Biomedical subjects

K L Knutson

Publications and source records attributed to K L Knutson.

23 records · Page 2Linked to original sources

PubMed1996

Regulation of distinct pools of protein kinase C delta in beta cells.

Previous studies from our laboratory have demonstrated the presence of several isoforms of protein kinase C (PKC), Ca(2+)-independent and Ca(2+)-dependent, in both whole islets and tumor-derived beta cells. In the basal state, a major proportion of the isoform was found in the crude membrane fraction with smaller amounts found in both the cytosolic and cytoskeletal fractions. Whole islets showed a similar distribution of the isoform. These studies were done to analyze the effects of insulin secretagogues on the distribution of PKC delta to different cellular pools in isolated insulinoma beta cells. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), produced a transient association of PKC delta with the beta cell cytoskeleton along with sustained decreases in cytosolic enzyme and transient increases in membrane enzyme. Neither glucose nor carbachol could acutely affect the subcellular distribution of PKC delta. Oleic acid decreased the amount of the enzyme associated with the cytoskeleton and led to a sustained decrease of cytosolic enzyme and a transient increase in membrane enzyme. Oleic acid was also able to prevent the increase in cytoskeletal enzyme induced by PMA. Both oleic acid and PMA potentiated glucose-induced insulin release but oleic acid, in contrast to PMA, was unable to initiate insulin release in the presence of substimulatory concentrations of glucose. These data demonstrate that different activators of PKC may have different effects on localization of the enzyme within the cells and suggest that there are at least three apparently distinct pools of PKC delta within the beta cell which may be important in insulin secretion or other aspects of beta cell function.

Animals↗

PubMed1994

Commercial synthesis of alpha-D-cellobiosyl bromide heptaacetate.

A detailed commercial process for the synthesis and purification of the activated disaccharide, alpha-D-cellobiosyl bromide heptaacetate (1) was developed. Reaction of alpha-D-cellobiose octaacetate (CBO) with HBr in glacial acetic acid or in glacial acetic acid/methylene chloride combination affords alpha-D-cellobiosyl bromide heptaacetate in high yield and excellent quality. Process variables such as reaction solvent, reaction time, reaction temperature, HBr stoichiometry, isolation methods and product purification options were optimized for large-scale synthesis. alpha-D-Cellobiosyl bromide heptaacetate was successfully prepared in a commercial manufacturing plant.

Acetates↗

PubMed1994

Effect of protein kinase C on the plasma membrane calcium pump in purified beta cells.

The effect of protein kinase C activation on (Ca(2+)-Mg2+)-ATPase and 45Ca2+ uptake in purified plasma membranes and membrane vesicles from beta cells was examined. PKC activation was achieved by incubating cells for 10 or 30 min in 100 nM or 1 microM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and evident by translocation of the alpha-isoform from the cytosolic to the membrane fraction. (Ca(2+)-Mg2+)-ATPase had a Km for Ca2+ of 0.56 +/- 0.17 microM and the Vmax was 120 +/- 12 nmol/min*mg protein in membranes from cells treated with TPA, while it was 0.66 +/- 0.14 microM and 135 +/- 19 nmol/min*mg protein, respectively, in its absence. In inside-out vesicles 45Ca2+ uptake had a Km for Ca2+ of 79 +/- 19 nM and a Vmax of 1.68 +/- 0.43 nmol/min*mg protein in the presence of TPA. In the absence of TPA, the Km was 71 +/- 17 nM, and the Vmax was 1.59 +/- 0.39 nmol/min*mg protein, respectively. It is concluded that in beta cells PKC activation does not regulate (Ca(2+)-Mg2+)-ATPase activity or Ca2+ transport directly.

Animals↗

PubMed1994

Identification and subcellular characterization of protein kinase-C isoforms in insulinoma beta-cells and whole islets.

Protein kinase-C (PKC) represents a growing family of serine/threonine kinases, which include both Ca(2+)-dependent and Ca(2+)-independent members. To evaluate the expression of PKC isoforms in insulin-secreting beta-cells, purified beta-cells from a glucose-sensitive rat insulinoma were fractionated into cytosolic, crude membrane, and cytoskeletal/nucleoskeletal fractions. Protein samples from each fraction were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to nylon membranes. The blots were then analyzed with antibodies specific for the alpha, beta, gamma, epsilon, zeta, and delta isoforms. In addition, expression was analyzed in whole isolated rat islets. Expression of all except the gamma isoform was detected in the insulinoma-derived beta-cells. Expression of the alpha, beta, and epsilon isoforms was confined predominantly to the cytosolic fractions. The delta isoform could be detected in all three of the subcellular fractions, whereas the zeta isoform was present in approximately equal amounts in both the cytosolic and crude membrane fractions. The delta isoform could be eluted from the cytoskeletal/nucleoskeletal fraction with 1% Triton X-100. All of the isoforms detected in the insulinoma-derived beta-cells were also detected in whole isolated islets. It is concluded that rat insulinoma beta-cells and whole islets express numerous isoforms of PKC, including both Ca(2+)-dependent and Ca(2+)-independent isoforms, which may be important in the various signal transduction processes of insulin secretion, proinsulin biosynthesis, and insulin gene expression.

Animals↗

PubMed1993

In vitro analysis of the accumulation and toxicity of inorganic mercury in segments of the proximal tubule isolated from the rabbit kidney.

Cellular accumulation and toxicity of inorganic mercury were studied in suspensions (1 mg protein/ml buffer) of proximal tubular segments isolated from the kidneys of rabbits. Mercuric chloride containing trace amounts of radiolabeled inorganic mercury (203Hg2+) was added to the buffer to produce a concentration of inorganic mercury ranging from 0.1 to 10 microM. Significant release of lactate dehydrogenase (LDH) and significant decreases in oxygen consumption (QO2), which were used as indices of cellular injury, were detected only when the tubules were in the presence of 10 microM inorganic mercury. At this concentration of inorganic mercury, cellular release of LDH increased and QO2 decreased significantly between the 1st and 4th hr of exposure, by which time most of the proximal tubular cells were necrotic. Maximal cellular content of inorganic mercury was attained within the first 5 min of exposure, during which time nearly 70% of the inorganic mercury in the bath was removed. Accumulation of mercury was more gradual when the tubules were exposed to 0.1 microM inorganic mercury. Addition of 40 microM glutathione, cysteine, or bovine serum albumin to the bath provided the segments of the proximal tubule with complete protection from the toxic effects of 10 microM inorganic mercury. The rate of uptake of inorganic mercury was also significantly decreased. By the end of 4 hr of exposure only about 30% of the content of mercury in the bath was abstracted. These findings indicate that isolated segments of proximal tubules take up inorganic mercury very rapidly and subsequently become intoxicated. They also show that when compounds containing free sulfhydryl groups are in the presence of inorganic mercury in the bath, the rate of uptake of inorganic mercury is significantly decreased and the tubules are provided protection from the toxic effects of the inorganic mercury.

Animals↗