Response of headgear release mechanisms to nonaxial force application.

Safety products have been developed to help reduce the incidence of trauma caused by headgear. Previous studies have reported the characteristics of breakaway-type headgear release mechanisms with axial force application. Not all accidental releases are triggered by an axial force and it is necessary to understand the characteristics of these mechanisms with nonaxial force application. Thirteen headgear release mechanisms were tested as part of a complete headgear system. With the system attached to a plaster head and neck model a tensile force was applied to the system at 30 degrees to the sagittal plane at 2 rates. The force of activation at release and the distance traveled were determined and analyzed statistically. Force values ranged from 4.6 to 36.7 pounds and face bow travel before release ranged from 0.97 to 3.42 inches. No consistent pattern of rate dependence was observed. Several devices demonstrated the desirable combination of low force and face bow travel at release.

The thyroid hormone receptor-beta-selective agonist GC-1 differentially affects plasma lipids and cardiac activity.

Thyroid hormones influence the function of many organs and mediate their diverse actions through two types of thyroid hormone receptors, TRalpha and TRbeta. Little is known about effects of ligands that preferentially interact with the two different TR subtypes. In the current study the comparison of the effects of the novel synthetic TRbeta-selective compound GC-1 with T3 at equimolar doses in hypothyroid mice revealed that GC-1 had better triglyceride-lowering and similar cholesterol-lowering effects than T3. T3, but not GC-1, increased heart rate and elevated messenger RNA levels coding for the I(f) channel (HCN2), a cardiac pacemaker that was decreased in hypothyroid mice. T3 had a larger positive inotropic effect than GC-1. T3, but not GC-1, normalized heart and body weights and messenger RNAs of myosin heavy chain alpha and beta and the sarcoplasmic reticulum adenosine triphosphatase (Serca2). Additional dose-response studies in hypercholesteremic rats confirmed the preferential effect of GC-1 on TRbeta-mediated parameters by showing a much higher potency to influence cholesterol and TSH than heart rate. The preferred accumulation of GC-1 in the liver vs. the heart probably also contributes to its marked lipid-lowering effect vs. the absent effect on heart rate. These data indicate that GC-1 could represent a prototype for new drugs for the treatment of high lipid levels or obesity.

Structural characterization of a higher plant calmodulin : spinacia oleracea.

Calmodulin is a eukaryotic calcium binding protein which has several calcium-dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH(2)-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH(2)-terminal half of the molecule, while nine of the 13 identified differences are in the COOH-terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins.

Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins.

Calmodulin is the name proposed for a multifunctional, calcium binding protein whose presence has been detected in a number of eukaryotic cells. In the studies summarized here, calmodulin has been isolated from spinach leaves (Spinacea oleracea), characterized, and compared to vertebrate calmodulins. Quantitative recovery data for a rapid-isolation protocol demonstrate that calmodulin is a major constituent of spinach leaves. Spinach calmodulin is indistinguishable from vertebrate calmodulins in phosphodiesterase activator activity using vertebrate brain phosphodiesterase and in quantitative immunoreactivity using antiserum made against vertebrate calmodulin. However, spinach calmodulin is really distinguished from vertebrate and invertebrate calmodulins in electrophoretic mobility and in amino acid composition. Spinach calmodulin, like vertebrate calmodulins, lacks tryptophan and contains 1 mol each of N epsilon-trimethyllysine and histidine per 17000 g of protein. In contrast to vertebrate calmodulins, spinach calmodulin has only one tyrosinyl residue and has a threonine/serine ratio of 1.3. While amino acid compositions indicate differences between spinach and vertebrate calmodulins, isolation and characterization of tryptic peptides containing the single histidinyl and N epsilon-trimethyllysyl residues and both prolinyl residues indicate that these regions in spinach calmodulin are similar to the corresponding regions in vertebrate calmodulin. These studies more fully define the general and specific characteristics of calmodulins and indicate that calmodulin structure is not as highly conserved among all eukaryotes as it is among vertebrates and invertebrates.

Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures.

Calmodulin, a multifunctional calcium-modulated protein, has been isolated from spinach leaf tissue and from spinach leaf messenger RNA translation products. The translation protein and the spinach leaf protein have been partially characterized and compared to vertebrate calmodulins. Spinach leaf calmodulin will quantitatively activate bovine brain phosphodiesterase and will undergo a calcium-dependent shift in electrophoretic mobility similar to that of bovine brain calmodulin. In the presence of Ca(2+) the spinach and brain proteins comigrate, but in the presence of chelators they do not. A polyadenylylated RNA fraction has been isolated from spinach leaf tissue and translated in a wheat germ cell-free translation system. The calmodulin synthesized in vitro has been isolated by using calcium-dependent affinity chromatography on phenothiazine-Sepharose conjugates. The translation protein comigrates with spinach calmodulin during polyacrylamide gel electrophoresis whether in the presence or the absence of Ca(2+). The translation protein also undergoes a calcium-dependent mobility shift identical to that of spinach calmodulin. Amino acid analysis of the translation calmodulin indicates that it does not contain N(epsilon)-trimethyllysine, an amino acid residue that is characteristic of all calmodulins previously examined. These studies suggest that N(epsilon)-trimethyllysine is not required for the calcium-dependent interaction of calmodulin with phenothiazines and indicate the potential utility of phenothiazine-Sepharose conjugates as affinity-based adsorbents in biological and biochemical investigations.

Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography.

A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is +/- 5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biochemical analyses.

Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes.

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.