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Large scale quantities of phosphodiester and phosphorothioate oligonucleotides are synthesized on an aminopolyethyleneglycol derivatized polystyrene (TentaGel) support. Efficient, automated synthesis up to 1 mmole scale is achieved with phosphoramidite nucleoside monomers and 5-ethylthiotetrazole activator.

Study design This study was designed to determine the current experience in the use of chymopapain injection in the treatment of herniated intervertebral discs by analyzing reports appearing between 1985 and 1993. Forty-five clinical studies included 7,335 patients treated worldwide, some including comparisons to open laminectomy/discectomy and others to percutaneous discectomy. Objectives Because controversy persists after 30 years of clinical use of chymopapain, the results of current experience should establish the efficacy for those who want to consider chemonucleolysis as a treatment for a herniated nucleus pulposus. Summary of background data There is the suggestion that use of other than conservative treatment is made only to achieve a better result in the short term. The selection of type of treatment will depend on contraindications, with failures of chemonucleolysis found largely in those having spinal stenosis or sequestrated discs. Worker compensation patients respond less successfully than those with better motivation. Methods The 45 studies were analyzed for determination of successful outcome and divided into 16 with more than 100 patients, 13 with less than 100 patients and 16 with comparison to other treatments. Results Individual success rates exceeded 60% whereas cohort total averaged 76%. In studies comparing chemonucleolysis with open discectomy, success rate averaged 76.2% as compared with 88% for open surgery. In two other studies, percutaneous discectomy was less successful than chemonucleolysis. Where included, duration of hospitalization showed less time and thus less costs for chemonucleolysis. Return to work complications showed time off slightly less for chemonucleolysis than for laminectomy. Conclusions Chemonucleolysis, though somewhat less effective than open discectomy, can be successfully and safely used in about four of five carefully selected patients without the trauma, risks, and subsequent fibrosis associated with lumbar disc surgery.

Two high-strength low-alloy (HSLA) families, acicular-ferrite steels and pearlite-reduced steels, contain microalloying additions of vanadium and niobium. Vanadium, niobium, and titanium combine preferentially with carbon and/or nitrogen to form a fine dispersion of precipitated particles in the steel matrix. This article summarizes the metallurgical effects of vanadium, niobium, molybdenum, and titanium. The metallurgical fundamentals were first applied to forgings in the early 1970s. The ultimate strength of first- and second-generation microalloy steels is adequate for many engineering applications, but these steels do not achieve the toughness of conventional quenched and tempered alloys under normal hot-forging conditions. Third-generation microalloy steels differ from their predecessors in that they are direct quenched from the forging temperature to produce microstructures of lath martensite with uniformly distributed temper carbides. Without subsequent heat treatment, these materials achieve properties, including toughness, similar to those of standard quenched and tempered steels.

SummaryRats fasted for periods of 24, 48, or 72 hr showed no changes in plasma glucagon concentration despite lowered blood glucose concentration. Islets isolated from both fed and fasted animals elicited biphasic glucagon and insulin secretion when stimulated with arginine and glucose, respectively. There were no differences in glucagon secretion rates between islets prepared from fed and fasted animals. Significant reductions in both the first and second phases of insulin secretion were observed in islets prepared from fasting animals.The authors wish to thank Ms. Carol Grimme and Ms. D. Barr for typing the manuscript, and Dr. A. Pagliara for anti-glucagon serum. This work was made possible by Public Health Service Grant AM-165 34.

Glucagon secretion and its control have been studied in perifused isolated islets of Langerhans of the rat. It was shown that a low concentration of glucose per se does not cause increased glucagon secretion, but that at low glucose concentrations the amino acid arginine stimulates a biphasic secretory response. Such amino acid stimulated glucagon secretion can be suppressed by increasing the glucose content of the perifused media from 1.67 to 5.5 or 16.7 mM; insulin secretion is also then increased. Since high concentrations of added porcine insulin (10 mU/ml) did not affect amino acid stimulated glucagon secretion at low glucose concentration, it was concluded that high concentrations of glucose and not insulin secreted in response to that glucose are probably responsible for suppression of glucagon secretion. At low concentrations of glucose, epinephrine (2.5 X 10(-7) M) also stimulated glucagon secretion. It is concluded that isolated rat islets of Langerhans can be used for the study of glucagon secretion in vitro, and that substances appearing in the blood in vivo at low glucose concentrations are probably responsible for increased glucagon secretion under conditions associated with hypoglycemia.

This investigation was initiated to characterize the stimulation of insulin secretion by phenazine methosulfate (PMS). Islets of Langerhans, isolated by the collagenase method from normal rats and rats pre-injected with either streptozotocin or 6-aminonicotinamide, were exposed to PMS under various experimental conditions and insulin secretion in response to PMS, glucose and pyridine nucleotides was determined. Insulin releasing action of PMS was dose-, time- and temperature-related, occurred in the absence of glucose, and was inhibited by epinephrine, but not by mannoheptulose. In the perifusion system, the pattern of response induced by PMS was spike-like release reaching a maximum in 5 min and declining rapidly to half-maximal value in 10 min. After exposure of islets to beta-cytotoxin either in vivo or in vitro, complete reversal of the cytotoxic effect was obtained with PMS which induced release of insulin in both normal and beta-cytotoxin islets pre-treated. It is concluded that islets depleted of coenzymes could still secrete insulin in response to a reactive proton donor, which might act by substituting for coenzymes and that the immediate action of beta-cytotoxins does not completely arrest the secretory mechanisms in islets of Langerhans.

The distribution of insulin injected into normal rats and guinea pigs as the free hormone or as insulin-antibody complexes was studied by extraction of immunoreactive insulin and by following the fate of125I- or131I-labeled insulin in the plasma, liver, kidneys and spleens of the injected animals. Injected free hormone rapidly disappears from the plasma to accumulate transiently in the liver, and, to a greater extent, in the proximal convoluted tubules of the kidneys. When insulin-antibody complexes formedin vitro are injected, the hormone disappears more slowly from the plasma and concentrates in the liver and spleen where it can be demonstrated by autoradiography in reticulo-endothelial cells; little or none is found in the kidneys. After injection into insulin-immunized guinea pigs, the hormone disappears very slowly from the plasma and accumulates almost exclusively in the liver with no evidence of concentration by any particular cell type; little or none is found in the kidneys or spleen.

The effect of streptozotocin (STZ) on the activity of the pentose phosphate shunt in islets was studied. Isolated rat islets were pre-incubated with glucose (1.7 mM) alone or with streptozotocin (STZ) or N-methyl-N-nitrosourea (MNU). The effects of these pretreatments on glucose metabolism and insulin secretion were assessed during subsequent incubation with either (1.14C), (6.14C). or (U.14C). glucose (16.7 mM) alone or plus phenazine methosulfate (PMS). Islets pretreated with STZ (1.5 mM) metabolized less (1.14C) and (U.14C). glucose. The order of inhibition by STZ of (14C)-glucose metabolism by islets was: (1.14C). greater than (U.14C). greater than (6.14C)-glucose. Whereas PMS (0.5 mM) increased the metabolism of both (U.14C). and (1.14C)-glucose, the metabolism of (6.14C)-glucose by STZ-pretreated islets was not increased by PMS. In a separate series of experiments, the total NADP+ + NADPH, but not the NAD content of the islets decreased after 2 min exposure of islets of STZ. At 30-min exposure, the levels of both pyridine coenzymes and that of 6-phosphogluconate were significantly decreased. The level of NADP+ + NADPH in islets was decreased more than the level of NAD. Insulin secretion was suppressed by the nitrosoureas. PMS (0.5 mM) increased the level of NADP+ + NADPH content of islets and augmented insulin secretion. It is concluded that the pentose phosphate pathway is inhibited on brief exposure of islets to STZ or MNU. Such inhibition may contribute to the suppression of insulin secretion caused by these nitrosoureas.

SummaryRelative to body weight, the plasma volumes of 60 normal guinea pigs fell from 5.2 to 3.7 ml/100 g body wt as body weight increased from about 600 to 1200 g. Lower values reported by others are attributed to differences in methodology. Slightly higher plasma volumes were found in 37 insulin-immunized guinea pigs. After exsanguination at the time of death, trapped plasma accounted for 10 to 20% of the weights of the livers, kidneys, and spleens, more being trapped when death occurred before exsanguination could be effected.

Adrenergic drugs (epinephrine, norepinephrine and isoproterenol) inhibit insulin secretion from pieces of rat pancreas incubated with glucose (150 mg/100 ml). This inhibitory effect appears to be mediated through alphareceptors for it is blocked by alpha- but little affected by beta-blocking agents. Cholinergic drugs (acetyl choline and carbamyl choline) have a stimulant effect using media containing glucose in low concentrations (100 mg/100 ml), and this effect is suppressed by simultaneous addition of atropine. These observations suggest that the autonomic nervous system may play a role in the control of insulin secretion under physiological conditions. (Endocrinology 80: 975, 1967)

The insulin-binding potency of guinea pig anti-insulin serum (GPAIS) is determined following reaction for 30 minutes at room temperature between solutions containing GPAIS and mixtures of unlabeled and131I-labeled insulin. Insulin which has not reacted with the antibodies is extracted from solution by a suspension of finely divided cellulose. From the radioactive contents of supernatant solutions from reaction mixtures containing the sample, an excess of non-precipitating (reference) GPAIS, and the buffered diluent, the potency of the sample can be determined. Factors affecting the assay procedure are considered and it is shown that, within defined limits, results are reproduceable and the method sensitive and rapid. The technique has potential uses for the study of insulin-antibody reactions and has practical applications in the production and rational use of GPAIS.

A method is described in detail for the assay of insulin in plasma or serum. It involves titration with labeled insulin of reactive insulin antibodies remaining after preincubation of the sample under assay or of standard solutions of insulin with an excess of guinea pig anti-insulin serum. Labeled insulin bound by antibodies in the assay is precipitated in the presence of alcohol (75 per cent, v/v) at room temperature, unbound labeled insulin then remaining in solution. The technic is simple and assays can be carried out in a few hours. It is shown that individual results are amenable to statistical assessment and suggested that the sensitivity of the method is only limited by the specific activity of the labeled insulin used.

A consistent immune response can be induced in guinea pigs with a single injection of insulin incorporated in a viscous water-in-oil emulsion. A minimum dose of insulin and the presence of a bacterial adjuvant appear essential. Under comparable experimental conditions, bovine insulin is shown to have approximately the same antigenic but much more immunogenic activity than porcine insulin. Very slow disappearance of circulating antibodies in actively immunized animals is thought to be due to sustained antibody synthesis. When administered daily for 30 days methotrexate, cyclophosphamide and predinisolone reduce or abolish the immune response to insulin, but 6-Mercaptopurine and Imuran are ineffective in relatively toxic doses.

Repeated injections of insulin in an adjuvant containing heavy mineral oil and lanolin produces very variable effects on antibody production in guinea pigs. Incorporation of H. pertussis vaccine in the adjuvant used for initial injections raises the proportion of active producers of antibodies and rapidly increases the levels of insulin antibodies in their serum. This adjuvant appears to be more effective than the conventional Freund's adjuvant but results in high mortality if given frequently. Using a simple method of antibody assay, it is possible to segregate nonproductive animals at an early stage and so increase the yield of highly active anti-insulin serum.

Glucagon has recently been shown to have a direct lipolytic action on liver (Bewsher and Ashmore 1966). Increased hepatic lipolysis, plus enhanced availability of plasma fatty acids, causes an elevation of acetyl CoA and fatty acyl CoA levels (Williamson, Herczeg, Coles and Danish 1966). Such elevated levels of acetyl CoA presumably account for the increased rate of ketogenesis observed with glucagon. Acute effects of insulin deficiency, induced by administration of guinea pig anti-insulin serum (AIS) to rats, are similar to effects produced by glucagon: elevation of plasma glucose, free fatty acid, and ketone levels, plus an increased rate of gluconeogenesis (Wagle and Ashmore, 1963 ; Wagle and Ashmore, 1964 ; Tarrant, Mahler, and Ashmore, 1964) . These findings have led to the suggestion that the early metabolic changes in experimental insulin deficiency may be induced by hormones with actions normally counterbalanced by insulin (Wright, 1965) . Such hormones are glucagon, epinephrine, ACTH and corticosteroids (Mahler, Stafford, Tarrant and Ashmore, 1964 ; Jungas and Ball, 1963) . Data presented in this paper supports the concept that the lipolytic, ketogenic, and gluconeogenic effects of glucagon are revealed in the insulin-deficient rat. It is suggested that stimulation of gluconeogenesis, both with glucagon and with AIS, is secondary to enhanced lipolysis and is mediated through facilitation of pyruvic carboxylase by elevated hepatic levels of acetyl CoA.

In diabetes abnormally high rates of gluconeogenesis have been demonstrated by various methods. Here it is confirmed that in rats which have been fasted or treated with alloxan, changes occur in hepatic enzyme activity which are compatible with an adaptation to increased rates of gluconeogenesis; there are increased glutamic-pyruvic and glutamic-oxalacetic transaminase and glucose-6-phosphatase activities and reduced lactic dehydrogenase activity. Comparable effects were demonstrated in the livers of rats killed in a diabetic state twenty-four to sixty hours after injection of guinea pig anti-insulin serum, with the exception that glutamic-pyruvic transaminase activity was not increased and glutamic-oxalacetic transaminase activity was increased when expressed per liver protein or per body weight, but there was no change in the activity of the total liver. This finding provides suggestive evidence that increased gluconeogenesis is also characteristic of this experimental diabetic syndrome produced by anti-insulin serum.

The diabetic syndrome induced in rats by guinea-pig anti-insulin serum is complicated by interstitial pancreatitis similar in several respects to that seen in some infants born of diabetic mothers. It is characterized by lesions in the exocrine portion of the gland which include eosinophilic infiltration (73 per cent), edema (54 per cent), and focal areas of necrosis (15 per cent) or hemorrhage (14 per cent). Its appearance is not related to the severity of the induced diabetes and its nature suggests that it is allergic in origin. Other histological findings are consistent with previous observations and similar to those encountered in human and other forms of experimental diabetes.

After three injections at weekly intervals of insulin in a water-in-oil emulsion containing H. pertussis vaccine, guinea pigs were given the same inoculum containing no vaccine at intervals of four weeks. At the first bleeding after eight weeks, most animals yielded serum binding more than 1.0 U. insulin per milliliter; the mean binding capacities of sera from animals in four out of seven groups exceeded 4.5 U. insulin per milliliter. Over eighteen months, five groups totalling 136 animals yielded 3,000 ml. serum, the pooled sera from each group binding 2.7 to 4.8 U. insulin per milliliter. It is concluded that use of H. pertussis vaccine offers a reliable method for the production of large volumes of potent anti-insulin serum.

SummaryIsolated islets obtained 1 to 2.5 hours after injection of guinea pig anti-insulin serum into normal rats secrete insulin in vitro at an abnormally high rate in response to glucose. Twenty-four hours after induction of the insulin-deficiency, the absolute rate of insulin secretion is subnormal, but is still increased in relation to the low insulin content of these islets. This sequence of changes is seen in pancreatic tissue removed from spontaneously diabetic animals and suggests that this system could be used as a model for the study of islet function during the development of insulin-deficiency.

Addition of glucose (16.7 mM) to isolated pieces of rat pancreas increased insulin secretion 5.4-fold over basal secretion rates. Ether at 1, 1.5 and 2 MAC inhibited this insulinogenic effect of glucose in a dose-related manner by 5, 18 (P less than 0.01) and 29 (P less than 0.01) per cent, respectively.

Previous studies have shown that halothane inhibits glucose-stimulated insulin secretion. This study was designed to determine whether the mechanism of inhibition involves a reduction in glucose metabolism. The effects of halothane on glucose (16.7 mM)-stimulated insulin secretion and glucose oxidation were studied in isolated rat pancreatic islets. Halothane, 0.11 mM (0.5 MAC), 0.22 mM (1.0 MAC), and 0.33 mM (1.5 MAC), inhibited glucose-stimulated insulin release in a dose-related manner by 5.2 per cent (NS), 21.0 per cent (P < 0.05), and 32.6 per cent (P < 0.01), respectively. At the 0.33 mM (1.5 MAC) concentration, halothane did not significantly inhibit the oxidation of 6-14C-glucose to 14CO2, although higher concentrations of halothane did result in significant inhibition. The data suggest that halothane's inhibitory effect on glucose-stimulated insulin secretion is not due to interference with glucose oxidation.

SummarySomatostatin at a final concentration of 1 μg or 10 μg/ml did not affect glucose stimulated oxidation of either D-[14C]1 or D-[14C]6 glucose, but inhibited both insulin release and cyclic AMP accumulation by isolated rat islets of Langerhans.

SummaryThe reactions of porcine proinsulin and its degradation products with insulin antibodies in guinea pig anti-insulin serum can be shown to vary with the immunological system used for study. From the results obtained with two such systems it is concluded that the connecting chain of amino acids in the molecule of proinsulin must mask sites on the insulin molecule which are capable of reacting with insulin antibodies.

The sulfonylureas, tolbutamide, tolazamide and acetohexamide significantly diminish uptake of insulin by the isolated perfused rat liver, whereas the nonhypoglycemic metabolite of tolbutamide, carboxytolbutamide, has no such effect. These results suggest that the sustained action of the sulfonylureas may in part be due to reduced hepatic uptake of endogenously secreted insulin.

A method is described for the assay of insulin. It involves pre-incubation of insulin under assay with an excess of guinea pig anti-insulin serum and subsequent incubation of the partially neutralized serum with an excess of unlabeled and/or I-131-labeled bovine insulin. If no more than about half of the antibodies in the serum are neutralized during pre-incubation, then the amount of bovine insulin bound during incubation decreases linearly and in proportion to the amount of pre-incubated insulin. Such a linear relationship was obtained with nine samples of pooled serum from uve groups of guinea pigs and was shown to be applicable over a wide range of serum and insulin concentrations. Since neutralized acid-alcohol affected the assay only when present in relatively high concentrations, the method has been applied to neutralized acid-alcohol extracts of pancreatic tissue without removal or excessive dilution of the alcohol. It is applicable at concentrations of insulin to be found in blood and is therefore a potentially simple and rapid method which would not require the use of labeled insulin of high specific activity. The uses and limitations of the method are discussed.

SummaryIsolated Islets of Langerhans from the rat's pancreas were incubated in media containing varying combinations of glucose, rat insulin, and guinea pig anti-insulin serum (GPAIS). Neither insulin nor GPAIS modified the rate at which insulin was secreted in response to glucose. The concept that insulin in the fluids surrounding Islet tissue can inhibit secretion of insulin by the β-cells is discussed in the light of these results.

THE antigenic properties of insulin are now well recognized, but no simple method has yet been described for the routine detection and assay of insulin antibodies. The method described here is based on the classical observation by Berson, Yalow, Bauman, Rothschild and Newerly1. They showed that during chromato-electrophoresis on filter paper, 131I-labelled insulin bound by insulin antibodies in human serum moves with the β-γ-globulins, while free insulin remains fixed at the point of application of the serum-insulin mixture on the filter paper. When an excess of unlabelled insulin mixed with a trace of labelled hormone is added to the anti-insulin serum, the excess insulin is adsorbed from solution by cellulose and the amount bound by the antibodies is measured in the supernatant solution. We have used this assay system for serum obtained from guinea-pigs treated with bovine insulin2 and capable of neutralizing insulin at rates ranging from 0.1 to 3.5 units per ml. serum. It can, however, be adapted for the detection and assay of antibody concentrations lower than this; it has not yet been used for anti-insulin sera of other animals or of man.

Precipitation of insulin by alcohol and some aspects of the “back-titration” method of insulin immunoassay were investigated. In the absence of insulin antibodies, alcohol has a definite nonspecific precipitant action on labeled insulin, an action which is most marked when alcohol is added in high concentration or when the medium contains much protein. This nonspecific precipitation of radio-iodinated insulin is not affected by the presence of high concentrations of unlabeled insulin; this is not explained but has no effect upon the assay system. The “back-titration” method of insulin assay is more sensitive and reproducible than the “competitive” technic when the latter is carried out under similar brief conditions of incubation. The “back-titration” method is shown to be specific for insulin, proinsulin being the only other hormonal substance tested and found to interfere with the assay system. The assay is not affected by small changes in pH about neutrality, can be carried out with pooled anti-porcine or anti-bovine insulin serum, and is not species-specific. Results obtained with human plasma are comparable with those found by the method of Morgan and Lazarow. Expected amounts of insulin are recovered over a wide range of plasma dilutions. For routine purposes, illustrated in a series of glucose tolerance tests, the “backtitration” method has proved practical, rapid, and reproducible.

Publisher Summary This chapter discusses hormone antibodies in endocrinology. This account of the hormonal antibodies has touched on aspects of widely differing disciplines and has, of necessity, covered many topics to some of which full justice may not have been done. There is no doubt, however, that hormone antibodies are now being found, studied, and used under so many circumstances that detailed knowledge of them will become more and more important in the future. It seems right that this account should end, therefore, with some specific observations of potential general interest. Of the human protein hormones, pituitary and placental gonadotropins have been used to treat cases of impotence and amenorrhea following hypophysectomy and to facilitate pregnancy in women with secondary amenorrhea. Most of the existing information concerning hormone transport in the blood comes from studies of insulin, and on the subject of insulin transport there is considerable controversy.

SummaryInsulin secretion provoked by glucose in the pancreas of the rat is not modified by addition of methylprednisolone to the incubation medium. Secretion by pancreatic tissue of the normal rat is reduced (to 60%) by prior adrenalectomy and increased (by 50%) after treatment with cortisol for 2 or 5 days. Treatment with cortisol for 3 days prevents the effect of adrenalectomy. These alterations are not associated with significant changes in the insulin content of the pancreas, and suggest that gluco-corticoids increase the sensitivity of the insulin secretory mechanism of the beta cells to glucose either directly or indirectly in a chronic process.

Insulin secretion by pancreatic tissuein vitro in response to glucose was related to plasma glucose and plasma insulin levels during life, and to the degree of granulation of the islets of Langerhans in 22 normal, 6 diabetic and 8 intermittently glycosuric Chinese Hamsters. Secretion was directly related to the insulin content of the pancreas and to the ratio of insulin to glucose in plasma, irrespective of the metabolic state of the animal, but was lowest in diabetic animals and highest in those with intermittent glycosuria. The relevance of these findings to current theories concerning “prediabetes” is discussed.

When insulin is incubated in a buffered medium with small pieces of pancreatic tissue from the normal rat, it is rapidly destroyed. The lytic substance released by the tissue into the medium destroys both insulin and glucagon, but has no effect upon growth hormone, large protein molecules, or upon insulin which is already bound by antibodies in guinea pig anti-insulin serum (GPAIS). When GPAIS is added to the medium, it traps added or secreted insulin before the lytic substance has time to destroy it. Thus, pancreatic insulin secretion in vitro is estimated from the fall in insulin-binding capacity of known amounts of GPAIS added to the medium at the beginning of incubation. The method appears to be specific, since in the presence of glucose no neutralization of GPAIS occurs using pancreatic tissue from the alloxan-diabetic rat or normal tissue incubated in the presence of mannoheptulose. Insulin secretion is directly related to the weight and size of pieces of incubated tissue, and occurs at a constant r...

The principal methods currently used to measure insulin secretion are briefly reviewed. From observed changes in concentration of immunoreactive insulin in plasma, insulin secretion in vivo can be more accurately assessed and smaller changes in secretion rate detected using pancreatic rather than systemic venous samples of blood. Measurement of secretion in vitro is complicated by the release from acinar tissue of a substance which destroys secreted insulin and similar polypeptides but whose action can be reduced or circumvented. When interpreting the effects of stimulants and inhibitors, it has also to be remembered that they could act indirectly through their actions upon other tissues of the body or upon cells other than the beta-cells of the pancreatic islets. These and other factors should be taken into account when interpreting data obtained by the several practical methods now used to study the insulin secretory process.

Crystals of the title compound (1) are monoclinic, space group B21/c, with Z= 8 in a unit cell of dimensions: a= 21.69 ± 0.03, b= 13.32 ± 0.02, c= 8.55 ± 0.01 A, β= 85.4 ± 0.1°. The structure was determined by Patterson and Fourier methods from photographic data and refined by full-matrix least-squares techniques to R 0.080 for 748 independent reflections. The m-bromophenyl substituent is titled 44° from the planar oxazolone ring, whereas the trifluoroacetyl group is coplanar with it. Bond angles and distances suggest that an aromatic oxazolium oxide structure is not a good representation.