Iothalamate stimulates hydroperoxide formation by soybean lipoxygenase

Sodium iothalamate produced a dose dependent increase in basal oxygen consumption when soybean lipoxygenase was incubated with arachidonic acid in 0.1M borate buffer pH 9.0. The increase in oxygen consumption was associated with an increase in absorbance at 234 nm indicating an increased conjugated diene formation. The stimulation of 02 consumption was demonstrated to be due to an increase in 1500H arachidonic formation. The increase in 1500H arachidonate formation could be blocked by mannitol which is an inhibitor of the lipoxygenase enzyme. N-methyl glucamine (meglumine) which is added to some preparations of iothalamate, was also able to suppress the increase in hydroperoxide formation in a dose dependent fashion.     

Enhancement of acetaldehyde-protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.     

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.     

Effect of polyamines on ADP-ribosylation by chick-embryo-liver nuclei

Effects of polyamines on poly(ADP-ribose) formation and DNA synthesis in the chick-embryo-liver nuclei were investigated. When 14-day chick-embryo-liver nuclei were incubated with [3H]NAD in the presence of 1 mM spermine, 2.5 mM spermidine, or 3.5 mM putrescine, a 9-fold increase in poly)ADP-ribose) formation was observed. Nuclei treated with nuclease showed high poly(ADP-ribose) synthetase activity as spermine-treated nuclei. However, no further increase in the polymer formation by polyamines was detected in the nuclease-treated nuclei. We found that an increase in the polymer formation by spermine was the result of an increase in both chain length and chain number of the polymer at 2.3- and 6-fold, respectively. The major ADP-ribosylated proteins were determined as two non-histone proteins of Mr 130 000 and 70 000. The experiment of DNA synthesis with nuclei ADP-ribosylated in the presence of spermine showed a 7-fold increase in [3H]dTMP incorporation into the acid-inaoluble fraction. A similar stimulation was also found with nuclei treated with other polysmines, spermidine and putrescine, in the presence of NAD. These results indicate that DNA synthesis in growing tissues containing polyamines at high levels, such as is the case with tumors and the fetus, is stimulated by polyamine-mediated ADP-ribosylation of the nuclear proteins.     

Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.     

Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alpha-hemoglobin to membrane

Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.     

Arachidonic acid metabolism in TNS-induced chronic and immunologic enteritis in rats, and the effect of 5-ASA

Inflammation of the rat distal intestine was induced by intradermal sensitization and subsequent multiple intrajejunal challenge with the hapten 2,4,6-trinitrobenzenesulphonic acid (TNBS) via an implanted catheter. The time course of the inflammatory reaction was followed by determination of the enteritis score and measurement of in vitro eicosanoid formation of homogenates of the gut after 0, 1, 2, 4, 7, 14 and 21 days of local daily challenge with 0.08% TNBS. There was a small initial increase of eicosanoid formation, reached at days 1 and 2, followed by a significant increase in metabolism of arachidonic acid on day 21. Although at day 1 a four-fold increase in inflammation score was reached, no further significant changes were observed during the following 3 weeks. The greatest increase in metabolite formation was observed in prostanoids TxB(2), PGE(2). and PGD(2) and the 5-lipoxygenase product LTC(4), whereas minor changes were found for LTB(4) and other lipoxygenase products such as 12- and 15-HETE. The formation of these metabolites was already inhibited by low-dose 5-aminosalicylic acid (5-ASA), given orally twice daily during the 3 weeks challenge period, while the enteritis score was affected dosedependently.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions.

The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway.     

On the correlation between light-induced protein fluorescence changes and the formation of metarhodopsin III465 in bovine photoreceptor disk membranes

The correlation between the absorption spectral changes and the increase in protein fluorescence after short illumination of suspensions of bovine photoreceptor disk membrane fragments was investigated. A comparison of the kinetics of the thermal formation of rhodopsin photoproducts with those of the increase in fluorescence indicates a close correspondence between the thermal formation of metarhodopsin III₄₆₅ and the light-induced fluorescence increase. This result suggests that a conformational change, probably involving a decrease in the polarity of the environment of tryptophan residues, occurs in association with the formation of metarhodopsin III₄₆₅.     

Influence of oxygen and substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes

Summary The influence of different states of oxygen and alkane substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes has been investigated. At sufficient oxygen and substrate supply a nearly equimolar ratio between the formation of biomass and alkane oxidation was observed. About 45% of the carbon source utilized was incorporated into the biomass. Strong oxygen limitation decreased protein formation and carbon incorporation into the biomass with a simultaneous increase in CO₂ formation, whereas periodic changes of oxygen supply only caused a decrease in carbon incorporation into the biomass and an increase in CO₂ formation. During cultivation in the presence of an inert hydrocarbon (pristane) it was found that carbon limitation and oxygen saturation diminished the formation of total and nitrogen-containing biomass, whereas carbon and oxygen limitation reduced the formation of total biomass.Offprint requests to: P. Riege     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Effect of dexamethasone on mannolipid synthesis by hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. Mannose incorporation into dolichol monophosphate mannose in homogenate and microsomal fraction of the hepatocytes was increased 2-fold over the controls 24 h after induction of inflammation by turpentine injection. Incubation of hepatocytes from both control and inflamed rats with 0.1-10 microM-dexamethasone produced a 1.5-fold increase of dolichol phosphate mannose formation, whereas, 100 microM-dexamethasone decreased its formation. The increase in the ratio of dolichol phosphate mannose formation in inflamed over controls was virtually eliminated when the cell homogenate assay mixtures included 30 nmol of exogenous dolichol phosphate. This supports the earlier suggestion that the increase in the enzyme activity in inflammation could be due to higher concentrations of endogenous dolichol phosphate [ Coolbear & Mookerjea (1981) J. Biol. Chem. 256, 4529-4535]. In contrast, the increase in the ratio of dolichol phosphate mannose formation between dexamethasone-treated and untreated hepatocytes remained unchanged when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the increase in glycosylation of dolichol phosphate in dexamethasone-treated hepatocytes is probably due to the increased mannosyltransferase activity, rather than due to higher concentrations of endogenous dolichol phosphate in these cells.     

Increased Radiation Resistance of Vegetative Bacillus pumilus

A 4.5-fold increase in vegetative cell radiation resistance of Bacillus pumilus E601, the internationally recognized biological standard for irradiation sterilization, was obtained by the repeated passage of resistant survivors through successive sublethal doses of (60)Co irradiation. This increase in resistance was accompanied by a corresponding increase in spore resistance through the seventh irradiation passage. By the fifteenth passage, the ability for spore formation was lost. Other effects noted by the successive irradiation dosages included loss of motility and pellicle formation, and changes in the Gram reaction, cell morphology, and colonial morphology. Increased resistance was also accompanied by an increased nutritional requirement for specific amino acids. Radiation resistance was not transferred from vegetative cells to spores.     

Morphine Tolerance and Physical Dependence: Reversal of Opioid Inhibition to Enhancement of Cyclic AMP Formation

Abstract: This laboratory has previously demonstrated that the μ-selective opiate receptor agonist sufentanil can produce a naloxone-reversible increase or decrease in the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus, depending on the concentration of opioid used. On the basis of these results, it was suggested that μ-opiate receptors are positively as well as negatively coupled to adenylyl cyclase. In the present study, the effect of chronic morphine exposure, in vivo, on the magnitude of electrically stimulated formation of cAMP and its modulation by sufentanil was investigated. In chronic morphine-treated preparations, the magnitude of electrically stimulated cAMP formation, while in the presence of an inhibitory (10⁻⁶ M ) concentration of sufentanil, is indistinguishable from the formation that occurs in opiate-naive preparations (in the absence of exogenous opioid). This indicates that the negative modulation of stimulated enteric cAMP formation by sufentanil manifests tolerance. Paradoxically, however, in "addicted tissue" the magnitude of the increase in cAMP formation produced by electrical stimulation in the presence of a previously inhibitory concentration of sufentanil is significantly larger than in its absence. Thus, the equivalence between the magnitude of stimulation-induced increase in cAMP formation observed in naive versus tolerant/dependent tissue, while in the presence of sufentanil, is due to the ability of an originally inhibitory concentration of opioid to enhance or facilitate stimulated formation of cAMP. It is suggested that tolerance/dependence to the opioid inhibition of stimulated cAMP formation results not only from the loss of inhibitory potency but also from its reversal to enhancement.     

Effect of double infection of cowpox virus-infected cells with paramyxovirus (Sendai virus) on formation of cowpox virus-specific cell surface antigen.

The formation of cowpox virus-specific cell surface antigen (CPV S-ag) was significantly enhanced by double infection with HVJ (Sendai virus). Simultaneous double infection, superinfection with HVJ and superinfection with CPV of cells persistently infected with HVJ similarly enhanced the formation of CPV S-ag, while pre-infection with HVJ was ineffective. To be effective, cells must be infected at a m.o.i. of greater than or equal to 1.0 and HVJ gene functions had to be expressed. The HVJ-infected cell extracts had an ability to accelerate uncoating (or degradation) of CPV, causing an early increase and a subsequent decrease in the infectivity of CPV. This activity reached a maximum 4--6 hr after HVJ infection, the increase paralleling enhancement of the total activity of several cellular enzymes. Addition of puromycin abolished the increase of these activities and the formation of CPV S-ag. Thus, the double infection with HVJ of CPV-infected cells induces an enhancement of CPV S-ag formation presumably as a consequence of activation of cellular enzymes which in turn accelerates uncoating of CPV.     

Spectral power of theta and alpha EEG-bands in preschool children with different level of set plasticity

Spectral power of theta and alpha EEG-bands at different stages of visual set was studied in 5-7-year-old children. Children with a plastic set had greater alpha-band spectral power values than children with a rigid set. At set formation stage children with a rigid set displayed an increase of theta-band EEG-power, which is a manifestation of "immature" arousal reaction. Children with a plastic set displayed at set formation stage an increase of alpha-band spectral power in bilateral occipital brain areas. Dynamics of EEG spectral power differed in children before and after 6 years of age. At set formation stage 5-6-year-olds displayed an above-mentioned "immature" arousal reaction, and 6-7-year-olds--an increase of alpha-band spectral power in occipital brain areas. At set actualization stage 6-7-year-olds displayed an increase of alpha-band spectral power in right posterior brain areas. At set extinction in this group a bilateral decrease of alpha-band spectral power in mid-frontal areas was observed. An importance for a quick set-shifting of certain brain areas' involvement in the process of visual perception is discussed.