful when lesions identified radiographically are to be lavaged.

Animals↗
PubMed1994

Body position and direction preferences in horses during road transport.

It has been hypothesised that horses have a preference for facing backward in a trailer during road transport in order to minimise shifts of body weight due to accelerations and decelerations. To determine if horses have preferences for facing forward vs. backward in a horse trailer, the authors analysed the percentages of time horses spent in different body positions and directions while standing in a moving or parked horse trailer. Body positions and directions of 8 Thoroughbred geldings were videotaped while horses were transported singly and untethered in a 4-horse stock trailer over a 32 km route of country roads; or while the same horses were untethered in the same trailer stationary in a parking lot. Analysis of the logit-transformed percentages of time horses spent in different directions indicated that they spent significantly more time facing backward when the trailer was in motion, but not when it was parked. Several horses displayed strong individual preferences for the directions they faced during road transport.

Animals↗
PubMed1994

Variability control in speech production tasks performed by adults and children.

Eighteen adults and 12 children, ranging from 7 to 11 years of age, participated in a study that investigated their abilities to control the temporal variability of their speech. For both the adults and the children, few substantive differences in performance were observed when considering control data versus findings from an experimental task in which they were specifically instructed to be as consistent as possible in producing various stimuli. Although some subjects did show reduced variability in the experimental condition, there was little evidence that such decreases typically represented more than random effects. In general, the results suggest that when producing multiple repetitions of words and short phrases, adults and 7-11-year-old children are essentially as consistent as they can be, whether specifically attempting to minimize variability or not. The basis for this could be that even in control conditions, subjects may perform at or near optimal levels of consistency across productions.

Adult↗
PubMed1994

Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

ABO Blood-Group System↗
PubMed1994

Human red cell Aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia.

Channel-forming integral protein (CHIP) is the archetypal member of the Aquaporin family of water channels. Delayed CHIP expression was shown recently in perinatal rat (Smith, B. L., R. Baumgarten, S. Nielsen, D. Raben, M. L. Zeidel, and P. Agre. 1993. J. Clin. Invest. 92:2035-2041); here we delineate the human patterns. Compared with adult, second and third trimester human fetal red cells had lower CHIP/spectrin ratios (0.72 +/- 0.12, 0.94 +/- 0.22 vs 1.18 +/- 0.11) and reduced osmotic water permeability (0.029, 0.026 vs 0.037 cm/s); CHIP was already present in human renal tubules by the second trimester. A patient with a novel form of congenital dyserythropoietic anemia (CDA) with persistent embryonic and fetal globins and absent red cell CD44 protein was studied because of reduced CHIP-associated Colton antigens. Novel CDA red cells contained < 10% of the normal level of CHIP and had remarkably low osmotic water permeability (< 0.01 cm/s), but no mutation was identified in Aquaporin-1, the gene encoding CHIP. These studies demonstrate: (a) unlike rat, human CHIP expression occurs early in fetal development; (b) red cell water channels are greatly reduced in a rare phenotype; and (c) disrupted expression of red cell CHIP and CD44 suggests an approach to the molecular defect in a novel form of CDA.

Adult↗