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S A Lewis

Publications and source records attributed to S A Lewis.

At least 163 records · Page 9Linked to original sources
PubMed1985

Three expressed sequences within the human beta-tubulin multigene family each define a distinct isotype.

This paper describes the isolation and complete sequence of a novel expressed human beta-tubulin gene (beta 2). The sequence is compared with that of two other expressed human beta-tubulin genes (M40 and 5 beta). All are encoded by four exons. Though the boundaries of each exon are absolutely conserved among the three genes, the intervening sequences differ considerably in size and sequence content. Two of the genes (M40 and 5 beta) contain one (M40) or ten (5 beta) members of the middle repetitive Alu family sequences within one of their intervening sequences. Comparison of the amino acid sequences encoded by each gene reveals a high level of homology overall, though there is significant divergence between the carboxy termini of two of the genes. The pattern of expression of each beta-tubulin gene has been studied in several different human cell lines using unique non-crosshybridizing probes derived from the 3' untranslated regions. Two of the genes, M40 and beta 2, are expressed at varying levels in all of the cell lines examined, though the level of expression of one of these genes parallels the other in most cases. The third gene, 5 beta, is detectably expressed only in cells of neural origin. Thus, distinct human beta-tubulin isotypes are encoded by genes whose exon size and number has been conserved evolutionarily, but whose pattern of expression may be regulated either co-ordinately or uniquely. Of the approximately 15 sequences contained in the human beta-tubulin multigene family, nine have now been sequenced fully. The overall composition of the multigene family and the evolutionary relationships among its various members are discussed.

Amino Acid Sequence↗
PubMed1985

Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium.

This paper reviews the properties and regulation of single amiloride-sensitive Na+ channels in the apical membrane, and Cl- and K+ channels in the basolateral membrane of rabbit urinary bladder. According to fluctuation analysis, there is an average of one amiloride-sensitive Na+ channel for every 40 micron2 of apical membrane. Each Na+ channel passes 0.7 pA of current under normal, short-circuit conditions. Apical channels are hydrolysed by the endogenous enzyme urokinase, which is released into the urine by the kidney. After exposure to urokinase, the Na+ channel loses its amiloride sensitivity, and eventually becomes unstable in the membrane. The selectivity and kinetic properties of single anion and K+ channels in the basolateral membrane were also studied using the patch clamp technique. The properties of these channels are discussed in terms of the regulation of transepithelial Na+ transport.

Amiloride↗
PubMed1985

The electrical basis for enhanced potassium secretion in rat distal colon during dietary potassium loading.

Previous studies in rat distal colon provide evidence for an active absorptive process for potassium under basal conditions, and for active potassium secretion during chronic dietary potassium loading. The present studies were performed with conventional and potassium-selective microelectrodes to determine the electrical basis for the increase in transcellular (active) potassium secretion observed during potassium loading. Compared to control tissues, potassium loading resulted in a 5-fold increase in transepithelial voltage (VT) and a 52% decrease in total resistance (RT) in the distal colon. The rise in VT was due to a decrease in apical membrane resistance and an increase in basolateral membrane voltage from -45 +/- 2 mV (cell interior negative) in control to -56 +/- 2 mV (p less than 0.001) in potassium loaded tissues. This difference in basolateral membrane voltage reflected in increase in intracellular potassium activity from 86 +/- 4 mM to 153 +/- 12 mM (P less than 0.001). In control tissues, the sequential mucosal addition of the sodium channel blocker amiloride (0.1 mM) and the potassium channel blocker tetraethylammonium chloride (TEA: 30 mM) produced no effect on the electrical measurements. However, in potassium loaded tissues, amiloride and TEA produced transepithelial changes consistent with inhibition of apical membrane conductances for sodium and potassium, respectively, reflected by increases in the resistance ratio, alpha (ratio of apical to basolateral membrane resistances). These data indicate that the decrease in apical membrane resistance during potassium loading was caused by an increase in apical membrane conductance for both potassium and sodium.

Amiloride↗
PubMed1985

Outcome of pregnancy in the rat with mild hyperphenylalaninaemia and hypertyrosinaemia: implications for the management of "human maternal PKU".

In attempting to determine the effects of mildly elevated maternal phenylalanine (Phe) blood levels on the developing fetal rat brain, a dietary supplement of Phe was given, under taste cover of Aspartame. Phe and tyrosine (Tyr) levels were mildly elevated throughout pregnancy without evidence of malnutrition. Mild hyperphenylalaninaemia with concurrent hypertyrosinaemia induced in rats prior to conception resulted in microcephaly and lasting behavioural problems in the offspring, specifically hyperactivity and learning difficulties. Dams fed Tyr to produce Tyr levels equivalent to the Phe-fed animals showed only the learning difficulties among the offspring. alpha-Methyl Phe, a Phe hydroxylase inhibitor, fed in conjunction with Phe, at the level relevant to these experiments, resulted in raised Tyr levels and does not provide a better method of determining whether mildly elevated maternal Phe levels alone, or Phe and Tyr in combination, cause the abnormality found in the offspring of Phe-supplemented dams. Therapeutic addition of Tyr to diets of mothers with even mild hyperphenylalaninaemia should be approached with caution as mild co-elevation of Phe and Tyr in the fetus may be harmful. In the face of such a possible therapeutic dilemma alternatives, such as dietary additions of other essential amino acids to limit fetal brain damage, need to be explored.

Animals↗
PubMed1985

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder.

The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl- on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (VT) decreased rapidly, transepithelial resistance (RT) increased, and calculated short-circuit current (Isc) decreased. Calculated ENa was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconate VT decreased, RT increased and Isc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. Calculated ENa fell substantially (from 130 to 68 mV on average). Ba2+ (3 mM) reduced calculated ENa in Cl- Ringer's but not in gluconate Ringer's. With replacement of serosal Cl- by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented and ENa did not fall. Replacement of serosal isosmotic Cl- medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl-per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased.

Animals↗
PubMed1985

Single anion-selective channels in basolateral membrane of a mammalian tight epithelium.

Basolateral membrane chloride permeability of surface cells from rabbit urinary bladder epithelium was studied using the patch-clamp technique. Two types of anion-selective channel were observed. One channel type showed inward rectification and had a conductance of 64 pS at-50 mV when bathed symmetrically by saline solution containing 150 mM chloride; the other resembled high-conductance voltage-dependent anion channels (VDACs). Both channels had the selectivity sequence Cl-approximately equal to Br-approximately equal to I- approximately equal to SCN- approximately equal to NO3- greater than F- greater than acetate greater than gluconate greater than Na+ approximately equal to K+ and were sensitive to the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Basolateral chloride conductance in urinary bladder is apparently due to the 64 pS anion channel, which is active at physiological potentials. Imperfect selectivity of this channel against cations might also account for the low, but finite, sodium permeability of the basolateral membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe.

A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The clone was isolated from a mouse brain cDNA library by low-stringency cross-hybridization with a cDNA probe encoding mouse glial fibrillary acidic protein (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). The identity of NF68 was established by hybrid selection using mouse brain polyA+ mRNA, and cell-free translation of the selected mRNA species. The cell-free translation product co-migrated with authentic 68,000-mol-wt neurofilament protein on an SDS/polyacrylamide gel, and was immunoprecipitable with a monospecific rabbit anti-bovine neurofilament antiserum. In addition, DNA sequence analysis of NF68 showed 90% homology at the amino acid level compared with the sequence of the porcine 68,000-mol-wt neurofilament protein. At high stringency, NF68 detects a single genomic sequence encoding the mouse 68,000-mol-wt neurofilament protein. Two mRNA species of 2.5 kb and 4.0 kb are transcribed from the single gene in mouse brain. The level of expression of these mRNAs remains almost constant in postnatal mouse brains of all ages and, indeed, in the adult. At reduced stringency, NF68 detects a number of mRNAs that are expressed in mouse brain, one of which encodes the 150,000-mol-wt neurofilament protein. The NF68 probe cross-hybridizes at high stringency with genomic sequences in species as diverse as human, chicken, and (weakly) frog, but not with DNA from Drosophila or sea urchin.

Animals↗
PubMed1985

Five mouse tubulin isotypes and their regulated expression during development.

We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M alpha 2) that encode alpha-tubulin and three (M beta 2, M beta 4, and M beta 5) that encode beta-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two alpha-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the beta-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three beta-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse beta-tubulin isotype and a chicken beta-tubulin isotype. The significance of the interspecies conservation of tubulin isotypes is discussed in relationship to microtubule function. We have used non-cross-hybridizing 3' noncoding region probes from the five cloned mouse tubulin cDNAs to study the developmental expression of each isotype in various mouse tissues. M alpha 1 and M beta 2 are expressed in an approximately coordinate fashion, and their transcripts are most abundant in brain and lung. M alpha 2 and M beta 5 are ubiquitously expressed and to a similar extent in each tissue, with the greatest abundance in spleen, thymus, and immature brain. In contrast, M beta 4 is expressed exclusively in brain. Whereas the expression of the latter isotype increases dramatically during postnatal development, transcripts from all four other tubulin genes decline from maximum levels at or before birth. Tissue-specific development changes in the abundance of tubulin isotype-specific mRNAs are discussed in relationship to organogenesis in the mouse.

Age Factors↗
PubMed1985

Temporal expression of mouse glial fibrillary acidic protein mRNA studied by a rapid in situ hybridization procedure.

A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week.

Animals↗
PubMed1985

Coronary angioplasty of multiple vessels: short-term outcome and long-term results.

Experience with percutaneous transluminal coronary angioplasty (PTCA) of multiple vessels was reviewed to assess short-term outcome and long-term results. PTCA of multiple vessels was performed in 100 of the initial 500 patients (20%) who underwent PTCA at the Medical College of Virginia between July 1979 and August 1984. Eighty-nine percent had class 3 or 4 angina, and 66% had unstable angina. Two-thirds had severe stenosis of two vessels or major branches and one-third had three-vessel disease. One or more significant lesions were dilated in two vessels in 84 patients, in three vessels in 14 patients, and in four vessels in two patients. PTCA of 273 lesions (2.7/patient) was attempted (range two to eight per patient) with angiographic success in 250 lesions (91.6%). Primary success (angiographic and clinical improvement) was achieved in 95 of 100 patients (95%); 84% had success in multiple vessels, and 79% had success in all attempted lesions. Complications occurred in 11 patients (11%); four patients (4%) underwent urgent bypass surgery and four additional patients (4%) had myocardial infarction. Long-term results were assessed in 44 patients with primary success who had follow-up of more than 1 year (mean 26 months) after multiple-vessel PTCA. Twenty-eight patients (64%) remain event-free and improved and 48% are event-free and asymptomatic. Clinical recurrence developed in 15 patients (34%); four had sustained improvement with repeat PTCA, three remain improved with medical therapy, and eight (18%) have undergone bypass surgery during follow-up. One patient (2.3%) developed late myocardial infarction, and deaths have occurred in the follow-up cohort.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Studies of sodium channels in rabbit urinary bladder by noise analysis.

Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (Isc) and correspondingly two components of apical Na+ entry, one amiloride-sensitive (termed IA and the A channel, respectively) and one amiloride-insensitive (IL and the leak pathway, respectively). The leak pathway gives rise to l/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na+ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses ("punching") increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel. Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism of l/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts with PNa/PK approximately 30 and loses selectivity in stages until PNa/PK reaches the free-solution mobility ratio (approximately 0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na+ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia.

Amiloride↗
PubMed1984

Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: an electrophysiological study.

Mammalian urinary bladder undergoes, in a 24-hour period, a series of slow fillings and rapid emptying. In part the bladder epithelium accommodates volume increase by stretching the cells so as to eliminate microscopic folds. In this paper we present evidence that once the cells have achieved a smooth apical surface, further cell stretching causes an insertion of cytoplasmic vesicles resulting in an even greater apical surface area per cell and an enhanced storage capacity for the bladder. Vesicle insertion was stimulated by application of a hydrostatic pressure gradient which caused the epithelium to bow into the serosal solution. Using capacitance as a direct and nondestructive measure of area we found that stretching caused a 22% increase in area. Removal of the stretch caused area to return to within 8% of control. An alternate method for vesicle insertion was swelling the cells by reducing mucosal and serosal osmolarity. This perturbation resulted in a 74% increase in area over a 70-min period. Returning to control solutions caused area to decrease as a single exponential with an 11-min time constant. A microtubule blocking agent (colchicine) did not inhibit the capacitance increase induced by hypoosmotic solutions, but did cause an increase in capacitance in the absence of a decreased osmolarity. Microfilament disrupting agent (cytochalasin B, C.B.) inhibited any significant change in capacitance after osmotic challenge. Treatment of bladders during swelling with C.B. and subsequent return to control solutions increased the time constant of the recovery to control values (22 min). The Na+-transporting ability of the vesicles was determined and found to be greater than that of the apical membrane. Aldosterone increased the transport ability of the vesicles. We conclude that some constituent of urine causes a loss of apical membrane permeability. Using electrophysiological methods we estimated that the area of cytoplasmic vesicles is some 3.3 times that of the apical membrane area. We discuss these results in a general model for vesicle translocation in mammalian urinary bladder.

Animals↗