Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate ⁴⁵Ca²⁺ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na⁺ + K⁺)-ATPase (plasma membrane marker) and Ca²⁺-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions.⁴⁵Ca²⁺ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na⁺ + K⁺)-ATPase or NADH oxidase activities.Accumulation of ⁴⁵Ca²⁺ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Presence of digitalis-like factor in mammalian plasma

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.     

Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex.

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate 45-Ca-2+ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na-++K-+)-ATPase (plasma membrane marker) and Ca-2+-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions. 45-Ca-2+ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na-++K-+)-ATPase or NADH oxidase activities. Accumulation of 45-Ca-2+ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Evidence that mammalian lignans show endogenous digitalis-like activities

Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma-butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man.     

Characterization and scatchard binding analysis of adrenal digitalis-like material.

Culture medium conditioned by the adrenocortical tumor cell line Y-1 has been fractionated by HPLC to determine whether the ouabain-like binding activity (OLB) of this medium co-purifies with digoxin immunoreactivity. Furthermore, we have evaluated whether this medium contains the ability to inhibit the ion translocating action of the sodium pump and how this material purifies on HPLC. These activities are not homogenous, however, maximal activity in each assay was found in the same HPLC fraction. The OLB of this fraction was not reduced by alkaline hydrolysis or trypsinization, heating in air at 150 degrees C did reduce activity. Scatchard binding analysis of this fraction indicated the presence of a single class of specific competitive inhibitor of the binding of radiolabelled ouabain to human erythrocytes. The inhibitor showed a Hill coefficient of unity.     

Relationships among endogenous digitalis-like factors in essential hypertension

Elements of a hypothesis that relate endogenous digitalis-like factors to both natriuretic hormone and hypertension are briefly reviewed. The stimulus for secretion of these factors appears to involve a tendency toward a state of extracellular fluid volume expansion as a consequence of an inherited or an acquired defect in renal function. Several studies implicate the brain and, in particular, the hypothalamus in the control of the secretion. The digitalis-like factors are thought to act by partial inhibition of active sodium transport, thereby promoting increased intracellular levels of Na+ and Ca2+ in a variety of cell types. In the kidney, inhibition of sodium transport leads to a compensatory natriuresis to correct the tendency for volume overload. In smooth muscle, the inhibition of sodium transport will indirectly increase intracellular calcium levels. The increased availability of Ca2+ will elevate muscle tone and increase peripheral vascular resistance. Also presented are criteria that may be used to characterize digitalis-like activity in samples and extracts obtained from purification procedures. Finally, we review our measurements of the 6-h integrated plasma levels of digitalis-like factors and other hormones for normotensive subjects and patients with essential hypertension. The data indicate the presence of two classes of digitalis-like factors with potentially different roles in electrolyte metabolism and hypertension.     

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.     

Evidence for the existence of the same endogenous digitalis-like factor in several mammalian species

1. Endogenous digitalis-like activity was studied comparatively in four mammalian species: guinea pig, dog, cow and rat. 2. Water extracts were prepared from guinea pig, dog, cow and rat hearts and assayed by ouabain radioreceptorassay, digoxin radioimmunoassay and digitoxin radioimmunoassay. Extracts were further analysed by fractionation by gel permeation chromatography with Sephadex G-25. 3. A similar behaviour was observed with the four species in the three assays. Extracts displaced tritiated ouabain binding to its receptor and labeled digoxin analogue binding to antidigoxin antibodies in a competitive manner. Displacement of labeled digitoxin analogue to antidigitoxin antibodies did not follow Michaelis-Menten kinetics. IC50 ratios between assays were similar for the four species studied. 4. Extracts from the four species exhibited a similar pattern when fractionated with Sephadex G-25. Endogenous digoxin-like immunoreactivity eluted after the salts, suggesting that the active material is of a molecular weight of less than 1000. 5. Results suggest that a similar endogenous factor endowed with digitalis-like characteristics is present in all mammalian species.     

Plasma volume expansion increases lysophosphatidylcholine and digitalis-like activity in rat plasma

A circulating factor with digitalis-like activity has been proposed to play a role in the regulation of plasma volume. Lysophosphatidylcholine has been found to be active in many assays for digitalis-like activity. To examine the relationship between plasma digitalis-like activity and plasma lysophosphatidylcholine, the effect of plasma volume expansion with saline on the plasma levels of phospholipids and on the ability of delipidated extracts of plasma to displace tritiated ouabain from the digitalis receptor was determined. Lysophosphatidylcholine was elevated after 15, 30, and 120 minutes of volume expansion but was decreased at 60 minutes. Phosphatidylcholine was decreased at 15, 60, and 120 minutes. Plasma sphingomyelin was not altered at any time point. The ability of plasma to displace tritiated ouabain was increased only at the 60 minute time point. These results indicate that the increase in digitalis-like activity in volume expanded states is mediated by a combination of at least two factors, lysophosphatidylcholine and another factor whose digitalis-like activity is not related to the surfactant actions of a lipid.     

Evidence for the contribution of LTR retrotransposons to C. elegans gene evolution

LTR retrotransposons may be important contributors to host gene evolution because they contain regulatory and coding signals. In an effort to assess the possible contribution of LTR retrotransposons to C. elegans gene evolution, we searched upstream and downstream of LTR retrotransposon sequences for the presence of predicted genes. Sixty-three percent of LTR retrotransposon sequences (79/124) are located within 1 kb of a gene or within gene boundaries. Most gene-retrotransposon associations were located along the chromosome arms. Our results are consistent with the hypothesis that LTR retrotransposons have contributed to the structural and/or regulatory evolution of genes in C. elegans.     

Evidence for the contribution of humic substances to conditioning films from natural waters

The presence of humic substances in conditioning films deposited on solid surfaces from natural waters was investigated using electron impact (EI), chemical ionization (CI) and secondary ion time-of-flight mass spectrometry (TOFSIMS). EI and CI spectra of a freshwater sample from a pond in Centennial Park, Sydney, Australia, showed a high degree of similarity with spectra of humic acids purchased from Fluka and Sigma as well as with reference humic acid and fulvic acid from the International Humic Substances Society, suggesting that most of the organic matter in the pond water was of humic origin. All the complex electron impact mass spectra feature series of high-intensity ions separated by 14 Da or 18 Da, which can be attributed to CH(2) and OH(2) respectively. Thermal desorption profiles of all samples generated by EI and CI were qualitatively similar. The secondary desorption peaks were less well-defined in CI compared to EI. Positive ion thermal desorption profiles displayed a two-step ionisation, with a sharp and well-defined initial desorption peak at t～50s, followed by a broader desorption peak with a maximum intensity at t ～ 100 s post-heating. The Centennial Park natural organic matter (NOM) differed from the other humic fractions in having two additional broad desorption peaks between the two described previously, and a less-defined initial peak. Infrared spectroscopy showed that proteinaceous matter in the lake water was insignificant in comparison with functional groups indicative of humic substances. TOFSIMS characterization showed almost identical spectra for Aldrich humic acid and Centennial Park NOM in the high mass region of 2000 Da to 3000 Da. Each spectrum contains approximately 25 groups of ion peaks, separated by 74 Da from group to group. Each group is composed of 6 or 7 individual peaks. The spectral features are consistent with a macromolecular structure of humic acid where aromatic rings are joined to the macrostructure via aliphatic spacer molecules.     

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Environmental bacteriophage-host interactions: factors contribution to natural transduction

Over the past two decades the potential for the exchange of bacterial genes in natural environments through transduction (bacteriophage-mediated gene transfer) has been well established. Studies carried out by various laboratories throughout the world have demonstrated that both chromosomal and plasmid DNA can be successfully transduced in natural environments ranging from sewer plants to rivers and lakes. Transduction has been shown to take place in the gills of oysters and the kidneys of mice. Model studies have demonstrated the ability of transduction to maintain genetic material in bacterial gene pools that would otherwise be lost because of negative fitness. Thus, transduction may affect the course of bacterial evolution. Identification of natural transduction has led to the investigation of the dynamics of bacteriophage host interactions in natural aquatic environments and to the exploration of various environmental factors that affect virus-host interactions. Two important environmental factors which affect virus-host interactions are the metabolic state of the host and the exposure of the host to DNA-damaging stresses such as solar UV light. Recent researches on these two areas of virus-host relationships are reviewed.     

Involvement of endogenous digitalis-like factors in voluntary selection of alcohol by rats.

This study tested the hypothesis that endogenous digitalis-like factor (DLF) is involved in the development of alcohol dependence in rats. In 33 male Wistar rats in conditioned place preference (CPP) experiment, ethanol evoked increase in time spent in the ethanol-associated compartment (702+/-82 in ethanol-treated vs. 426+/-86 sec in the controls). Digoxin pretreatment (125 microg/kg, i/p) did not affect the time spent in the water-associated compartment (476+/-80 sec), but prevented the acquisition of ethanol CPP (385+/-112 sec in ethanol-paired side, P<0.05). In a two bottle choice test, where rats (n=6 per group) chose between drinking water and 9% ethanol, immunization against two putative DLFs, marinobufagenin and ouabain (MBG and OLC) resulted in a 60% increase of ethanol consumption. Acute intragastric administration of 9% ethanol to the rats was associated with increased OLC in cerebrospinal fluid, and stimulated urinary excretion of MBG and OLC. Thus, in rats, digoxin, which mimics the effects of DLFs, suppresses the free choice of alcohol, while immunization against DLFs is associated with alcohol seeking behavior.     

Evidence for an upper limit to mitotic spindle length

Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.     

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Major contribution of epididymis to alpha-glucosidase content of ram seminal plasma

Acid alpha-glucosidase and L-carnitine (a well-known epididymal marker) were measured in rete testis and epididymal fluids of adult male rams. These fluids were collected by selective catheterization or by a micropuncture technique, respectively. Both parameters remained at a low and constant level in rete testis and all along caput and corpus epididymidis. Then they increased at equivalent rates in cauda epididymidis to much higher levels than those in seminal plasma (5 mU/mg protein and 10 mM, respectively). An optimum pH study of alpha-glucosidase activity in these fluids showed two well-separated peaks in rete testis and caput epididymal fluids around pH 4 and 7, respectively, but only a single peak at pH 4 in cauda epididymidis that was comparable to the one in seminal plasma. Sucrose density gradient fractions analyzed for their enzyme content in the absence or presence of sodium dodecyl sulfate (1% w/v), a selective inhibitor of acid alpha-glucosidase activity, allowed the demonstration of two enzyme forms at pH 6.8 in rete testis fluid sedimenting in the 7S and 4S regions of the gradient, while a unique 4S form was encountered in cauda epididymidis and in seminal plasma. Although the fate of the minor 7S component of the rete testis fluid in its epididymal transit is presently unknown, similarities between the enzyme in cauda epididymidis and seminal plasma are strong enough to support the hypothesis that epididymis contributes primarily to the acid alpha-glucosidase content of ram seminal plasma.