Comparison of Dinamap 8100 with sphygmomanometer blood pressure measurement in a prepubertal diabetes cohort.

OBJECTIVE: To compare the conventional sphygmomanometer with the semiautomated Dinamap 8100 (Critikon, Tampa, FL, USA) for the measurement of blood pressure in prepubertal children with insulin-dependent diabetes mellitus. METHODOLOGY: Blood pressure was measured using both methods in 61 prepubertal children (aged 8-13 years) on 189 occasions over 4 years. The measurements were compared using the Bland-Altman plot. Tracking correlations of blood pressure centiles over time were analyzed by the general estimating equation. RESULTS: Accuracy criteria of the Association for the Advancement of Medical Instrumentation were met and a British Hypertensive Society 'B' grading was reached. Differences in systolic and diastolic blood pressure were found between the two methods (P < 0.01). For systolic blood pressure, common correlations were 0.54 (Dinamap) and 0.51 (sphygmomanometer) and for diastolic blood pressure were 0.33 and 0.42, respectively. CONCLUSION: The Dinamap 8100 is an acceptable alternative in clinic practice and research for prepubertal children.

Production and isolation of guinea pig IgE antibody.

Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the importance of IgE antibodies in both early- and late-onset hypersensitivity responses, has heightened interest in production, separation, and isolation of this immunoglobulin class from the guinea pig. IgE antibodies were produced by treatment of strain 13 guinea pigs with cyclophosphamide followed by injection with S. aureus enterotoxin. Serum was obtained and the globulin fraction isolated by addition of caprylic acid then ammonium sulfate. Immunoglobulins were separated into classes using fast protein liquid chromatography (FPLC) employing a Mono Q column and a linear gradient of 0.01-0.3 M Na,K phosphate buffer, pH 7.5 (buffer B). IgG eluted in two major peaks. IgG2 was not retained on the column and emerged with the starting buffer; IgG1 was eluted with 15-20% buffer B. IgE, detected as heat labile homocytotropic antibody, was found in the fraction eluting with 30-35% buffer B. The elution profile of the guinea pig immunoglobulins was predicted from the pattern obtained with immunoglobulin classes from other species. This chromatographic procedure enabled rapid isolation of immunoglobulin classes from guinea pig sera and effectively separated IgG1 from IgE, the two classes associated with hypersensitivity reactions.

Tetraplex formation of a guanine-containing nonameric DNA fragment.

A combination of spectroscopic and calorimetric techniques has been used to characterize the structures formed by a family of short, guanine-containing DNA single strands of the form d[GGTTXTTGG], X = A, C, G, T. In 1 molar NaCl at low temperatures, these molecules do not behave like single strands, but rather exhibit properties consistent with tetraplex formation. The standard state enthalpies, entropies, and free energies for formation of each tetraplex have been measured, as have preliminary nuclear magnetic resonance (NMR) spectra. In 1 molar KCl, the melting behavior of the structure or structures is more complex than in 1 molar NaCl. This observation may be related to the recently proposed "sodium-potassium switch."

Chemical synthesis and cloning of the human growth hormone releasing factor gene.

Human growth hormone releasing factor (hGRF) gene has been synthesized and cloned. The sequence of the synthetic hGRF gene, consisting of preferred codons for expression in E. coli, was designed with the aid of computer programs. Six segments with lengths ranging from 39 to 51 nucleotides were synthesized by solid-phase phosphoramidite method. The entire gene of 141 base pairs was constructed by enzymatic ligation of all synthetic segments and then cloned into plasmid pUC-19. The positive colonies were confirmed by the screening of ampicillin resistance, inactive beta-galactosidase, and analyzing by use of restriction enzymes and dot-blot hybridization. The cloned gene was sequenced by M13 dideoxynucleotide chain termination method and proven correct.

Intra- and intermolecular reactions of 4,4'-diisocyanatodiphenylmethane with human serum albumin.

Diisocyanates are highly reactive industrial chemicals that have been shown to possess toxic activity, including potential for allergic sensitization. To assist in diagnosis of sensitization, immunoassays for diisocyanate-specific antibodies are performed; such assays require preparation of diisocyanate-containing hapten-protein conjugates. Conditions were investigated for formation of conjugates yielding varying degrees of hapten binding. Relative concentrations of haptens and proteins were varied as were pH, temperature, and time of reaction. Quantitation of 4,4'-diisocyanatodiphenylmethane (MDI) binding with human serum albumin (HSA) was assessed by absorbance of the isolated conjugates at 250 nm after determination of the molar extinction coefficient for MDI. At pH 7.4 and 37 degrees C, the binding reaction was found to be biphasic with binding of 5-6 mol of MDI groups/mol of HSA within the first minute, followed by incorporation at a rate of 0.16 mol/min during the next 2 h. Evaluation of reaction products using SDS-PAGE revealed extensive inter- and intramolecular cross-linking of HSA by MDI. Intramolecular cross-linking was accompanied by an increased migration of conjugates from an initial molecular mass of 66 kDa, typical of HSA, to a molecular mass of 44 kDa. The change in migration was also produced by using disuccinimidyl tartarate (DST) as hapten and was eliminated when DST was cleaved with sodium periodate. It was attributed to altered protein shape. Conditions that favored binding of MDI with HSA were a high relative concentration of MDI:HSA, a pH of 9.4, and a temperature of 37 degrees C. Under such conditions it was calculated that 53 mol of MDI were bound per mole of HSA after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Diisocyanate antigens that detect specific antibodies in exposed workers and guinea pigs.

Evaluation of the immunologic contribution to the pathogenesis of isocyanate lung disease necessitates preparation of isocyanate-protein conjugates to detect anti-isocyanate antibodies. The preparation and characterization of the conjugates were described in the preceding paper in this issue. Sera were obtained from two guinea pigs immunized with 4,4'-diisocyanatodiphenylmethane (MDI) and from three workers with occupational exposure to MDI. By use of Western blot analysis, guinea pig IgG antibodies were best detected by the monomeric component of MDI-guinea pig serum albumin. ELISA additionally indicated that conjugates which contained a high density of hapten detected greater amounts of antibody than did conjugates containing low amounts of hapten. The same procedures were then used to assess the amount of MDI-specific IgG and IgE antibody in sera from symptomatic workers. Effective MDI-HSA antigens were those that were monomeric and had high haptenic content. Highly substituted conjugates of monoisocyanates (phenyl isocyanate and p-tolyl isocyanate) with serum albumins were also effective in detecting antibodies to MDI. These results indicate the importance of the composition of isocyanate conjugate antigens in detecting antibodies to diisocyanates and suggest that standards be developed for preparation and characterization of these diagnostic serologic reagents.

An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in Escherichia coli.

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on glycerol.

Experimental evolution of a novel pathway for glycerol dissimilation in Escherichia coli.

Wild-type Escherichia coli utilizes glycerol aerobically through an inducible pathway mediated by an ATP-dependent kinase and a glycerol 3-phosphate dehydrogenase which is a flavoprotein. A mutant, strain ECL424, employing a novel pathway for glycerol utilization was isolated. The novel pathway is mediated by an NAD-linked dehydrogenase and a dihydroxyacetone specific enzyme II of the phosphoenolpyruvate phosphotransferase system. This study describes the selection from strain ECL424, a derivative which grows more rapidly on glycerol. The derivative, strain ECL428, produces twice the parental levels of both the dehydrogenase and the enzyme II during growth on glycerol. The function of the dehydrogenase in wild-type cells is unknown, although hydroxyacetone (acetol), 3-hydroxy-2-butanone (acetoin), and 1-amino-2-propanone are gratuitous inducers. The induction can be prevented by glucose whose effect can be cancelled by external cyclic AMP. The effects of hydroxyacetone, glucose, and cyclic AMP are attenuated in the two mutants in which the dehydrogenase is produced at high basal levels. The dihydroxyacetone specific enzyme II is inducible by the substrate in both wild-type and mutant strains and serves for growth on the triose.

Glycerol kinase as a substitute for dihydroxyacetone kinase in a mutant of Klebsiella pneumoniae.

With dihydroxyacetone as the sole source of carbon and energy, constitutively synthesized glycerol kinase of the glp system supported aerobic growth of Klebsiella pneumoniae mutants lacking the inducible dihydroxyacetone kinase of the dha system. Glycerol kinase had an apparent Km of 0.01 mM for its physiological substrate and 1 mM for its surrogate substrate. However, the growth rate on dihydroxyacetone of cells relying on glycerol kinase increased with the concentration of the carbon and energy source up to 50 mM, suggesting that permeation is rate limiting.