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Biomedical subjects

L Best

Publications and source records attributed to L Best.

At least 55 records · Page 3Linked to original sources
PubMed1996

Activation of an anion conductance and beta-cell depolarization during hypotonically induced insulin release.

The perforated patch technique was used to study the effects of hypotonic extracellular solutions on membrane potential and whole-cell currents in intact rat pancreatic beta-cells. A 30% reduction in osmolarity resulted in activation of an outwardly rectifying CI(-)-selective conductance in rat beta-cells. This conductance was inhibited by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Exposure to a hypotonic medium also led to a transient stimulation of electrical activity accompanied by cell swelling and a gradual return towards control volume. These effects were also associated with the generation of an inward current at a holding potential of -70mV, and a stimulation of insulin release from intact perifused islets. All of the above effects were inhibited by DIDS. It is suggested that the stimulation of insulin release by hypotonic solutions results from activation of a volume-sensitive anion conductance generating an inward current and leading to a subsequent depolarization of the beta-cell.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Changes in 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein-, fura-2 and autofluorescence in intact rat pancreatic islets in response to nutrients and non-nutrients.

Intracellular pH (pHi) was measured in intact rat islets loaded with the dye 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein. Raising the concentration of glucose from 3 to 13 mM caused a modest, gradual increase in pHi (500:450 fluorescence ratio). The addition of 20 mM lactate caused a gradual decline in pHi which reversed upon withdrawal of lactate. In contrast, the weak acids propionate and acetate (20 mM) induced a rapid, pronounced fall in pHi followed by a gradual recovery. Upon removal of the weak acid, a marked, reversible alkalinization occurred. The addition of 20 mM NH4Cl caused a pronounced intracellular alkalinization, followed by recovery. The subsequent removal of NH4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pHi. Epifluorescence at 350 and 380 nm excitation, and the 350:380 fluorescence ratio, an index of cytosolic [Ca2+] ([Ca2+]i), were measured in islets loaded with the calcium indicator fura-2. Approximately 30% of the total fluorescence was estimated to be derived from NAD(P)H autofluorescence. Addition of KCl or acetylcholine to fura-2 loaded islets raised and lowered, respectively, the 350 and 380 signals, thereby causing marked increases in the 350:380 ratio. Neither KCl nor acetylcholine affected cellular NAD(P)H autofluorescence in non-loaded islets. An increase in glucose concentration caused an increase in both the 350 and 380 fluorescence signals and also in the 350:380 ratio. Qualitatively similar, although smaller changes were observed when Ca2+ was omitted from the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1995

Bacteriology of urinary tract stones.

It has been reported that up to half of renal stones and associated urine specimens have been positive on culture, and that up to 50% of such stones contain magnesium ammonium phosphate. In a prospective study using infrared and wet chemical analysis, we found positive cultures in only 7 of 132 renal, 5 of 105 ureteral and 6 of 21 bladder stones obtained surgically and handled with sterility. Of the culture positive calculi only 43% from the kidney, none from the ureter and 50% from the bladder contained detectable magnesium ammonium phosphate. However, magnesium ammonium phosphate was detectable in 20% of renal, 2% of ureteral and 27% of bladder stones with negative cultures. Of the culture positive renal and ureteral calculi 42% were predominantly calcium phosphate and 17% were predominantly calcium oxalate. For culture negative stones 25% and 51% from the kidney, and 15% and 82% from the ureter were composed of predominantly calcium phosphate and calcium oxalate, respectively. Among the culture positive stones, related positive urine cultures were noted in 100% of the renal, 20% of the ureteral and 50% of the bladder cases, compared to 26%, 10% and 27%, respectively, of culture negative calculi. The same organism was found in the stone and urine in only 38% of the cases. The lower frequency of positive urine cultures, of stones with magnesium ammonium phosphate, and especially of culture positive renal and ureteral stones (5%) than in previous reports suggests that stone culture may be of less value than indicated previously, except for bladder calculi and large renal stones, such as the branched type.

Adult↗
PubMed1994

A possible role for glyceraldehyde transport in the stimulation of HIT-T15 insulinoma cells.

D-Glyceraldehyde was transported into HIT-T15 cells at a linear rate for approx. 2 min and appeared to be unsaturable up to a concentration of 50 mM. Evidence was obtained for an electrogenic component of uptake of the triose. The rate of D-glyceraldehyde transport was also reduced in the absence of Na+, suggesting that a component of uptake was Na(+)-linked. Transport of D-glyceraldehyde could be prevented by N-ethylmaleimide but not significantly by p-chloromercuribenzenesulphonic acid, L-glyceraldehyde, nor by a number of inhibitors of known transport systems. However, D-glyceraldehyde transport was inhibited by alpha-cyano-4-hydroxycinnamate, an inhibitor of some anion transport systems. D-Glyceraldehyde caused a marked depolarization of HIT-T15 cells accompanied by a rise in cytosolic [Ca2+] and [Na+] and a gradual intracellular acidification. The glyceraldehyde-induced rise in cytosolic [Na+] and intracellular acidification, but not the depolarization or rise in cytosolic [Ca2+], were reduced by dithiothreitol and 5-aminoguanidine, compounds which form chemical adducts with alpha-ketoaldehydes. Incubation of HIT cells with either D- or L-glyceraldehyde resulted in the formation of large amounts of D-lactate, the end product of methylglyoxal metabolism via the glyoxalase pathway. It is suggested that the depolarizing action of glyceraldehyde is the result, at least in part, of its electrogenic transport, probably via Na(+)-coupled entry into HIT cells involving an unidentified transport system. The intracellular acidification and a component of the increase in cytosolic [Na+] may be largely due to the presence of one or more dicarbonyl contaminants in the glyceraldehyde preparation.

Coumaric Acids↗
PubMed1994

Intracellular pH, cytosolic calcium concentration and electrical activity in RINm5F insulinoma cells.

The addition of L-lactate or acetate to RINm5F cells caused a transient intracellular acidification, an increase in [Ca2+]i and induced electrical activity. The subsequent withdrawal of lactate or acetate resulted in an intracellular alkalinization with no apparent changes in [Ca2+]i nor electrical activity. Intracellular alkalinization and acidification by application by application and withdrawal of NH4Cl were both accompanied by transient increases in [Ca2+]i in the absence of electrical activity. The induction of electrical activity by lactate was associated with the appearance of inward whole cell currents. Changes in intracellular pH may affect [Ca2+]i though not necessarily by altering plasma membrane potential. The inward currents associated with lactate application may represent an organic anion conductance contributing towards the stimulation of electrical activity by organic acids.

Acetates↗
PubMed1994

Renal cell carcinoma in von Hippel-Lindau disease: another piece of the puzzle.

The association of von Hippel-Lindau disease and renal call carcinoma is well recognized. However, the most appropriate surgical approach in patients with von Hippel-Lindau disease and renal carcinoma remains controversial. We examine this controversy and report on our experience. Our cohort consisted of 28 affected members of three von Hippel-Lindau kindreds. Screening of these individuals consisted of an abdominal ultrasound (US) in their early teens and a baseline computerized axial tomography (CT) scan in their late teens. To date, nine patients have been found to have renal cell carcinoma. In three of these patients, the surgical specimen revealed multiple small foci of "clear cells" characteristic of renal cell carcinoma that had not been detected by careful radiological assessment prior to surgery. In two patients, these lesions were located at the opposite pole from the radiologically-detected tumor. Nephron-sparing surgery, therefore, risks leaving potential tumor that may be responsible for mortality. Conversely, radical procedures condemn a patient to life-long dialysis and/or transplantation with associated risk and morbidity. The potential for the metachronous emergence of contralateral renal cell carcinoma, however, justifies efforts to preserve normal tissue in patients harboring radiologically-detected lesions; however, we need to know with some certainty the natural history of the correct management of this disorder.

Journal Article↗
PubMed1994

Tying it all together: integrating a hospital-based health care system through case management education.

Recognizing the importance of the case manager as a system integrator, United Health Services, Inc. (UHS), a hospital-based health care system located in upstate New York, implemented several diverse case management models. Case managers were working in a variety of settings, often in isolation. It was determined that a system-wide case management education program would accomplish two goals: (a) provide all case managers within the UHS system with similar case management practice skills and language, and (b) provide an opportunity for case managers to meet, share role responsibilities and common case management issues, and use each other as resources. With input from leadership throughout the UHS system, a 4-week case management education program was developed and presented. Participants included multidisciplinary staff who had case management responsibilities within the system. Sessions were taught by UHS staff experts in a number of different disciplines. A teaching guide and manual were developed to supplement the didactic material. Feedback from the program was provided via written participant evaluation and follow-up discussions.

Health Occupations↗
PubMed1993

Stimulation of HIT-T15 insulinoma cells by glyceraldehyde does not require its metabolism.

The addition of the triose D-glyceraldehyde (5-20 mM) to HIT-T15 hamster insulinoma cells caused a rapid, marked depolarisation of the plasma membrane accompanied by a pronounced intracellular acidification, an increase in the cytosolic free calcium concentration [Ca2+]i and enhanced secretion of insulin. D-glyceraldehyde did not reduce the rate of efflux of 86Rb+ from loaded perifused cells. All of the above effects of D-glyceraldehyde were also observed in response to L-glyceraldehyde. The changes in membrane potential and intracellular pH (pHi) caused by D-glyceraldehyde were unaffected by the glycolytic inhibitor iodoacetate, by K(+)-channel blockers (tolbutamide and tetraethylammonium), or by inhibitors of the transport of lactate (alpha-fluorocinnamate), alanine (methylaminoisobutyrate) or glucose (phloretin, phlorrizin). The glyceraldehyde-induced depolarisation and acidification were also observed in the absence of extracellular Ca2+ or Na+. The increase in [Ca2+]i evoked by D-glyceraldehyde was reversed by removal of Ca2+ from the medium. The formation of lactate by HIT-T15 cells was not significantly increased by addition of 10 mM D-glyceraldehyde or L-glyceraldehyde. In contrast, 10 mM glucose caused an approximately fourfold rise in lactate production. The oxidation of D-glyceraldehyde by HIT-T15 cells was also extremely modest compared to glucose oxidation by these cells. These results suggest that the stimulation of HIT-T15 cells by either D-glyceraldehyde of L-glyceraldehyde does not require metabolism of the triose within the cell and may not involve closure of nucleotide-sensitive K+ channels. We propose that the electrogenic transport of glyceraldehyde across the plasma membrane, possibly via H+ cotransport, might lead to depolarisation and hence to Ca2+ entry into the cell.

Animals↗
PubMed1992

Stimulation of insulin secretion by glucose in the absence of diminished potassium (86Rb+) permeability.

Two inhibitors of the nucleotide-sensitive K+ (KATP) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86Rb+ efflux. In the presence of either 500 microM tolbutamide or 100 microM quinine, a reduced rate of efflux of 86Rb+ was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86Rb+ efflux. It is concluded that a net reduction in beta-cell K+ permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the beta-cell in addition to the closure of K+ channels.

Animals↗
PubMed1992

2-Ketoisocaproate transport in insulin-secreting cells.

The transport of the nutrient secretagogue 2-ketoisocaproate (KIC) was studied in isolated rat pancreatic islets and in the HIT-T15 insulinoma cell line using an oil-filtration technique. In both islets and HIT-T15 cells, KIC uptake was a slow process, not reaching equilibrium within 10 min KIC transport was not dependent upon Na+ in the medium, was not inhibited by alpha-cyano-4-hydroxycinnamate nor by 2-amino-2-norborane carboxylic acid (BCH) and did not appear to be electrogenic. Evidence was obtained to suggest that KIC uptake occurred via passive diffusion into the cell of the undissociated acid species. This possibility was supported by the apparent unsaturability of KIC uptake in HIT-T15 cells. Addition of 10-30 mM KIC to dispersed islets cells or HIT-T15 cells produced a rapid intracellular acidification. In islets, the rate of transport of 10 mM KIC was comparable with oxidation rate of the keto-acid suggesting that uptake could be rate-limiting factor for KIC oxidation and thus stimulated insulin release. However, in HIT-T15 cells, the rate of uptake of KIC greatly exceeded the oxidation rate. The low rate of KIC oxidation could explain the poor secretory response of HIT-T15 cells to KIC.

Animals↗
PubMed1992

Effects of cyclosporin A on induced HIT cell alkalinization.

We studied whether therapeutic doses of cyclosporin A (CsA) modify the effects of nutrient and non-nutrient stimuli on pHi, in the insulin-secreting beta-cell line HIT-T15. Glucose caused a transient acidification, followed by alkalinization. CsA failed to block this alkalinization. PMA elicited a gradual alkalinization by a protein kinase C mediated mechanism which is not inhibited by CsA. The depolarization with high K+ was associated with a rise in pHi. CsA was able to completely block this increase in pHi. Ionomycin induced a rapid cytosolic alkalinization partially inhibited by CsA. We conclude that in HIT-T15 cells, therapeutical doses of CsA inhibit the Ca(2+)-dependent pathway of Na+/H+ antiport activation but not protein kinase C activation of this exchanger.

Animals↗
PubMed1992

Lactate transport in insulin-secreting beta-cells: contrast between rat islets and HIT-T15 insulinoma cells.

The transport of L- and D-lactate into rat pancreatic islets and HIT-T15 insulinoma cells was studied by measuring uptake of 14C-labelled substrate at room temperature and by following changes in intracellular pH (pHi) in islets and HIT-T15 cells loaded with 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). Uptake of L-lactate into HIT-T15 cells was rapid, reaching equilibrium after 5 min with an apparent Km value of 4.8 mM. Transport was markedly inhibited by alpha-cyano-4-hydroxycinnamate, alpha-fluorocinnamate, quercetin and p-chloromercuribenzenesulphonate (pCMBS), and was enhanced in citrate medium. Uptake of D-lactate was less rapid, apparent equilibrium not being reached within 10 min. In contrast to HIT-T15 cells, rat pancreatic islets showed greatly reduced rates of transport of L- and D-lactate together with a correspondingly lower degree of inhibition by alpha-cyano-4-hydroxycinnamate. The addition of L- or D-lactate to HIT-T15 cells, but not dispersed islet cells, resulted in a marked and rapid intracellular acidification followed by a gradual recovery. In both HIT-T15 cells and isolated islets, the rates of transport of both L- and D-lactate in the presence of alpha-cyano-4-hydroxycinnamate were significantly greater in a depolarising K+ medium compared to the normal Na+ medium. These observations suggest that native rat islet cells have considerably reduced activity of the lactate-/H+ transport system compared to HIT-T15 insulinoma cells. There is evidence in both cell types of an additional electrogenic pathway for lactate which might play a role in coupling lactate efflux to beta-cell depolarisation.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Acute stimulation of pancreatic islets by inhibitors of lactic acid transport.

The transport of L-lactate into rat pancreatic islets and RINm5F insulinoma cells was inhibited by alpha-cyano-4-hydroxycinnamate, alpha-fluorocinnamate, quercetin and by p-chloromercuribenzene-sulphonic acid. The addition of each of these compounds to perifused islets resulted in an immediate, marked stimulation of insulin release. Enhanced insulin secretion was accompanied by a similarly rapid and pronounced increase in the rate of 45Ca2+ efflux from pre-loaded, perifused islets. In general, these stimulatory effects were most pronounced in the presence of a threshold concentration of glucose (5 mM) in the perifusion medium. In islets pre-loaded with 86Rb+, the addition of alpha-fluorocinnamate or quercetin caused a modest diminution in efflux rate whilst enhanced rates of 86Rb+ outflow were apparent in the presence of 5 mM glucose. It is suggested that these inhibitors of lactic acid transport stimulate the beta-cell, at least in part, by increasing the intracellular: extracellular lactate gradient, thereby promoting the electrogenic efflux of endogenous lactate from the cell.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Studies of the mechanism of activation of HIT-T15 cells by lactate.

L-Lactate, D-lactate, propionate and acetate (all 20 mM) caused a marked intracellular acidification in HIT-T15 cells loaded with 2'7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF), followed by recovery to more alkaline values. The effects of L- and D-lactate, but not propionate or acetate, were inhibited by 5 mM alpha-fluorocinnamate. Both L- and D-lactate caused a marked depolarisation and rise in cytosolic [Ca2+] in HIT cells as assessed by oxonol-V and quin2 fluorescence, respectively. Propionate had similar, though less marked, effects, whereas acetate exerted only a modest influence on membrane potential and cytosolic [Ca2+]. The rate of oxidation of L-lactate by HIT cells greatly exceeded that of D-lactate. alpha-Fluorocinnamate delayed, but did not prevent, the effects of L-lactate on HIT cell membrane potential or cytosolic [Ca2+]. L-lactate diminished the rate of efflux of 86Rb+ from preloaded HIT cells. Inhibition of calcium- and nucleotide-sensitive K+ channels with tetraethylammonium and tolbutamide also reduced the 86Rb+ efflux rate, and prevented any further reduction in response to L-lactate. However, such inhibition of K+ channels did not prevent a further depolarisation and rise in cytosolic [Ca2+] upon the subsequent addition of lactate. It is suggested that the activation of HIT-T15 cells by lactate is not the result of intracellular acidification or increased metabolic flux, and does not require diminished K+ permeability. An alternative mechanism is based upon the possible electrogenic flux of lactate across the plasma membrane.

Acetates↗
PubMed1991

The role of RAP1 in the regulation of the MAT alpha locus.

The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, [sequence: see text], and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2. We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region. Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription. Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA. These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.

Base Sequence↗