Diphtheria toxin induces leakage of acidic liposomes

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Long-term amphetamine treatment attenuates or reverses the depression of neuronal activity produced by dopamine agonists in the ventral tegmental area

Neuronal activity was recorded from the ventral tegmental area (VTA) of immobilized, locally anesthetized rats on the day immediately following long-term treatment (twice daily for 6 consecutive days) with saline, 1.0 or 5.0 mg/kg $\text{d}$-amphetamine ($\text{d}$-AMPH). Each rat was challenged intravenously with $\text{d}$-AMPH (beginning with 0.0625 mg/kg) or with 0.005 mg/kg apomorphine. Treatment with $\text{d}$-AMPH significantly reduced the ability of this drug to inhibit VTA activity. In fact, nearly half of the neurons in the high-dose treatment group were excited by $\text{d}$-AMPH, whereas only 20% of control neurons showed this response. Moreover, apomorphine routinely accelerated firing rate in the VTA following treatment with 5.0 mg/kg $\text{d}$-AMPH but this response was never observed in control neurons, not even in those that were excited by $\text{d}$-AMPH. Thus, tolerance appears to develop to the ability of dopamine agonists to inhibit VTA activity and this effect may be mediated, at least in part, by a subsensitivity of inhibitory dopamine autoreceptors.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

Rate sensitivity of blood pressure to hypoxia

A biochemical kinetic model is used to describe changes in mean arterial blood pressure in dogs to three different rates of fall of arterial partial pressure of oxygen. The model is a linear loop with one variable rate coefficient (parametric control) which has been previously shown to characterize the rate sensitivity to presented stimuli. A three component model was identified under a least squares criterion and it showed that a unique (stimulation independent) representation can be obtained which can serve as a conceptual framework for the study of this phenomenon.     

5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex

The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN----InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.     

Stimulation of adenylate cyclase by adenosine and other agonists in mesenteric artery smooth muscle cells in culture

An adenosine-sensitive adenylate cyclase has been characterized in cultured mesenteric artery smooth muscle cells. N-Ethylcarboxamide-adenosine (NECA), N-Methylcarboxamide-adenosine (MECA), L-N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine (2-cl-Ado) all stimulated adenylate cyclase in a concentration dependent manner. NECA was the most potent analog (EC50, 1 microM), whereas PIA (EC50, 15 microM), 2-Cl-Ado (EC50, 15 microM) and MECA (EC50, 24 microM), were less potent and had efficacies relative to NECA of 0.61, 0.61 and 0.65, respectively. Adenosine showed a biphasic effect: stimulation at lower concentrations and inhibition at higher concentrations, whereas 2' deoxyadenosine only inhibited adenylate cyclase activity. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentration and was blocked by 3-isobutyl-l-methylxanthine (IBMX) and 8-phenyltheophylline (8-PT). Adenylate cyclase from these cultured cells was also stimulated by other agonists such as epinephrine, norepinephrine, prostaglandins, dopamine, NaF and forskolin. The stimulation of adenylate cyclase by isoproterenol, epinephrine and norepinephrine was blocked by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol and flupentixol all inhibited dopamine-stimulated adenylate cyclase activity. In addition, the stimulation by an optimal concentration of PIA was additive or almost additive with maximal stimulation caused by catecholamines and prostaglandins. These data indicate the presence of adenosine (Stimulatory "Ra"), catecholamine and prostaglandin receptors in mesenteric artery smooth muscle cells and suggest that these agents may exert their physiological actions through their interaction with their respective receptors coupled to adenylate cyclase.     

Comparison of the binding of chicken alpha-macroglobulin and ovomacroglobulin to the mammalian alpha 2-macroglobulin receptor

Chicken alpha-macroglobulin (alpha M) and ovomacroglobulin were purified by Ni+2 chelate chromatography. These proteins had similar subunit structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chicken alpha M bound 1.0 mol and ovomacroglobulin bound 0.8 mol 125I-trypsin per mol inhibitor, respectively. Ovomacroglobulin cleared rapidly from the circulation of mice, and the clearance was inhibited by asialoorosomucoid, but native chicken alpha M cleared slowly (t 1/2 greater than 1 h). After reaction with trypsin, this alpha-macroglobulin cleared rapidly (t 1/2 = 3 min), and this clearance was inhibited by a 1000-fold molar excess of human alpha 2M-methylamine. Ovomacroglobulin-trypsin did not inhibit the binding of 0.2 nM 125I-labeled human alpha 2M-methylamine to mouse peritoneal macrophages in vitro, but chicken alpha M reacted with trypsin inhibited the binding by 50% at 1.9 nM. A kappa I of 1.1 nM was calculated for the binding of chicken alpha M-trypsin to the mammalian alpha-macroglobulin receptor. This affinity is comparable to that obtained with human and bovine alpha 2M.     

Characterization of a long-term T-helper-cell line which produces IL-2 and induces immunoglobulin secretion

Six OKT4+ human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.     

Macrophage-activating factor for cytotoxicity produced by a human T-cell hybridoma

Human MAF-C (macrophage-activation factor for cytotoxicity)-producing hybridoma H2-E3-5 was prepared by somatic cell fusion of PHA-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphatic leukemia cells (CEM). The following activities were assayed: (1) macrophage-migration-inhibitory factor (MIF), (2) macrophage-activation factor for glucose consumption (MAF-G), (3) macrophage-activation factor for O2-formation (MAF-O), and (4) macrophage-activation factor for cytotoxicity (MAF-C). After anion-exchange chromatography, MAF-C activity could be distinguished from MIF and MAF-O activities. It is shown that MAF-C is not the same as MAF-G from the culture supernatants of CEM 11, a parent cell line of H2-E3-5. Furthermore, MAF-C from H2-E3-5 culture supernatants activated differentiated macrophages but not monocytes.     

Stimulation of prostaglandin E2(PGE2) secretion by benzylpenicilloyl (BPO) carrier conjugates in human peripheral blood lymphocytes

The conjugates of the main determinant of penicillin, benzylpenicilloyl (BPO), with various protein carriers can stimulate mononuclear cells from human peripheral blood lymphocytes (PBL) to increase PGE₂ secretion. Thus, BPO bound to either human γ-globulin (BPO-HGG), bovine γ-globulin (BPO-BGG), and keyhole limpet menocyanin (BPO-KLH) increased the PGE₂ level 8–20 times above the level produced by stimulation with the carriers alone. Since in previous studies it was shown that preincubation with BPO-HGG can suppress the thymidine uptake after subsequent stimulation with BPO-KLH, we investigated if the induction of this unresponsiveness could be caused by PGE₂. The results of this study show that: (a) although the amount of PGE₂ secreted by the stimulation of BPO-HGG was 20-fold greater than the basic level, this amount is not sufficient to suppress significantly the thymidine uptake; (b) preincubation of PBL with higher than physiological concentration of externally added PGE₂ caused significant suppression of thymidine uptake after stimulation with BPO-KLH, but the addition of physiological concentration of PGE₂ did not show the same effect; (c) preincubation with BPO-HGG had no effect on PGE₂ level after subsequent challenge with BPO-KLH; and (d) inhibitor of PGE fails to influence the suppression of BPO-KLH proliferation induced by BPO-KLH.     

Occurrence of hydroxyproline in a toxin from the marine snail Conus geographus

A 22-residue peptide toxin from the venomous marine snail Conus geographus (L.) was found to have a most unusual amino acid composition: Lys₄, Arg₃, $\text{1}\text{2}\text{Cys}{}_6$, Asx₂, Glx₂, Thr, Ala, plus three residues of trans-4-hydroxyproline. Absence of Gly and Pro indicates that the hydroxyproline must be in sequences different from those in which hydroxyproline occurs in collagen and other proteins.     

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of amino acid levels in rat blood obtained by catheterization and decapitation

A novel, sensitive and highly resolving amino acid analysis procedure was developed and used to compare two methods of obtaining blood from experimental animals. The procedure, utilizing a meter long microbore HPLC column containing spherical cation-exchange resin and fluorescence detection following postcolumn reaction with o-phthaldialdehyde, was shown to reliably measure forty-one primary amine components in rat plasma.Comparison of values from blood obtained by decapitation and by catheterization documented the significant artifactual influence of the decapitation procedure on approximately half of the measured constituents.     

Effects of dietary saturated, n-6 and n-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats

Effects of the dietary administration of saturated fat and of n-6 and n-3 polyunsaturates on blood pressure, prostaglandin metabolism in small vessels, tissue fatty acid distribution and urinary PGE₂ excretion were compared. Rats were divided into three groups. Diets contained 10% hydrogenated cocunut oil (HCO), 10% safflower oil (SFO) or 10% cod liver oil (CLO) added to a basic fat free diet for 10 weeks. Systolic blood pressure was increased in the CLO group animals. Urinary PGE₂ excretion was decreased in the HCO and CLO groups as compared to that in the SFO group animals. PGE₂, 6-keto-PGF₁ and thromboxane (Tx) B₂ outflow from isolated perfused mesenteric arterial beds were extremely decreased in the CLO group animals, and to a lesser extent in the HCO group as compared to the SFO animals. In the tissue phospholipid, 20:3n−9/20:4n−6 ratios were increased in the HCO group indicating essential fatty acid deficiency, and n-6 and n-3 polyunsaturates were elevated in the SFO and the CLO group animals respectively. Arachidonic acid concentration was highest in the SFO group, while there was no significant differences between the HCO and the CLO group. These results suggest that dietary fatty acid manipulation effects urinary PGE₂ excretion and PGI₂, PGE₂ and TxA₂ synthesis in mesenteric arterial beds and also changes the tissue fatty acid distribution. Furthermore, n-3 polyunsaturates caused an extreme reduction of 2-series PGs synthesis in small resistance vessels.     

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt

When injected into rats, a certain amount of mannitol is taken up by the liver and is associated with sedimentable structures. Isopycnic centrifugation in a sucrose gradient shows that a large part of mannitol is present in mitochondria, what remains is located in the lysosomes. The hypotonic release of mannitol present in organelles shows that the polyol is shared between mitochondria and lysosomes. The trapping of mannitol in lysosomes could result from the heterophagic or autophagic function of the lysosomes; the mechanism of its accumulation in mitochondria is still unexplained.     

Islet-activating protein, pertussis toxin, inhibits Ca2+-induced and guanine nucleotide-dependent releases of histamine and arachidonic acid from rat mast cells

Islet-activating protein (IAP) suppressed Ca2+-induced histamine release and phospholipase A2 activation in Gpp(NH)p-loaded mast cells. Since the guanine nucleotide-binding protein involved in adenylate cyclase inhibition is known to lose its function upon being ADP-ribosylated by IAP, the nucleotide-binding protein is likely to mediate Ca2+-linked biosignalling leading to histamine secretion.     

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH₂S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the $\text{in}\text{vitro}$ activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro¹, D-Phe², D-Trp^3,6]LH-RH antagonist was completely inhibitory at 30 ng $\text{in}\text{vitro}$ and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed $\text{in}\text{vivo}$ antiovulatory activity at the 250 μg level.