Studies on the immediate and delayed leucocytosis elicited by brief (30-min) strenuous exercise

Eight healthy male volunteers exercised for two 30-min sessions starting 3 h apart on an electronically braked cycle ergometer at a work load (mean 155.9 W, SD 33.4 W) which required an oxygen consumption that was 70% of their maximal rate of oxygen uptake. Venous blood samples were taken through an indwelling cannula over a period of 6 h beginning shortly before the first bout of exercise and were analysed for routine haematological parameters and for lactate, noradrenaline, adrenaline and cortisol. Both bouts of exercise induced an immediate leucocytosis due to rises in lymphocytes and neutrophils but only the first exercise bout induced a substantial delayed neutrophilia. In at least five subjects, changes in lymphocyte and platelet numbers were correlated (Spearman's rank procedure, P less than 0.05) with simultaneous changes in the plasma concentrations of lactate, noradrenaline and adrenaline over the 6-h period studied. Increases in the plasma concentration of cortisol due to exercise correlated positively with the percentage changes in neutrophil numbers at 3 h and 6 h. These results are consistent with the suggestion that the immediate and delayed leucocytosis induced by exercise are mediated respectively by catecholamine and by cortisol.     

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M _r of 60 000 and is composed of two identical subunits of M _r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K _mvalues for DL-isocitrate adn NADP⁺ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn²⁺ or Mg²⁺ for activity. NAD⁺ cannot substitute for NADP⁺. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn²⁺ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C. Offprint requests to: J. J. Perry     

The effects of endogenous or exogenous catecholamines on blood respiratory status during acute hypoxia in rainbow trout (Oncorhynchus mykiss)

Summary An extracorporeal circulation of rainbow trout (Oncorhynchus mykiss) was utilized to continuously monitor the rapid and progressive effects of endogenous or exogenous catecholamines on blood respiratory/acid-base status, and to provide in vivo evidence for adrenergic retention of carbon dioxide (CO₂) in fish blood (cf. Wood and Perry 1985). Exposure of fish to severe aquatic hypoxia (final P _wO₂=40–60 torr; reached within 10–20 min) elicited an initial respiratory alkalosis resulting from hypoxia-induced hyperventilation. However, at a critical arterial oxygen tension (P _aO₂) between 15 and 25 torr, fish became agitated for approximately 5 s and a marked (0.2–0.4 pH unit) but transient arterial blood acidosis ensued. This response is characteristic of abrupt catecholamine mobilization into the circulation and subsequent adrenergic activation of red blood cell (RBC) Na⁺/H⁺ exchange (Fievet et al. 1987). Within approximately 1–2 min after the activation of RBC Na⁺/H⁺ exchange by endogenous catecholamines, there was a significant rise in arterial PCO₂ (P _aCO₂) whereas arterial PO₂ was unaltered; the elevation of P _aCO₂ could not be explained by changes in gill ventilation. Pre-treatment of fish with the -adrenoceptor antagonist phentolamine did not prevent the apparent catecholamine-mediated increase of P _aCO₂. Conversely, pre-treatment with the -adrenoceptor antagonist sotalol abolished both the activation of the RBC Na⁺/H⁺ antiporter and the associated rise in P _aCO₂, suggesting a causal relationship between the stimulation of RBC Na⁺/H⁺ exchange and the elevation of P _aCO₂. To more clearly establish that elevation of plasma catecholamine levels during severe hypoxia was indeed responsible for causing the elevation of P _aCO₂, fish were exposed to moderate hypoxia (final P _wO₂=60–80 torr) and then injected intraarterially with a bolus of adrenaline to elicit an estimated circulating level of 400 nmol·l^-1 immediately after the injection. This protocol activated RBC Na⁺/H⁺ exchange as indicated by abrupt changes in arterial pH (pHa). In all fish examined, P _aCO₂ increased after injection of exogenous adrenaline. The effects on P _aO₂ were inconsistent, although a reduction in this variable was the most frequent response. Gill ventilation frequency and amplitude were unaffected by exogenous adrenaline. Therefore, it is unlikely that ventilatory changes contributed to the consistently observed rise in P _aCO₂. Pretreatment of fish with sotalol did not alter the ventilatory response to adrenaline injection but did prevent the stimulation of RBC Na⁺/H⁺ exchange and the accompanying increases and decreases in P _aCO₂ and P _aO₂, respectively. These results suggest that adrenergic elevation of P _aCO₂, in addition to the frequently observed reduction of P _aO₂ are linked to activation of RBC Na⁺/H⁺ exchange. The physiological significance and the potential mechanisms underlying the changes in blood respiratory status after addition of endogenous or exogenous catecholamines to the circulation of hypoxic rainbow trout are discussed.Abbreviations P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen tension - P _da dorsal aortic pressure - pHa arterial pH - P _wO₂ water oxygen tension - RBC red blood cell - V _f breathing frequency     

The interaction of troponin-I with the N-terminal region of actin

The interaction between troponin-I and actin that underlies thin-filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin-I (residues 96-116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375-387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin-I which has inhibitory activity interacts with the N-terminal region of actin. The surface of contact of the inhibitory segment of troponin-I with actin involves two regions of the N-terminal of actin. These are located between residues 1-7 and 19-44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation.     

Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes

The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline-stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP.     

Fine needle aspiration biopsy of metastatic melanoma. A morphologic analysis of 174 cases

The prognostic and therapeutic decisions in cases of metastatic melanoma depend upon the morphologic documentation of metastatic disease, which may rapidly and accurately be done by fine needle aspiration (FNA) biopsy of clinically suspicious lesions. The tumor cells derived from malignant melanomas demonstrate a wide range of appearances, however, and other neoplasms may be mimicked. Furthermore, additional neoplasms of other types are more frequent in melanoma patients: the possibility of a new primary tumor must be considered if the morphology of the tumor cells is uncharacteristic. Therefore, a study was undertaken to analyze the morphologic changes seen in FNA biopsy specimens from metastatic malignant melanoma and to determine which features could be the most useful in establishing a definitive diagnosis. A total of 174 consecutive cases, comprising 151 malignant aspirates and 23 inconclusive aspirates, were reviewed. The most significant features for identification of melanoma over other tumor types were the cell shape and nuclear position, the presence of numerous isolated neoplastic cells and occasional binucleated or multinucleated cells. Intracellular melanin in neoplastic cells was diagnostic when present, but it was absent in 60% of the cases. Macronucleoli and/or intranuclear cytoplasmic invaginations were characteristic but variable features. Morphology was also found to vary by site and cell type. Lung aspirates were less cellular and more likely to contain melanin. Aspirates of subcutaneous nodules were more often composed of spindle-shaped cells or of other variant cell types. Lymph node aspirates more often yielded epithelioid cells with macronucleoli and/or intranuclear invaginations. Spindle-cell melanomas usually demonstrated inconspicuous nuclei and rarely showed enlarged nucleoli. Epithelioid-cell tumors contained multinucleated cells and areas of cell wrapping more frequently than did spindle-cell tumors. The findings in this study emphasize that a full awareness of the spectrum of morphologic presentations of metastatic melanoma as well as of the clinical history are needed for greater precision in its diagnosis and for avoidance of the pitfall of misdiagnosing nonmelanomas with similar appearances.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

Characterization of a Ca2+-stimulated lipid peroxidizing system in the sea urchin egg

Addition of calcium chloride to an egg homogenate of Strongylocentrotus purpuratus stimulates O2 consumption which is not inhibited by millimolar cyanide. Results strongly suggest that Ca2+-stimulated O2 consumption is at least partially the result of polyunsaturated fatty acid oxidation. First, addition of arachidonic acid (AA), or other polyunsaturated fatty acids, to the homogenate enhance Ca2+-stimulated O2 consumption; this enhancement, by AA, being coupled to its oxidation to a hydroxy fatty acid. Second, calcium stimulates a lipase activity in the homogenate that is capable of releasing free fatty acids. Third, Ca2+-stimulated O2 consumption and AA oxidation have virtually identical calcium requirements and pH optima. The sequence of events then is that upon calcium addition to the homogenate, lipase activity is increased which liberates free fatty acids. At the same time calcium also activates a polyunsaturated fatty acid oxygenase, possibly lipoxygenase, that converts the free fatty acids to hydroxy fatty acids. The possible physiological importance of this reaction is underscored by the high affinity for Ca2+ [approximately 10(-7)M], an ion known to increase above the required levels at fertilization. The pH activity profile also suggests possible physiological modulation because a pH change of 6.8 increasing to 7.2, as suggested to occur after fertilization, yields almost a twofold increase in O2 consumption. Egg homogenates from many other invertebrate species have the ability to oxidize AA in a Ca2+-dependent fashion. For the investigated species, the presence of Ca2+-stimulated O2 consumption and AA oxidation correlates with the presence of cyanide insensitive respiration in the intact egg.     

Distribution and abundance of fishes in the San Francisco Bay estuary between 1980 and 1982

In 1980 a long-term study of the fishery resources of the San Francisco Bay estuary was initiated in an effort to delineate the importance of freshwater inflow to fish and invertebrate abundance and distribution in the bay. An analysis of the trawl data collected between January 1980 and December 1982 illustrates the influence of the timing and magnitude of freshwater inflows on fish fistribution and abundance in this estuary from the perspective of monthly, seasonal and annual time scales. Normally found in the delta, Suisun Bay and San Pablo Bay during periods of increased salinity, pelagic species moved downstream after the two peak flows studied, while demersal species usually found in Central San Francisco Bay moved upstream. Such upstream movements may be due in part to transport by strong density-driven currents.Timing and magnitude of monthly catches of some species varied on a seasonal cycle coincident with variations of freshwater inflow. Most species, especially the marine species, showed no consistent cycle of monthly catches. In the wet years of 1980 and 1982 the distributions of freshwater, estuarine and anadromous species were extended downstream into San Pablo, Central and South San Francisco Bays and some marine species, including the flatfish, were more abundant in the upstream areas. In the dry year of 1981 when bay salinities were higher, few marine species extended their distributions upstream into San Pablo and Suisun Bays. Jacksmelt was the only fish of the 15 most abundant species with its peak abundance in 1981. Most marine species were more abundant in the San Francisco Bay estuary in the wet years.     

Stimulation by epinephrine of the membrane transport of long chain fatty acid in the adipocyte

In isolated rat adipocytes, epinephrine rapidly stimulates the transport of long chain fatty acid across the plasma membrane. At a concentration of unbound oleate of 0.1 microM ([fatty acid]/[albumin] = 1) and 5 min exposure to the hormone, the minimal effective concentration of epinephrine is 0.03 and the optimal concentration 0.3 microM (0.01 and 0.1 microgram/ml). The stimulated rates are 5-10-fold the basal rate of influx or efflux. The hormone effect is on the transport process specifically as shown by isolation of the product of transport in either direction as unesterified fatty acid and inhibition by the transport inhibitors phloretin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. This effect of epinephrine on transport coordinates physiologically with lipase activation to bring about fatty acid release from adipose tissue.