Histochemical characterization of myotomal muscle in the grass pickerel, Esox americanus vermiculatus (LeSeuer), and the muskellunge, E. masquinongy (Mitchell)

A histochemical study of the myotomal muscles in the grass pickerel, Esox americanus vermiculatus , and the muskellunge, E. masquinongy , was performed using actomyosin ATPase and NADH diaphorase activities. Three fibre types, i.e., red, white and pink were distinguished on the basis of their enzyme activities. White muscle fibres comprised the bulk of the myotomal musculature. The relative proportion of red muscle fibres was greater in the caudal region than in more anterior regions of the body. Pink fibres formed only a few layers between red and white. These findings are discussed in relation to the possible functional significance of the muscle fibre types in swimming and feeding behaviour in these species.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

Inositol 1,4,5-trisphosphate and guanine nucleotides activate calcium release from endoplasmic reticulum via distinct mechanisms

A sensitive and specific guanine nucleotide regulatory process has recently been shown to rapidly mediate a substantial release of Ca2+ from endoplasmic reticulum within the N1E-115 neuronal cell line (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The relationship between this mechanism and Ca2+ efflux mediated by the intracellular regulator inositol 1,4,5-trisphosphate (IP3) has been investigated. Using saponin-permeabilized N1E-115 cells, studies reveal a number of distinctions between the activation of Ca2+ release mediated by GTP and IP3. Thus, the GTP-mediated Ca2+ release process is specifically activated by polyethylene glycol which increases both GTP sensitivity and the extent of GTP-activated Ca2+ release; in contrast, IP3-dependent Ca2+ release is unaffected by polyethylene glycol. The non-hydrolyzable GTP analogue guanosine 5'-O-(3-thio)triphosphate, which completely inhibits GTP-mediated Ca2+ release, does not alter release mediated by IP3. Decreasing the release temperature from 37 to 4 degrees C decreases IP3-activated Ca2+ release by only 20%, whereas the action of GTP on Ca2+ release is abolished at 4 degrees C. Activation of Ca2+ release by IP3 is completely inhibited by increasing free Ca2+ from 0.1 to 10 microM, whereas the fraction of GTP-dependent Ca2+ release (approximately 50% of ionophore-releasable Ca2+) remains unaltered with increasing free Ca2+. These distinctions between IP3- and GTP-mediated Ca2+ release indicate that the two effectors function via distinct mechanisms to activate Ca2+ release; however, they do not preclude the possibility that coupling between the two mechanisms can occur or that a common Ca2+-translocating pathway activated by both effectors exists.     

Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose)

Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP-ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA.     

Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor

The effect of self-phosphorylation on the protein-tyrosine kinase activity of the epidermal growth factor receptor has been investigated using immunoaffinity-purified protein. Enzyme was first incubated for various times with excess ATP to phosphorylate it to differing extents; the ability of the enzyme to phosphorylate exogenous peptide substrates was then measured as a function of its self-phosphorylation state. Increasing self-phosphorylation to 1.3-1.8 mol of phosphate mol-1 of epidermal growth factor receptor enhanced protein-tyrosine kinase activity 2-3-fold. Comparison of the kinetics of protein-tyrosine kinase activity at different ATP concentrations revealed significant differences between unphosphorylated and phosphorylated enzyme. At low levels of ATP, a double reciprocal plot of the protein-tyrosine kinase activity of the unphosphorylated enzyme was hyperbolic, suggesting that ATP may act as an activator of the enzyme. At higher ATP concentrations, where greater levels of self-phosphorylation occurred during the reaction, the kinetics appeared linear and similar to those of the phosphorylated enzyme. Dose-response studies using three different peptide substrates (angiotensin II, gastrin, and a synthetic peptide corresponding to the self-phosphorylation site in p60v-src) showed that exogenous substrates inhibit receptor self-phosphorylation. In each case, half-maximal inhibition was observed at a peptide concentration approximately equal to the substrate's Km. A kinetic analysis comparing peptide phosphorylation using unphosphorylated and prephosphorylated enzyme indicated that the self-phosphorylation site can act as a competitive inhibitor (alternate substrate) versus peptide substrates. These results suggest that self-phosphorylation of the epidermal growth factor receptor removes a competitive constraint so that exogenous substrates can be more readily phosphorylated.     

Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors.

Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ 'site' or 'component' is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP-regulatory-protein-catalytic-unit (Ni-C) interactions.     

Comparison of health problems related to work and environmental measurements in two office buildings with different ventilation systems.

A cross sectional survey investigating "building sickness" was carried out in two buildings with similar populations of office workers but differing ventilation systems, one being fully air conditioned with humidification and the other naturally ventilated. The prevalence of symptoms related to work was assessed by a questionnaire administered by a doctor. A stratified, randomly selected sample of workers was seen (84% response). Building sickness includes several distinct syndromes related to work, most of which were significantly more common in the air conditioned building than the naturally ventilated building--namely, rhinitis (28% v 5%), nasal blockage and dry throat (35% v 9%), lethargy (36% v 13%), and headache (31% v 15%). The prevalence of work related asthma and humidifier fever was low and did not differ significantly between the two buildings. An environmental assessment of the offices was performed to attempt to identify possible factors responsible for the differences in the prevalence of disease. Globe temperature, dry bulb temperature, relative humidity, moisture content, air velocity, positive and negative ions, and carbon monoxide, ozone, and formaldehyde concentrations were all measured. None of these factors differed between the buildings, suggesting that building sickness is caused by other factors.