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We examined the contribution of signal-transduction pathways to acetylcholine-induced insulin release in the clonal beta-cell line HIT-T15. To assess the importance of changes in cytosolic free Ca2+ [( Ca2+]i), we studied time courses of the effects of glucose and acetylcholine on [Ca2+]i and insulin release in quin 2-loaded HIT cells. Incubation in the presence of glucose (2 mM) resulted in a sustained increase in [Ca2+]i in HIT cells from 98 +/- 7 nM to 195 +/- 12 nM measured after 9 min, whereas subsequent addition of acetylcholine (50 microM) produced a transient increase in [Ca2+]i which reached a peak after 30 s (at 274 +/- 10 nM), returning to pre-stimulus levels after 3 min. In contrast, incubation of HIT cells with acetylcholine in the presence of glucose produced a sustained increase in insulin release over and above that stimulated by glucose alone; after 10 min acetylcholine had potentiated glucose-stimulated insulin release by an additional increment of 135%. The transient increase in [Ca2+]i induced by acetylcholine was dose-dependent, and was prevented by omission of glucose or extracellular Ca2+ from the incubation medium. It was also inhibited by inclusion of 50 microM-verapamil in the incubation medium (by 87 +/- 3%) or by decreasing the Na+ concentration in the medium (by 73 +/- 6%). To evaluate the role of the protein kinase C pathway, we have pretreated HIT cells with the phorbol ester 12-O-tetradecanoylphorbol acetate (TPA), to deplete the protein kinase C activity, and have compared their secretory activity with that of control cells. Protein kinase C activity was decreased by 73% in HIT cells cultured in the presence of 200 nM-TPA for 22-24 h. TPA pre-treatment also significantly decreased the insulin content of HIT cells, but had no effect on cell number or the increases in [Ca2+]i induced by glucose or acetylcholine. TPA-pre-treated cells responded comparatively less well to secretagogues than did control cells: glucose-stimulated insulin release was decreased by 40%, whereas potentiation by TPA was significantly decreased by 50% in comparison with control cells (P less than 0.05, n = 24). Acetylcholine (50 microM) potentiated glucose-stimulated insulin release by 61% in control cells. This effect was abolished in HIT cells pre-treated with TPA, whereas these cells still retained their normal secretory response to stimulation by forskolin. These data suggest that an early increase in [Ca2+]i may be important for the initial increase in insulin release induced by acetylcholine in HIT cells.(ABSTRACT TRUNCATED AT 400 WORDS)

We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal beta-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mM increased Cai by 36%, from 89 +/- 4 to 121 +/- 6 nM (mean +/- S.E.M., n = 23). Further raising the medium glucose concentration to 10 mM increased Cai to 139 +/- 6 nM. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mM K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 +/- 48 nM. The increases in Cai in response to glucose of K+ were blocked by the simultaneous presence of verapamil (50 microM). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.

Twelve hundred and fifty-one pregnant women were contacted and their infants followed to age 5 years. Nine hundred and fifty-one (76%) children completed the trial. Measurements were made of birthweight, length, head circumference, and triceps skinfold at 10 days, and of weight, height, head circumference, and triceps at 5 years. Throughout the entire period of the study half the families, selected at random, were supplied with milk tokens entitling the mother, while pregnant, and all children under age 5 years, to 1 pint of milk a day at half its current price. The tokens led to a small increase in milk purchases by the families and to a small increase in milk drunk by the children. However no effect was detected in any growth measurement either in the total group, or in a more "vulnerable" group of children from the largest families.

Extensive planning and coordination are the keys to successful implementation of a hospital-wide, computer-based patient information system. With user involvement, problems can be minimized and benefits realized. Nursing administrators can avoid common pitfalls by following the example of one hospital staff in meeting the challenge of computerization.

Two family constructs--prominence and interpersonal distance--are examined. Their origins are traced in the experimental and theoretical literature. The validity of each construct is investigated using data obtained from 15 families with a symptom-bearing child. Validity is supported in both cases through the occurrence of significant correlations among different measures of the same construct. It is suggested that the current distress of the families being studied may have increased the clarity of the prominence hierarchies and dyadic distances that emerged.

We have shown by genetic mapping, molecular cloning, and DNA sequencing that four Escherichia coli mutants, which express the adaptive response to alkylation damage constitutively, are mutated in the ada gene. All four mutant ada genes have two GC to AT transition mutations in the coding region and encode altered Ada proteins with two amino acid substitutions in the N-terminal domain. E. coli carrying the mutated ada genes on recombinant plasmids overexpressed both the mutated ada gene and the chromosomal alkA gene. This observation indicates that the mutant Ada proteins act as strong positive regulators of the ada and alkA genes in the absence of DNA alkylation. One mutant protein, Ada-11, was shown to be a strong activator of ada gene expression in a cell-free system. An altered pattern of tryptic digestion of the Ada-11 protein compared with the wild-type Ada protein suggested that it has a different conformation. One amino acid substitution, namely methionine residue 126 replaced by isoleucine, occurred in all four mutant Ada proteins, and this mutation alone was sufficient to convert the Ada protein into a strong activator of ada and alkA gene expression in vivo.

The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.

The hospital records of 951 children from a previously established birth cohort for which behavioral and extensive background information was available were checked for 3 years following the fifth birthday of the youngest child. The aim was to determine whether children with certain specific behavior disorders had a higher rate of injury than those without. Although the predicted relationships between overactive behavior, decreased concentration, and injury rate were not found, the hypothesis of an increased risk of injury for boys and for children with discipline problems was confirmed. Also, the association between male sex and injury remained after controlling for the behavior variables related to injury. In general, some support was found for the suggestion of increased risk of injury among children who have specific behavior problems, although, contrary to widespread belief, this relationship accounts for only a small percentage of the injuries found in the cohort. Moreover, the relationship is modified by maternal characteristics and by the sex of the child. Unexpectedly, encopresis and marked fears were found to be risk factors for injury, although these effects were relatively small.

Occipitoatlantoaxial malformation was diagnosed in a 19-month-old mare of Appaloosa breeding and in a 3-year-old Quarter Horse gelding. Both horses had abnormal head and neck carriage since birth, but neurologic deficits did not become evident clinically until the horses reached 2 and 3 years of age, respectively. Palpation and manipulation of the base of the skull and cervical area proved to be useful diagnostically. Movement could not be elicited at the atlantooccipital joint but, in comparison with clinically normal horses, the range of dorsoventral motion at the atlantoaxial joint was increased. Alternate subluxation and relocation of this joint generated audible clicking sounds. Radiography revealed symmetric atlantooccipital fusion, with modification of the atlas, atlantoaxial joint, and axis. These findings were confirmed at necropsy.

A recent case of ovarian pregnancy, the diagnosis of which is often retrospective, is presented. The discussion is based on current literature concerning the diagnostic difficulties associated with this condition.

The results of a randomized controlled trial of a single daily dose of acetyl salicylic acid (aspirin) in the prevention of reinfarction in 1,239 men who had had a recent myocardial infarct were statistically inconclusive. Nevertheless, they showed a reduction in total mortality of 12% at six months and 25% at twelve months after admission to the trial. Further trials are urgently required to establish whether or not this effect is real.

A clinical and functional study of rheumatoid elbows has been done to discover the magnitude and frequency of disabilities caused by elbow problems. Pain and weakness were found to affect all activities which were tested, with adjacent joint involvement, flexion deformity, and lack of supination causing significant disability. Instability was not a frequent problem. The frequency of functional disabilities, linked to severe pain, suggests that the development of an elbow joint replacement would be worthwhile.

Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca2+: at or below 10 microM Ca2+ no insulin secretion was evoked by glucose in freshly isolated islets. The threshold Ca2+ requirement was increased after culture of islets for 44 h. In both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for extracellular Ca2+. Thus potentiation of insulin release involves an increase in the sensitivity of the B cell to Ca2+.

We have examined the relationship between extracellular Ca2+, cytosolic free Ca2+ and insulin release in the clonal beta-cell line HIT-T15. Glucose-stimulated insulin release was dependent on the extracellular Ca2+ concentration in a dose-related manner; the threshold medium Ca2+ concentration for glucose-stimulated insulin release was 0.5 mM. Both forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased insulin release in the presence of glucose at all extracellular Ca2+ concentration tested (0.1-2.5 mM) but not in the absence of Ca2+. Thus, the threshold medium Ca2+ concentration for glucose-stimulated insulin release was reduced to 0.1 mM by forskolin or TPA. Step-wise increases in the medium Ca2+ concentration in the presence of an initiator of insulin release resulted in a dose-related increase in cytosolic free Ca2+. In the presence of 10 mM glucose, cytosolic free Ca2+ in HIT cells was increased from 60 +/- 5 nM in Ca2+-free medium to 290 +/- 46 nM in medium containing 2.5 mM Ca2+. The effects of increasing extracellular Ca2+ in the presence of 40 mM K+ were similar but considerably more pronounced. Inclusion of either TPA or forskolin in the incubation medium had no significant effect on the steady-state cytosolic free Ca2+ levels in the absence of glucose but in the presence of 10 mM glucose forskolin caused modest (11-18%) increases in steady-state cytosolic free Ca2+ levels at extracellular Ca2+ concentrations of 0.25 mM or above. In contrast, in the presence of glucose TPA significantly reduced the steady-state levels of cytosolic free Ca2+ by 17-21% at extracellular Ca2+ concentrations of 0.25 mM or above. These data provide further evidence that insulin release mediated by activation of beta-cell protein kinases involves primarily an increase in sensitivity of the secretory system to intracellular Ca2+.