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At least 307 records · Page 17Linked to original sources
PubMed1985

Inositol trisphosphate induces calcium release from nonmitochondrial stores i sea urchin egg homogenates.

This study presents evidence that inositol trisphosphate (IP3) releases Ca2+ from intracellular stores in sea urchin eggs. First, high voltage discharge was used to transiently permeabilize eggs and introduce IP3; the resultant induction of cortical reactions (a well characterized Ca2+-dependent event) provided indirect evidence that IP3 released Ca2+ from intracellular stores. Next, Ca2+ uptake and release from egg homogenates and homogenate fractions were monitored by both Ca2+ minielectrodes and the fluorescent Ca2+ indicator, quin-2. Both assay methods showed Ca2+ release upon IP3 addition, with a half-maximal response at 50-60 nM IP3 and maximal Ca2+ release at approximately 1 microM IP3. Homogenates were 300-fold more sensitive to IP3 than IP2, and Ca2+ release was 95% inhibited by the Ca2+ antagonist TMB-8 (3 mM). Fractionation by density gradient centrifugation showed that activities for Ca2+ sequestration and IP3 responsiveness co-purified with endoplasmic reticulum microsomes. Following an initial IP3 addition, homogenates were refractory (desensitized) to additional IP3. However, if homogenates were centrifuged and the vesicles resuspended in media lacking IP3, they would respond to added IP3, therefore, showing that desensitization is most likely due to the presence of IP3. This study also shows that the mechanism of IP3 action is inherent to the microsomes and ions present in the medium used, with no cytoplasmic factors being required. The stability of this microsome preparation and the purification obtained with density gradient centrifugation make this a promising system with which to further characterize the mechanism of IP3 action.

Adenosine Triphosphate↗
PubMed1988

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II.

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.

Adenosine Triphosphate↗
PubMed1978

Effect of thiocyanate and other univalent anions on the binding of thyroxine to rat liver nuclei and homogenate.

The binding of thyroxine (125I-T4) in rat liver homogenate and isolated nuclei was investigated in vitro. In homogenate, the binding of T4 was modified by thiocyanate ion (SCN-) added to the incubation mixture as potassium thiocyanate. The displacing effect of SCN- increased with increasing concentrations of this compound, the lowest effective dose being that which significantly displaced T4 from liver homogenate (105 pmol per one ml medium). Moreover, SCN- significantly increased the displacing effect of unlabelled T4. In contrast, however, no effect of SCN- was found on the binding of T4 in the isolated liver nuclei. In sonicated nuclei no effect of SCN- on T4 binding was found, but specific binding of T4 under these circumstances was impaired. Finally, in a homogenate a displacing effect of other univalent anions (BF4-, ClO4- and MnO4-) was found resembling that of SCN-. It is suggested that, in the liver homogenate, SCN- displaces T4 from non-specific binding sites. This view is also supported by the fact that in liver nuclei, where specific binding sites prevail, no effect of SCN- was observed.

Animals↗
PubMedDate not supplied

Long-term preservation of egg and tissue homogenates for determination of organochlorine compounds: freezing versus freeze-drying.

Storage of wet egg homogenates at temperatures from -18 degrees to -28 degrees C was more suitable for long-term preservation than freeze-drying. Changes in residue levels of heptachlor epoxide, oxychlordane, dieldrin, hexachlorobenzene,p,p'-DDE, mirex, and PCBs were not significant over a 3-year period in fresh herring gull egg homogenates stored at -18 degrees to -28 degrees C. Compounds with gas chromatographic retention times shorter than hexachlorobenzene vaporized during freeze-drying at a rate proportional to their volatility. Evaporative losses of components with vapor pressures less than hexachlorobenzene did not occur in naturally contaminated herring gull eggs after storage at room temperature for up to 1 year. Higher losses of all compounds, up to 25% for p,p'-DDE, occurred in freeze-dried whole-body herring gull homogenates. Easily dehydrochlorinated compounds were rapidly degraded in freeze-dried chicken egg homogenate at room temperature: The half-life of p,p'-DDT and p,p'-DDD was about 20 days, and that of alpha- and gamma-hexachlorocyclohexane was much less than 16 days. About one-third of oxychlordane in herring gull eggs was lost in 1 year under these conditions, but none was lost after freeze-drying when the homogenate was stored at -18 degrees to -28 degrees C.

Eggs↗
PubMed1979

[Mechanism of activation of acetylcholinesterase in brain tissue homogenates by detergents].

The mechanisms of activation of acetylcholinesterase (AChE) in the homogenates of rat and frog forebrains and medullae oblongatae under effects of non--ionic detergents (Triton X-100 and Tw-en 80) were studied. Titration of the active centers of enzyme by the highly selective phosphoorganic inhibitor HD-42 showed that the inhibitor-induced activation is due to the appearance of previously masked active sites of the enzyme. Activation of AChE in the homogenates of rat brain medulla oblongata was caused by lysis of the structural elements of nervous tissue which were not destroyed by homogenization. Another mechanism of AChE activation by the detergents (frog brain homogenate) is the destruction of the vesicular membrane structures present in the homogenate with AChE centers located on the inner surface of the vesicles.

Acetylcholinesterase↗
PubMed1967

Metabolism of lysophosphatidyl ethanolamine and lysophosphatidyl choline by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages.

A comparison has been made between the conversion of (32)P-labeled lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl choline (LPC) to their respective acylated and deacylated derivatives by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages. Synthesis of PE by both homogenates and of PC by macrophage homogenates proceeded to about the same extent and is attributed to direct acylation of the lyso compounds. At higher LPC concentrations formation of PC by leukocytes is far greater than by macrophages. The mechanism of this enhanced synthesis of PC, which is brought out by higher substrate concentrations, is believed to be a transfer of the acyl group of one LPC molecule to another. Under optimal conditions macrophage homogenates deacylated LPE to a greater extent than LPC, while the reverse was true for leukocyte homogenates. Albumin inhibited deacylation of LPC and its conversion to PC by leukocytes, perhaps by binding the substrate (2 moles of LPC per mole of albumin). Other effects of albumin-stimulation of deacylation and acylation of LPE by macrophages, inhibition of deacylation and acylation of LPE by leukocytes-remain unexplained.

Albumins↗
PubMed1984

[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase.

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.

Animals↗
PubMed1981

Effect of platelet homogenate on platelet production of bone-marrow-transplanted mice.

Haemopoiesis of mice was reconstituted with 1 X 10(6) bone marrow cells after 800 r total body irradiation. Then their megakaryocytopoiesis was inhibited by platelet homogenates given at different points of time on three subsequent days. The effect was measured by the peripheral platelet counts on the 14th day after transplantation when the platelet count of irradiated and marrow-transplanted CFLP animals was 410 X 10(9)/l, and that of CBA mice was 185 X 10(9)/1. Platelet homogenates prepared from 1-2.5 X 10(8) platelets daily given on days 1, 2 and 3 did not significantly reduce the peripheral platelet count estimated on the 14th day, while homogenates given on days 8, 9 and 10 reduced it markedly. Homogenates prepared from 0.7-1.0 X 10(6) white blood cells did not influence the 14th day platelet count. Thus, platelet homogenates exert their inhibitory effect at a late stage of platelet production, i.e. not by an inhibition of thrombopoietin or a thrombopoietin effect.

Animals↗
PubMed1981

Kinetics and mechanism of deuterium oxide-induced fluorescence enhancement of fluorescyl ligand bound to specific heterogeneous and homogeneous antibodies.

Comparative kinetics studies of ligand dissociation and D2O enhancement were performed with both heterogeneous and homogeneous anti-fluorescyl immunoglobulin G antibodies. Heterogeneous rabbit and homogeneous mouse (monoclonal) antibody preparations were purified by immunoadsorption and found to be pure IgG by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Relatively high affinities of all liganded antibody preparations were determined by dissociation rate studies, demonstrating comparatively long lifetimes for the dissociation of bound fluorescein. In addition, rabbit anti-fluorescyl preparations were found to display marked heterogeneity of off-rates while mouse monoclonal anti-fluorescyl preparations exhibited a single off-rate indicating homogeneity. D2O fluorescence enhancement studies showed that heterogeneous kinetics was observed with both heterogeneous and homogeneous antibody active sites. Temperature studies of ligand D2O enhancement and dissociation rates using homogeneous anti-fluorescyl antibodies revealed similar, yet different activation energies (22.7 +/- 0.8 cal and 20.2 +/- 0.3 cal, respectively) for both phenomena. The studies demonstrated that the anti-fluorescein antibody active site consists of both solvent accessible and relatively inaccessible components, and that the binding of ligand involves both exchangeable hydrogen atoms and other as yet unresolved interactions. The mechanism of D2O fluorescence enhancement is discussed in terms of its complexity involving heterogeneous rate mechanisms.

Animals↗
PubMed1981

The interaction of topically applied toluene and ambient temperature on O2 uptake of tissue homogenates.

These studies examined the role of toluene in temperature-cancer experiments where it was used as a solvent for benzo(a)pyrene (BaP). Toluene (0.08 ml/wk topically) and ambient temperature (cool Ta = 16 degrees C, normal Ta = 23 degrees C and warm Ta = 32 degrees C) interacted to affect O2 uptake of tissue homogenates. After 3.5 weeks of acclimation and 2 weeks of toluene (0.16 ml total), O2 uptake of muscle homogenates was elevated 32% for all Ta's; O2 uptake of skin homogenates was elevated 139% for all Ta's; however, O2 uptake of liver homogenates was elevated 665% in cool Ta, elevated 36% in normal Ta but depressed 50% in warm Ta. The pattern of liver homogenate O2 uptake of C greater than N greater than W is similar to that for tumorigenesis when BaP is dissolved in the toluene, although toluene alone is nontumorigenic. Common cellular mechanisms may be involved in the metabolism of toluene, the activation of BaP and in thermogenic acclimation.

Administration, Topical↗
PubMed1980

Sequential deiodination of thyroxine to 3,3'-diiodothyronine via 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine in rat liver homogenate. The effects of fasting versus glucose feeding.

The characteristics of thyroxine (T4) deiodination to 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) and of each of the latter to 3,3'-diiodothyronine (3,3'T2) were examined in rat liver homogenate. Each of the four reactions was enzymatic in nature, demonstrating pH and temperature optima, and tissue and time dependence. All reactions were considerably augmented (greater than 10-fold) by the presence of a thiol agent. At pH 7.2 with 2 muM T4 as substrate, rT3 generation was 3.3 +/- 0.44 (S.E.) and T3 formation was 4.8 +/- 0.57 pmol/min/100 mg of homogenate protein. Fasting for 72 h resulted in a significant inhibition of T4 deiodination, compared to that in the glucose-fed animals, in a 2% homogenate preparation. Enzyme activity for T4 to T3 was reduced by 54% (p less than 0.05) in the homogenate from the fasted rats. Fasting lowered the enzyme activity of T4 to rT3 by 56% (p less than 0.05). Although the monodeiodination of T3 to 3,3'-T2 was also significantly depressed (p less than 0.01) by fasting, rT3 deiodination to 3,3'-T2 was not. The in vitro additon of 5 mM dithioerythritol did not reverse the effect of fasting on any reaction. These results demonstrate that a 72-h fast significantly impairs the sequential deiodination of T4 in liver homogenate. The effect of fasting appears to be mediated mainly through a reduction in enzyme concentration rather than co-factor availability.

Animals↗
PubMed1996

Efficacy of Bio-Catalyzer alpha.rho no.11 (Bio-Normalizer) supplementation against peroxyl radical-induced oxidative damage in rat organ homogenates.

To better delineate the antioxidant potential of Bio-Catalyzer alpha.rho No.11 (Bio-Normalizer), a natural food supplement recently proposed as an antioxidant agent, we investigated the efficacy of Bio-Normalizer supplementation to protect rat organ homogenates against oxidative damage induced in vitro by peroxyl radicals generated in the hydrophobic or in the hydrophilic phase. Bio-Normalizer supplementation efficiently protected rat kidney homogenates against the accumulation of thiobarbituric reactive substances (TBARS), the formation of protein carbonyl derivatives and the depletion of alpha-tocopherol induced by peroxyl radicals generated from the hydrophobic azo-initiator 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN). It also protected the heart but not the liver or the brain homogenates. Bio-Normalizer supplementation did not have effect in any organ homogenates when peroxyl radicals were generated from the hydrophilic azo-initiator 2,2'-azobis (2-amidinopropane) dihydrochloride) (AAPH). In vitro direct addition of aqueous solutions of Bio-Normalizer to the organ homogenates was ineffective against AMVN or AAPH-induced oxidative damage. Our findings expand previous reports on the antioxidant activity of Bio-Normalizer. They confirm that supplemented Bio-Normalizer protects against peroxyl radical-induced oxidative damage and suggest that its antioxidant action depends on in vivo bioactivation, it is organ specific and it is limited to damage induced by peroxyl radicals generated in the hydrophobic phase.

Animals↗
PubMedDate not supplied

[Splitting of fructose diphosphate by muscle and tumor homogenates].

Effect of MgCl2 and ATP on the elevation in lactate, pyruvate and methylglyoxale content was studied on incubation of fructosediphosphate with muscle and tumor homogenates. Muscle homogenate accumulated lactate, pyruvate and methyglyoxale more intensively as compared with tumor homogenate. Pyruvate was formed only in presence of MgCl2 and ATP, although it did not practically affect the elevation of lactate in muscle homogenate. The data obtained suggest that tumor homogenate does not contain the enzymatic system involved in methylglyoxale formation.

Adenosine Triphosphate↗
PubMed1996

On the mechanical characterization of compact bone structure using the homogenization theory.

In a previous paper (Crolet et al., 1993, J. Biomechanics 26, 677-687), a modelling of the mechanical behavior of compact bone was presented, in which the homogenization theory was the basic tool of computation. In this simulation, approximations were used for the modelling of the lamellae and the osteons: the lamella and the osteon were divided into cylindrical sectors, each sector being approximated as a parallelepiped having a periodic structure (fibrous composite for the lamella, superimposition of plates for the osteon). The present study deals with a new model without these approximations. First, it can be proved that the homogenized elasticity tensor for a lamella, which has non-periodic structure, is obtained at each geometrical point as a homogenized tensor of a periodic problem. Similarly, for the osteonal structure, the components of the homogenized tensor are determined at each point as the result of a periodic homogenization. The software OSTEON, which is the computational method associated with this model, allows one to obtain a better understanding of the effects of many bony parameters. The obtained results are in accordance with experimental data.

Algorithms↗
PubMed1999

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.

Coumaric Acids↗
PubMed1999

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice.

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.

Allyl Compounds↗
PubMed1999

Longitudinal relaxation times of 129Xe in rat tissue homogenates at 9.4 T.

Longitudinal relaxation times of 129Xe were measured in homogenates of rat brain, kidney, liver, and lung at varying oxygenation levels as a means to assess the feasibility of magnetic resonance (MR) imaging of tissue using laser-polarized (LP) 129Xe as the signal source. The measured relaxation times ranged from 4.4 +/- 0.4 sec in deoxygenated lung homogenate to 22 +/- 2 sec in deoxygenated brain homogenate. When the LP gas is introduced to the subject via inhalation, these relaxation times are long enough to allow accumulation and subsequent MR imaging of LP 129Xe in tissues. Imaging of dissolved LP 129Xe will yield an intrinsic signal-to-noise ratio (SNR) that is approximately 3% of the proton intrinsic SNR. This relatively low intrinsic SNR is expected to be adequate for some tracer applications. T1 of 129Xe was found to depend on the oxygenation level of the tissue, and the effect of oxygenation is likely dependent on the amount of hemoglobin in the tissue homogenate.

Animals↗
PubMed2003

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system.

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.

Acetonitriles↗