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We examined the frequencies of autoantibodies to glutamate decarboxylase, GAD65, protein tyrosine phosphatase, IA-2/ICA512, and insulin, and of HLA class II markers in ICA-positive first-degree relatives of patients with type 1 diabetes. Our results indicate that while the presence of HLA susceptibility markers is associated with anti-islet autoantibodies, protective DQB1 markers do not absolutely prevent development of autoantibodies or progression to autoimmune diabetes.

A DNA-dependent ATPase has previously been purified from bacteriophage T4-infected Escherichia coli. A mutant phage strain lacking this enzyme has been isolated and characterized. Although the mutant strain produced no detectable DNA-dependent ATPase, growth properties were not affected. Burst sizes were similar for the mutant phage and T4D in polA1, recB, recC, uvrA, uvrB, uvrC, and various DNA-negative E. coli. UV sensitivity and genetic recombination were normal in a variety of E. coli hosts. Mapping data indicate that the genetic locus controlling the mutant occurs near gene 56. The nonessential nature of this gene is discussed.

Infection by bacteriophage T4 has previously been shown to cause a rapid inhibition of the host recBC DNase, an ATP-dependent DNase that is required for genetic recombination in Escherichia coli. We report here the partial purification of a protein ("T4 rec inhibitor") from extracts of T4-infected cells and some characteristics of the in vitro inhibition reaction with purified inhibitor and recBC nuclease. This inhibitory activity could not be purified from extracts of uninfected E. coli. Both the ATP-dependent exonuclease and DNA-dependent ATPase activities of recBC DNase are inhibited by T4 rec inhibitor. Experiments suggest that the inhibitor interacts with the nuclease in a stoichiometric manner. The biological significance of this inhibition is discussed with respect to control reactions in phage-infected cells.

The binding of 125I-insulin was determined using erythrocytes obtained from 11 subjects with Type I diabetes mellitus treated with continuous subcutaneous infusions of insulin for 1 year or more. The binding characteristics were compared to those for erythrocytes isolated from 12 normal subjects and 10 subjects with Type I diabetes mellitus treated with conventional daily injections of insulin. The total binding of 125I-insulin, receptor concentration, and high and low affinity binding constants were estimated using washed erythrocytes obtained from fasted subjects. The mean total specific binding for subjects treated with continuous subcutaneous infusion did not differ from that for conventionally treated diabetic subjects but was slightly lower than that for normal subjects at p less than 0.05. Receptor concentration did not vary significantly between the groups. High and low affinity binding constants were slightly lower in the group treated with continuous subcutaneous infusion. Both basal and diurnal plasma levels of free immunoreactive insulin were slightly but significantly elevated in both groups of diabetic subjects compared to that in normal subjects. Thus, in spite of the greater biological effectiveness of the continuous insulin infusion program in terms of glycemic control, the insulin-binding parameters, as well as the estimates of plasma free immunoreactive insulin levels, are consistent with modest and comparable degrees of hyperinsulinemia with both treatments.

The effect of cyclosporine (Cyclosporin A) on insulin binding to erythrocytes was investigated in Type 1 diabetes mellitus of recent onset. The subjects were drawn from a pilot study (The Canadian Open Study on the effects of immunosuppression with Cyclosporine in Type 1 Diabetes Mellitus) in which 50% of the patients demonstrated remission during one year of cyclosporine administration. Specific binding of 125I-insulin was examined before and after 3, 6, or 12 months of cyclosporine in different groups of patients. Those who maintained target control of blood glucose without exogenous insulin for two or more weeks were designated non-insulin requiring. Basal and intravenous glucagon-stimulated immunoreactive plasma C-peptide rose in all groups but to higher levels in non-insulin requiring groups. Insulin binding at tracer concentration, reflecting the number of insulin receptors, was initially normal but tended to decrease with duration of cyclosporine administration. This decrease was significant especially in groups which remained insulin-requiring throughout the study. The affinity of erythrocyte receptors was assessed by determining the insulin concentration required for 50% inhibition of 125I-insulin binding, the ID50. These values suggested that the affinity of insulin receptors was not affected in subjects attaining non-insulin requiring remission; however, in subjects remaining dependent on exogenous insulin, receptor affinity appeared to be adversely affected. Even in subjects who demonstrated complete remission, affinity was decreased during periods of dependence on exogenous insulin. After discontinuation of cyclosporine for one month or more, the mean daily insulin dosage increased and plasma C-peptide decreased. Insulin binding at tracer concentration was not affected but the apparent affinity was decreased after withdrawal of cyclosporine. These results suggest that insulin action at the receptor may be affected by the administration of cyclosporine. The number of insulin receptors appears to be decreased but whether this effect has an impact on insulin sensitivity remains to be seen. Receptor affinity appears to be affected mainly by exogenous insulin. Thus immunosuppression with cyclosporine in newly diagnosed Type 1 diabetes mellitus may have a modest adverse effect on insulin receptors; whether the benefits of cyclosporine treatment outweigh this risk is difficult to assess.

Eighteen lean adult volunteers with insulin-requiring diabetes mellitus attempted to achieve normoglycemia using continuous subcutaneous insulin infusion (CSII) or conventional insulin therapy (CIT) in a randomized crossover trial of 68 +/- 2.5 weeks (mean +/- SEM) duration. As reported (Diabetes Care 8: 447-55, 1985) the group with absent to low beta-cell function (C-peptide negative, n = 11) attained mean post-absorptive normoglycemia only during CSII vs CIT (p less than 0.05). Only following CSII was this without change in post-absorptive serum triglyceride concentrations (-4 +/- 5.6 vs 12 +/- 4.7 mg/dl; -0.04 +/- 0.6 vs 0.14 +/- 0.05 mM, p less than 0.05) or body weight (0.01 +/- 0.02 vs 0.05 +/- 0.01 kg/week, p less than 0.05). In the group with glucagon stimulated serum C-peptide 100-400 pmol/L (C-peptide positive) responses to CSII or CIT were equal. As total daily insulin dosage (0.05 +/- 0.04 U/kg/day) was the same under all conditions, to explain the efficacy of CSII, glucoregulatory hormone responses were examined. Pre- and post-test breakfast serum free immunoreactive insulin and plasma glucagon concentrations were essentially unaffected by C-peptide or treatment status. Erythrocyte 125I-insulin binding was decreased in the C-peptide negative group only during CSII (8.6 +/- 0.5 vs 10.1 +/- 0.7%, p less than 0.005); C-peptide positive group receptor binding was consistently low (8.2 +/- 0.8, 8.4 +/- 0.9%). During CIT using intermediate-acting insulin post-lunch peripheral venous insulin failed to rise (p less than 0.05), but in the C-peptide positive group, on the basis of C-peptide responses to breakfast an undetected rise and fall of portal venous insulin was assumed to coincide with each meal. Thus, only during CIT in the C-peptide negative group, which received on average 6.4/wk/subject fewer pre-meal regular insulin boluses (p less than 0.01), was the frequency of meal-related change in portal insulinemia decreased. Consistent meal-related fluctuations in portal insulinemia inherent in CSII hepatocytes sensitized by a post-receptor mechanism to the suppressive effects of insulin on glucose output and thus were indirectly responsible for the observed improvement in glycemic control and lipid metabolism in the C-peptide negative group.

Studies have been made of the effect of the DNA delay mutations of bacteriophage T4 on growth and genetic recombination in a number of Escherichia coli hosts. DNA delay mutations in genes 39, 52, 58 (61), and 60 result in abnormally high recombination frequencies. These high recombination frequencies are discussed in the context of other observations.

Ordinarily, enzymes that catalyze bimolecular reactions involving DNA and nucleoside triphosphates are assayed in terms of the predicted changes in DNA. However, by assay of the enzymes by changes in nucleoside triphosphates, new enzymes may be sought without prior knowledge of the role of DNA in the reaction. This approach was used to isolate two new enzymes from Escherichia coli and one from T4 phage-infected E. coli. In this communication, the general application of this technique and its specific use in the isolation of two DNA-dependent ATPases from E. coli are described. Both enzymes catalyze a DNA-dependent cleavage of ATP to ADP and Pi. The two enzymes can be distinguished by their characteristic activities with different DNAs and synthetic polydeoxynucleotides. Neither of the enzymes shows endonuclease or exonuclease activity in conventional assays. These reactions were studied by isotope-exchange experiments. The discovery of these enzymes attests to the effectiveness of this method to seek new reactions involving nucleic acids. Moreover, our results suggest that other enzymes of nucleic acid metabolism may be found and purified by this new approach.