Contractile Proteins of a Benthic Fish.I. Effects of Temperature and Pressure on Myosin ATPase.

SYNOPSIS. Studies are described on the adenosine triphosphatase(ATPase) properties of myosin isolated from skeletal muscleof Coryphaenoides, a benthic fish captured at 2,200 meters depth.Ca²⁺-ATPase and EDTA-ATPase of Coryphaenoides myosin show thesame pH dependence as ATPase of mammalian myosin; however, ratesof ATP hydrolysis by Coryphaenoides myosin are only 5–10%of rates of ATP hydrolysis by rabbit skeletal myosin. Coryphaenoidesmyosin ATPase shows a decrease from Q₁₀ of 2.0 at 25°C toQ₁₀ of 1.4 a t 2°C, and undergoes irreversible denaturationat temperatures above 25°C. At pH 6.8 to pH 8.5, Coryphaenoidesmyosin ATPase undergoes activation by pressure at 25°C,but at 2°C shows negligible effect of pressure at valuesbelow 3,000 psi. The kinetic data on Ca²⁺-ATPase indicate valuesof 11 kcal/mole for H, –7.5 kcal/mole for TS, and –5.7cc/mole for V at 25°C, pH 7.6. Comparable data at 2°Cindicate values of 5 kcal/mole for H. –13 kcal/mole forTS, and negligible V. According to the results of 25°C,Ca²⁺-activatkm of myosin-ATP may involve disruption of fouror five hydrophobic or polar groups, presumably due to an "opening-up"of the myosin molecule at or near the site for ATP binding.It would also appear that Coryphaenoides myosin has undergonean adaptive change in the enzyme mechanism for ATPase such thatthe rate of ATP hydrolysis is relatively insensitive to pressureand temperature under conditions encountered by the living fish.     

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway

Certain angina and coronary artery disease forms do not respond to Ca²⁺ channel blockers, and a role for vasoactive eicosanoids such as PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-induced coronary smooth muscle contraction is Ca²⁺ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca²⁺), PGF₂ (10^-5 M) caused transient [Ca²⁺]_i increase followed by maintained [Ca²⁺]_i increase and 34% cell contraction. Ca²⁺ channel blockers verapamil and diltiazem (10^-6 M) abolished maintained PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca²⁺ influx, caused 36% cell contraction and [Ca²⁺]_i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

{delta}15N and {delta}13C Measurements of Antarctic Peninsula Fauna: Trophic Relationships and Assimilation of Benthic Seaweeds

Measurements of ¹³C, ¹⁵N, and C/N for a variety of Antarcticpeninsula fauna and flora were used to quantify the importanceof benthic brown algae to resident organisms and determine foodweb relationships among this diverse littoral fauna. ¹³C valuesranged from–16.8 for benthic algal herbivores (limpets)to –29.8 for the krill, Euphausia superba; the averagepooled value for brown macroalgae, including their attachedfilamentous diatoms, was–20.6. There was no correlationbetween biomass ¹³C or ¹⁵N with C/N content, and consequentlyboth ¹³C and ¹⁵N values were useful in evaluating trophic relationships.¹⁵N values of the fauna ranged from 3.1 to 12.5, with lowestvalues recorded in suspension feeders (e.g., bryozoans) andhighest values in Adelie penguins (12.5) collected in 1989.The comparatively lower ¹⁵N value for a Chinstrap penguin (6.9)collected in 1997 is attributed to the different dietary foodsources consumed by these species as reflected in their respective¹³C values. Significant amounts of benthic macroalgal carbonis incorporated into the tissues of invertebrates and fishesthat occupy up to four trophic levels. For many benthic andepibenthic species, including various crustaceans and molluscs,assimilation of benthic algal carbon through detrital pathwaysranges from 30 to 100%. Consequently, the trophic importanceof benthic brown algae may well extend to many pelagic organismsthat are key prey species for birds, fishes, and marine mammals.These data support the hypothesis that benthic seaweeeds, togetherwith their associated epiphytic diatoms, provide an importantcarbon source that is readily incorporated into Antarctic peninsulafood webs.     

{alpha}Melanocyte Stimulating Hormone: Chemical Nature and Mechanism of Action

Melanotropin (-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂,synthesized and secreted by the pars intermedia of the vertebratepituitary. This peptide hormone is derived from pro-opiomelanocortin,a precursor protein which contains within its structure thesequences of other melanotropic peptides (- and rß-MSH,corticotropin), and possibly other hormones. -MSH is the physiologicallyrelevant melanotropin secreted by the pituitary and in mostvertebrates plays the essential role in adaptive color changesthrough its action on integumental chromatophores. The initial actions of -MSH are mediated at the level of themelanocyte membrane and involve signal transduction from receptorto adenylate cyclase on the intracellular surface of the membrane.This results in elevated cytosolic cyclic AMP levels followedby melanosome dispersion within dermal melanocytes and melanogenesiswithin epidermal melanocytes. The action of -MSH on dermal melanocytesrequires calcium for transduction of signal and cyclic AMP production.Melanosome dispersion per se does not, however, require extracellularcalcium. Structure-function studies of -MSH analogues and fragmentshave provided important insights relative to the structuralrequirements of the hormone for receptor binding and transduction.Substitution of certain residues within -MSH has led to thedevelopment of melanotropins that exhibit extraordinary potencyand prolonged biological activity     

Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells

Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. ₁-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate G_q/11 signaling pathways that subsequently increase intracellular Ca²⁺ concentration ([Ca²⁺]_ic). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca²⁺]_ic. Avian tendon cells were analyzed for mRNA expression of ₁-adrenoceptors by RT-PCR. Avian tendons expressed the _1A- and _1B-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca²⁺-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca²⁺]_ic. KMD-3213, an _1A-adrenoceptor antagonist, significantly reduced the Ca²⁺ response. Other adrenoceptor antagonists had no effect on the Ca²⁺ response. The absence of extracellular Ca²⁺ also significantly reduced the response to NE, indicating that Ca²⁺ influx contributed to the rise in [Ca²⁺]_ic. This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca²⁺ response is coupled to the _1A-adrenoceptor subtype. -adrenoceptors; fibroblasts; catecholamines; tenocytes     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca²⁺ spark and Ca²⁺ wave frequency but reduced global intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (–40 mV) cells, TNF- increased the frequency and amplitude of Ca²⁺ spark-induced, large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient K_Ca current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks, and iberiotoxin, a K_Ca channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H₂O₂ that stimulates Ca²⁺ sparks and transient K_Ca currents, leading to a reduction in global [Ca²⁺]_i, and vasodilation. cerebrovascular circulation; ryanodine-sensitive Ca²⁺ release channel; Ca²⁺-activated K⁺ channel; reactive oxygen species; vasodilation     

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes

The intestinal brush border (BB) Na⁺/H⁺ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) by the cholinergic agonist carbachol and Ca²⁺ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca²⁺]_i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca²⁺ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca²⁺-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKC pseudosubstrate-derived inhibitor peptide. [Ca²⁺]_i elevation caused translocation of PKC from cytosol to membrane. PKC bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca²⁺-dependent manner. PKC and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca²⁺]_i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca²⁺]_i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC is not necessary for the Ca²⁺-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis. Na absorption; PDZ domains; signal complex     

Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6

Ca⁺-induced Ca²⁺ release (CICR) in the heart involves local Ca²⁺ signaling between sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) ₁-subunit (1C_YM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing 1C_YM and RyR2 were vesiculated and exhibited spontaneous Ca²⁺ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with 1C_YM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca²⁺ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca²⁺ transients that required extracellular Ca²⁺. Depolarization-induced Ca²⁺ release in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing 1C_YM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca²⁺ current densities. In addition, the gain of CICR in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local _1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca²⁺ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca²⁺ release and the incidence of spontaneous Ca²⁺ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6. heart failure; dihydropyridine receptor; excitation-contraction coupling     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Analysis of the diurnal change in osmotic potential in leaves of Fraxinus excelsior L.

The daily cycle of the transpiration rate, stomatal conductance,water potential, and the concentration of the main osmoticumidentified in ash leaves, malate and mannitol, were monitoredin a field on the Isere river plain. On sunny days, the stomatalconductance tends to remain close to its maximun value allowinga high transpiration rate and diurnal variations in leaf waterpotential, _w, which may fall as low as –2 MPa at solarnoon. These variations of _w are closely correlated with changesin malate, mannitol and the concentration of the well-knownosmoticum K⁺, which agree with the involvement of an osmoticadjustment to counteract the evaporative demand during daylighthours. How malate, mannitol and K⁺ contribute to the osmoticadjustment was analysed subsequently by comparing the solutepotential _s, evaluated by the Boyle-Van't Hoff relation, tothe osmotic potential measured by thermocouple psychrometry.These experiments have led us to suspect some errors in themeasurement of , presumably due to experimental artefacts andthe ability of Ca ²⁺ , present in high levels in leaves, toform chelates with malate once the cells have been decompartmentedby freezing and thawing. Since significant changes of Ca²⁺ alsooccurred during the diurnal variations of _w, the possible mechanismsby which Ca²⁺ may be implicated in controlling the water statusof the tree are discussed. Key words: Fraxinus excelsior L, osmotic adjustment, thermocouple psychrometry, malate, calcium     

Ca2+ influx through {alpha}1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine-sensitive, spontaneous global Ca²⁺ oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both _1S dihydropyridine receptors (DHPRs) and ryanodine-sensitive Ca²⁺-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global Ca²⁺ oscillations could be inhibited by addition of 0.1 mM CdCl₂/0.5 mM LaCl₃ or 5 µM nifedipine to the extracellular bathing solution. After either treatment, Ca²⁺ oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked Ca²⁺ oscillations, normal orthograde signaling between DHPRs and RyRs, such as that elicited by 80 mM KCl depolarization, was still observed. In addition, we showed that spontaneous Ca²⁺ oscillations were never present in cultured mdg myotubes, which lack the expression of _1SDHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the _1SDHPRs and RyR1, the initiation of Ca²⁺ oscillations in myotubes may be facilitated, in part, by the Ca²⁺ influx through the _1s-subunit of the DHPR. calcium-induced calcium release; dihydropyridine receptors; excitation-contraction coupling; ryanodine receptors; skeletal muscle     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

Studies on Hormone Recognition by Arthropod Target Tissues

Numerous experiments have implicated the crustacean hepatopancreasas a target of the molting hormone, and recent studies stronglysuggest that ß-ecdysone is the crustacean moltinghormone. For example ß-ecdysone can elicit moltingin niter-molt crustaceans and stimulates the synthesis of proteinand RNA in the hepatopancereas. When ³H--ecdysone is injectedinto premolt crayfish, it is efficiently excreted, but labelcan be detected in almost all tissues examined. To determinewhether target tissues contain specific "receptor" moleculeswhich distinguish those tissues that respond to molting hormonefrom those that do not, a series of in vivo and in vitro experimentswere conducted utilizing Sephadex chtomatography and sucrosedensity gradient ultracentrifugation. These studies reveal thepresence of two proteins in the cytosol fraction of the hepatopancreasthat bind label from the ³H--ecdysone. These proteins have approximatemolecular weights of 250,000 (11.3S) and 130,000 (6.35S), andthe larger may be an aggregate of the smaller. Micro-chemicalanalysis demonstrated that the label associated with these proteinsis no longer attributable to -ecdysone nor any ecdysone-likecompound thus far described. Two alternate structures are describedthat may represent the metabolite bound to the protein, andit is suggested that the binding protein-metabolite complexmay be the the active from of the molting hormone. Studies arealso described dealing witha hemolymph lipoprotein in silkmothsthat binds juvenile hormone and it is postulated that the watersoluble hemolymph lipoprotein-juvenile hormone complex is themeans by which the lipoidal juvenile hormone is transportedin the aqueous hemolymph.     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis     

Serotonin Directly Increases a Calcium Current in Swim Motoneurons of Aplysia brasiliana

Muscle fibers in the swim appendages of the mollusk Aplysiabrasiliana are innervated by cholinergic motoneurons. Serotonin(5-HT) causes an increase in amplitude of junctional potentialsand muscle contractions at this neuromuscular synapse. We studiedmotoneurons with intracellular current-clamp recording and single-electrodevoltage-clamp analysis to determine the effects of 5-HT on somaticcurrents in these presynaptic neurons. Serotonin was found tohave no effect on action potential duration in motoneurons bathedin normal seawater, and no effect of 5-HT could be detectedon K⁺ currents, indicating that 5-HT does not indirectly enhancecalcium currents by prolonging the action potential. Calciumcurrents were isolated by replacing extracellular sodium withTEA and adding tetrodotoxin and other K⁺-channel blockers. Underthese conditions motoneuron action potentials were greatly prolongedand could be blocked with Co²⁺ or Cd²⁺. Addition of 5-HT increasedthe duration of these Ca²⁺ spikes by about 35%. In motoneuronsstudied with voltage clamp, the amine produced a 58% increasein total inward calcium current. Use of the calcium channelblockers nifedipine, nimodipine, -conotoxin GVIA, and -agatoxinTK revealed that motoneurons express varying amounts of L-,N- and P-like calcium channels, but only an agatoxin-sensitive,P-type channel is sensitive to 5-HT. It is concluded that 5-HTacts directly to increase a P-type Ca²⁺ current during a normalspike. The resulting increase in intracellular calcium couldcontribute to an increase in transmitter release and accountfor the increase in junctional potentials in swim muscles.     

The Effect of Hydroxymethanesulphonate on Photosynthesis in Chlorella pyrenoidosa

Photosynthesis under conditions known to favour glycollate excretionby algae did not result in glycollate excretion in a strainof Chlorella pyrenoidosa unless an inhibitor of glycollate oxidase,-hydroxypyridin-2yl-methane sulphonate (-HPMS), was present.This inhibitor increased the total amount of glycollate presentin the supernatant from the cells during photosynthetic carbondioxide fixation and gave accumulation of ¹⁴C in glycollateduring ¹⁴CO₂ fixation under conditions favouring glycollatesynthesis. At pH 8.3 -HPMS did not stimulate photosynthetic¹⁴CO₂ fixation in C. pyrenoidosa as occurs with some algae.Photoassimilation of acetate was inhibited by -HPMS, and thiswas shown to result from acetyl-CoA synthetase inhibition by-HPMS.     

The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

The Adaptation of Enzymes to Pressure I. A Comparison of Trout Liver Fructose Diphosphatase with the Homologous Enzyme from an Off-Shore Benthic Fish

SYNOPSIS. At low temperature (3°C), in the absence of substrateand cofactor, trout liver fructose diphosphatase (FDPase) isinactivated by exposures to relatively low pressures. FDP andMg²⁺ protect against this inactivation; hence, maximum catalysisat pH 7.5 is pressure insensitive, while at more alkaline pH,it is markedly accelerated by pressure. The volume change ofactivation, V*, at saturating FDP and Mg²⁺ concentrations isabout –40 cm₃/mole. The apparent Km for FDP and the Kafor Mg²⁺ are markedly increased by pressure. At low FDP or Mg²⁺levels these kinetic properties outweigh V* in determining thereaction rate; hence, under these conditions, piessure retardscatalysis. Similarly, the K₄ for AMP is notably pressure sensitive.Comparable effects of pressure on the kinetic constants forliver FDPase from benthic Corypliaenoides are much less pronounced,suggesting that in these off-shore species enzyme-substrate,enzyme-cofactor, and enzyme-modulator interactions have beentailored through evolution for pressure independent catalyticfunction.