Biomedical subjects

H C Cable

Publications and source records attributed to H C Cable.

13 recordsLinked to original sources

PubMed2000

LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis.

Using an expression cloning system to discover novel genes involved in apoptosis, we identified a 326 bp bone marrow cDNA fragment (termed Je2) that suppresses, upon transfection, CD95-mediated apoptosis in Jurkat T cells. Sequence homology revealed that Je2 maps to 3p21.3, to an intronic region of the candidate TSG LUCA-15 locus. It represents, in fact, an antisense transcript to the 3'-UTR of two novel splice variants of this gene. Overexpression of sequence representing one of these splice variants (a 2.6 kb cDNA termed Clone 26), inhibited proliferation of Jurkat cells and sensitized them to CD95-mediated apoptosis. This study therefore implicates the LUCA-15 gene locus in the control of apoptosis.

Alternative Splicing↗

PubMed1996

Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

Base Sequence↗

PubMed1995

Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells.

1. Alpha-2-adrenoceptor agonists, such as noradrenaline, are potent inhibitors of insulin secretion, and it has been suggested that they control a late step in the pathway of exocytosis. We have investigated whether this could be related to a change in the extent of actin polymerization in the pancreatic B-cell, since actin microfilaments are implicated in regulating the access of secretory granules to the plasma membrane prior to exocytosis. 2. Cultured HIT-T15 pancreatic B-cells responded to noradrenaline with an increase in F-actin content, as judged by a rise in the fluorescence output after probing of the cells with phalloidin (a toxin which binds specifically to F-actin) conjugated to rhodamine. The response to noradrenaline was rapid, dose-dependent and sustained and could be reproduced by the highly selective alpha-2-agonist UK14,304. Examination of HIT-T15 cells by fluorescence microscopy after treatment with rhodamine-phalloidin, revealed a significant localization of F-actin immediately adjacent to the plasma membrane. The pattern of F-actin distribution in the cells was not altered dramatically by noradrenaline, although the intensity of staining close to the plasma membrane appeared to be slightly reduced. 3. The increase in F-actin content induced by noradrenaline and UK14,304 was inhibited significantly by the alpha-2-antagonist idazoxan but not by the alpha-1-selective antagonist prazosin. Pretreatment of HIT-T15 cells with pertussis toxin did not lead to any direct alteration in F-actin content, although the toxin significantly modified the responses induced by noradrenaline and UK14,304. In each case, cells incubated for 24 h with pertussis toxin responded to the alpha-2-agonist with an enhanced fluorescence output, indicating that F-actin levels had increased still further. This did not correlate with any gross change in the distribution of F-actin as judged by fluorescence microscopy. 4. The results demonstrate that alpha-2-adrenoceptors are coupled to control of actin polymerization in HIT-T15 cells. They suggest that regulation of F-actin formation could be a component of the mechanism by which alpha-2-agonists mediate inhibition of insulin secretion.

Actins↗

PubMed1994

Treatment of cultured pancreatic B-cells with streptozotocin induces cell death by apoptosis.

Treatment of cultured pancreatic B-cells (HIT-T15 and RINm5F) with the diabetogenic drug streptozotocin resulted in a significant increase in the number of cells that became detached from the substrate during a subsequent culture period. Examination of the detached cells by fluorescence microscopy after staining with acridine orange or by electron microscopy revealed evidence of chromatin condensation and margination. Isolation and fractionation of DNA from these cells revealed a pattern of oligonucleosomal fragmentation that was not evident in untreated cells. All of these features are characteristic of entry of the cells into apoptosis and the results suggest that the diabetogenic action of streptozotocin involves induction of apoptosis in pancreatic B-cells.

Animals↗

PubMed1993

Concentration-dependent effects of adrenaline on the profile of insulin secretion from isolated human islets of Langerhans.

The effects of the mixed alpha/beta-agonist adrenaline on insulin secretion from isolated human islets of Langerhans were studied. In static incubation experiments, adrenaline (0.1 nmol/l to 10 mumol/l) caused a concentration-dependent inhibition of glucose-induced insulin secretion from isolated human islets. However, perifusion experiments revealed that the time-course of the secretory changes induced by adrenaline was complex. When employed at a high concentration (1 mumol/l), adrenaline caused a sustained inhibition of glucose-induced insulin secretion, which could be relieved by the addition of the alpha 2-antagonist yohimbine (10 mumol/l). By contrast, infusion of adrenaline at a lower concentration (10 nmol/l), produced a large initial potentiation of glucose-induced insulin secretion. This response was, however, short-lived and followed by sustained inhibition of secretion, which could be relieved by yohimbine (10 mumol/l). The initial stimulation of insulin secretion provoked by 10 nmol adrenaline/l was abolished when islets were incubated in the presence of the beta-antagonist, propranolol (1 mumol/l), consistent with activation of beta-adrenoceptors. In support of this, treatment of human islets with the selective beta 2-agonist clenbuterol, was also associated with marked stimulation of insulin secretion. By contrast, each of two selective beta 3-agonists tested failed to alter insulin secretion from human islets. The results indicate that human pancreatic B-cells are equipped with both alpha 2- and beta 2-adrenoceptors which can affect insulin secretion. Adrenaline interacts with both of these but the alpha 2-response is predominant and can overcome the tendency of beta 2-adrenoceptors to potentiate insulin release.

Clenbuterol↗

PubMed1987

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro.

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

Acrylates↗

PubMed1986

Pinocytic capture and exocytosis of rat immunoglobulin IgG-N-(2-hydroxypropyl)methacrylamide copolymer conjugates by rat visceral yolk sacs cultured in vitro.

Rat immunoglobulin (IgG) was covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via glycylglycyl spacer. The resultant conjugate, free IgG and HPMA copolymer (containing a low percentage of tyrosinamide to facilitate radiolabelling) were radioiodinated, and their rates of pinocytic uptake, intracellular degradation and exocytic release by rat visceral yolk sacs cultured in vitro were determined. Free IgG was pinocytosed rapidly by the yolk sac and some IgG was subject to intracellular proteolysis. In comparison the IgG-HPMA copolymer conjugate was captured more slowly, but faster than unmodified HPMA. IgG was also exocytosed rapidly by the yolk sac following pinocytic capture and similarly IgG-HPMA copolymer had a much higher rate of release than unmodified HPMA. Measurement of tissue accumulation of 125I-labelled IgG-HPMA copolymer in the presence of increasing concentrations of non-radiolabelled IgG showed competition for membrane binding sites between the free, and polymer-bound immunoglobulin. These experiments indicate that immunoglobulins can be covalently bound to a soluble polymer developed as a drug-carrier in such a way that they can still interact with specific membrane receptors and they are subsequently subjected to specific cellular transport mechanisms.

Animals↗

PubMed1985

Characterization of the adsorptive pinocytic capture of a polyaspartamide modified by the incorporation of tyramine residues.

Previously it has been shown (Duncan, R., Starling, D., Rypácek, F., Drobník, J. and Lloyd, J.B. (1982) Biochim. Biophys. Acta 717, 248-254) that incorporation of tyramine residues into poly (alpha, beta-(N-2-hydroxyethyl]-DL-aspartamide (PHEA) greatly increases its rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro. Here we describe the relationship between the tyramine content (1.2-21.9 mol%) of modified PHEA and its rate of uptake by yolk sacs. Above a level of substitution of approximately 10 mol% the rate of uptake rises rapidly, and the concentration-dependence of capture is indicative of uptake by adsorptive pinocytosis. Serum proteins were shown to compete effectively for membrane binding sites, indicating a nonspecific interaction of PHEA-derivatives with the yolk sac membrane. PHEA derivatives of the same tyramine content, but of different mean molecular weights (Mr), were captured at the same rates.

Adsorption↗

PubMed1984

Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

2,4-Dinitrophenol↗

PubMed1982

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage.

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and 125I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[125I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of 125I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of Mr 120 000 was internalized by macrophages somewhat more rapidly than copolymer of Mr 46 000, but was excluded from the yolk sac.

2,4-Dinitrophenol↗

PubMed1982

Degradation of side-chains of N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal thiol-proteinases.

N-(2-Hydroxypropyl)methacrylamide copolymers bearing oligopeptide side-chains terminating in p-nitroaniline (NAp) were incubated with rat liver lysosomal enzymes in the presence of the thiol glutathione, and the rate of p-nitroaniline release was measured. Twelve of the 16 side-chains investigated were hydrolysed to release p-nitroaniline and in all but one case degradation was partially or totally inhibited by leupeptin. The effect of substrate concentration on the degradation of the most readily cleaved side-chain, -Ala-Gly-Val-Phe-NAp, was measured.

Acrylamides↗

PubMed1981

Effect of molecular size of 125I-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages.

Rates of pinocytosis of different molecular-weight distributions of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs and rat peritoneal macrophages were measured in vitro. Four preparations of mean molecular weights 50 000, 84 000, 700 000 and 7 000 000, were used. Macrophages captured the highest-molecular-weight preparation more rapidly than the other preparations. In contrast, rate of capture by the yolk sac decreased with increasing molecular weight. Incubations with a very-high-molecular-weight fraction derived from the 7 000 000-average-mol. wt. preparation clearly demonstrated that very large polymer molecules are not accumulated by the yolk sac, but are preferentially captured by macrophages. Analysis of the 125I-labelled poly(vinylpyrrolidone) internalized by the two cell types confirmed that low-molecular-weight material is preferred by the yolk sac, whereas the macrophage is less discriminating.

Animals↗

PubMed1981

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis.

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of 125I-labeled PVP and colloidal 198Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [14C]DIVEMA and 125I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.

Animals↗