Polymer scientists work to beef up biosensors.
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Antibodies↗
Biomedical subjects
G Graff
Publications and source records attributed to G Graff.
Preparation and purification of soybean lipoxygenase-derived unsaturated hydroperoxy and hydroxy fatty acids and determination of molar absorptivities of hydroxy fatty acids.
A method was developed for the preparation and purification of polyunsaturated hydroperoxy and hydroxy fatty acids from 18:2n-6, 18:3n-6, 18:3n-3, 20:3n-6, 20:3n-3, and 20:4n-6 with soybean lipoxygenase. This method involved incubation of unsaturated fatty acids in Tris-HCl buffer with soybean lipoxygenase and extraction of reaction products on C18 solid-phase columns. The yields of conversion of fatty acid substrate to oxygenated products were in all cases greater than or equal to 95%. C18 solid-phase extracted reaction products were purified by C18 HPLC, yielding 3-4 mg of products with purities of greater than or equal to 98%. Purified polyunsaturated hydroxy fatty acids were characterized by HPLC and mass spectral analysis and showed absorption spectra with maximum absorption occurring between 234.5 and 237.5 nm due to the presence of a conjugated diene function. Polyunsaturated hydroxy fatty acid potassium salts were converted to (4-bromobenzoyl)methyl esters by reaction with 2,4'-dibromoacetophenone in the presence of 18-crown-6. Molar absorptivities for the conjugated diene functions were determined by relating their absorbance to the absorbance contributed by the 4-bromobenzoyl chromophore and its molar absorption intensity of 17.6 X 10(3) (M.cm)-1. The molar absorptivities determined for 13-OH-9,11-18:2, 13-OH-6,9,11-18:3, 13-OH-9,11,15-18:3, 15-OH-11,13-20:2, 15-OH-8,11,13-20:3, 15-OH-11,13,17-20:3, and 15-OH-5,8,11,13-20:4 ranged from 23.2 x 10(3) to 24.6 x 10(3) (M.cm)-1. The molar absorbtivity values of 18.8 x 10(3) and 20.3 x 10(3) (M.cm)-1 determined for commercial, chemically synthesized 12-OH-5,8,10,14-20:4 [12-(S)-hydroxyeicosa-5,8,10,14-tetraenoic acid (12(S)-HETE)] and 5-OH-6,8,11,14-20:4 ((+/-)-5-HETE) were lower than that of soybean lipoxygenase-derived 15-OH-5,8,11,13-20:4 (15-HETE), which exhibited a molar absorptivity of 23.3 x 10(3) (M.cm)-1. The molar absorptivity values determined for 5-, 12-, and 15-HETE are considerably lower than 30.5 x 10(3) (M.cm)-1, which was reported previously.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Methylsulfamic acid esters. A new chemical class of oral antiarthritic agents.
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Animals↗
Antiinflammatory agents. 4. Syntheses and biological evaluation of potential prodrugs of 2-amino-3-benzoylbenzeneacetic acid and 2-amino-3-(4-chlorobenzoyl)benzeneacetic acid.
A series of potential prodrugs of 2-amino-3-benzoylbenzeneacetic acid (amfenac) and 2-amino-3-(4-chlorobenzoyl)benzeneacetic acid were synthesized and evaluated for their cyclooxygenase inhibiting properties, antiinflammatory potency, and gastrointestinal irritation liability. One compound, 2-amino-3-(4-chlorobenzoyl)benzeneacetamide, possessed a therapeutic index 1 order of magnitude greater than that of indomethacin.
Acetamides↗
AHR-15010--a novel anti-arthritic agent.
AHR-15010 (3-(2-methoxyphenoxy)-1,2-propanediol bissulphamate ester) is a compound of novel structure that displays anti-arthritic activity in adjuvant arthritis in rats. When given orally from days 18 through day 50, (excluding weekends) after adjuvant injection, AHR-15010, at doses of 3.16 to 100 mg kg-1, produced significant anti-inflammatory activity and reduced the severity of the hind paw joint lesions as monitored by X-ray analysis. AHR-15010, however, has no acute anti-inflammatory activity in the Evans Blue-carrageenan pleural effusion assay in rats, has no analgesic activity in mice, and has no activity in a classic, delayed-type, hypersensitivity assay in mice or in a cotton pellet granuloma test in rats. These data, in conjunction with biochemical data showing that AHR-15010 has no prostaglandin synthetase inhibiting activity suggest that AHR-15010 is an anti-arthritic with a unique mechanism of action. AHR-15010 is a carbonic anhydrase inhibitor. Data are presented that suggest that AHR-15010 and acetazolamide, a prototype carbonic anhydrase inhibitor, may present novel approaches to the treatment of arthritis.
Animals↗
Optical spectra and kinetics of reactions of prostaglandin H synthase: effects of the substrates 13-hydroperoxyoctadeca-9,11-dienoic acid, arachidonic acid, N,N,N',N'-tetramethyl-p-phenylenediamine, and phenol and of the nonsteroidal anti-inflammatory drugs aspirin, indomethacin, phenylbutazone, and bromfenac.
A combination of cyclooxygenase activity assays, rapid spectrophotometry and pre-steady-state, steady-state, and transient-state kinetics is used to characterize further the properties of prostaglandin H synthase. 13-Hydroperoxyoctadeca-9-11-dienoic acid is used as oxidizing substrate and the effects of the following compounds are examined: arachidonic acid, N,N,N',N'-tetramethyl-p-phenylenediamine, phenol, diethyldithiocarbamate, and the nonsteroidal anti-inflammatory drugs aspirin, indomethacin, phenylbutazone, and Bromfenac. The order of reactivity of four of these substrates, predominantly with compound II of prostaglandin H synthase, is N,N,N',N'-tetramethyl-p-phenylenediamine greater than phenol greater than indomethacin approximately phenylbutazone. Aspirin exhibits no effect. Arachidonic acid causes inactivation. Diethyldithiocarbamate acts as a reducing substrate for the oxidized forms of prostaglandin H synthase. Bromfenac appears to act both as a protective agent and inhibitor.
Animals↗
1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor.
In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.
Animals↗
[Peripheral nerve repair: value of biological glues and epiperineural suture in late interventions. Experimental study in rats].
In order to approximate as close as possible genuine clinical conditions, the sciatic nerve of the rat was divided and then repaired after a delay of one or seven days either by application of a biological glue or by an epiperineural suture technique. The metabolic activity of the sciatic nerve Schwann cells - whether located in the distal or the proximal ends - and that of the (fast acting) white gastrocnemius and (slow acting) red soleus muscle were assessed using 32P-radiolabeled acid-soluble phosphates. Delayed repair, as judged by our biochemical criteria, was equivalent whatever method, biological glue or suture, was used. The frozen and lyophilized forms of the biological glue provided similar results.
Animals↗
[Prophylactic mezlocillin therapy in cesarean section].
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Bacterial Infections↗
Inhibition of blood platelet aggregation by dioxo-ene compounds.
Compounds with a conjugated oxo-ene-oxo system were tested for inhibition of blood platelet aggregation. All compounds with this structure in trans configuration were effective inhibitors of aggregation induced by thrombin and by arachidonic acid. While the oxo-trans-ene-oxo system is prerequisite for such activity, other structural features of the compounds may be varied without loss of activity. Inhibition is exemplified by 9,12-dioxo-trans-10- and 10,13-dioxo-trans-11-octadecenoic acids and their methyl esters, by 11,14-dioxo-trans-12- eicosenoic acid, by 4,7-dioxo-trans-5- decene and by trans- dibenzoylethylene . The half-inhibition concentrations are in the order of 2-6 microM, with complete inhibition at 8-20 microM. According to experiments with the inhibiting 9,12-dioxo-trans-10-octadecenoic acid, the normal oxygenation of exogenous arachidonic acid by platelets is not affected but the thrombin-induced internal release of this acid seems to be abolished by the inhibitor. The inhibition of aggregation in the presence of exogenous arachidonic acid and its products suggests that the inhibitor also interferes with other events leading to aggregation. By implication from other properties of the oxo-trans-ene-oxo system, reaction with SH groups may be a mechanism for inhibition.
Arachidonic Acid↗
Natural abundance 13C NMR spectra of human muscle, normal and diseased.
13C NMR spectra of human surgical muscle samples have been obtained at 50 and 118 MHz. Numerous sharp peaks in the 13C spectrum have been assigned to carbon atoms of soluble metabolites and fatty acyl chains of neutral fats and membrane-bound phospholipids. Comparisons have been made of 13C NMR spectra of normal and diseased muscles after removal of neutral fat by extraction with isopentane. Creatine, lactic acid, and phospholipids are not removed from muscles by isopentane. The most striking difference between 13C NMR spectra of isopentane-extracted normal and diseased muscle samples was the size of the residual 30.5 ppm methylene carbon resonance, that is, small in normal muscles and in nonspecific muscle diseases, but large in myogenic and neurogenic muscle diseases. In addition, differences were also found between normal and diseased muscles in their creatine content and their ability to produce lactic acid. By deoxycholate treatment of isopentane-extracted diseased muscle it is estimated that about one-fifth of the total phospholipids are highly mobile. T1 and NOE measurements indicated that the differences in peak height for the 30.5 ppm resonance between normal and diseased muscle are due only to difference in the amount of highly mobile fatty acyl chains in the muscle and not due to differences in relaxation parameters. 13C NMR of isopentane-extracted muscle appears to permit differentiation of normal from diseased muscle and, within diseased muscle, grading of the severity of the disease.
Biopsy↗
Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate.
Phospholipase C from human platelets was found to catalyze the Ca2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4'-phosphate (DPI), and phosphatidylinositol 4',5'-bisphosphate (TPI) at Ca2+ concentrations from 150 microM to 5 mM. Both DPI and TPI inhibited the hydrolysis of [2-3H]inositol-labeled PI (250 microM) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI/PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1,2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI/PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (greater than or equal to 95%) and overall formation of 1,2-diacylglycerol (greater than or equal to 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a Ki of about 10 microM. The Ka for Ca2+ in PI hydrolysis was profoundly increased from 5 to 180 microM when TPI (36 microM) was included with PI (250 microM). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 mM. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI/PI and TPI/PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI/PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by greater than or equal to 75%. Changes in 33P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33P label of both TPI and DPI (15 s) followed by an increase in [33P]phosphatidic acid but no change in [33P]PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI/PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1,2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1,2-diacylglycerol from inositol phospholipids.
Blood Platelets↗
Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids.
Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.
Fatty Acids, Unsaturated↗
The alkylacyl and alkenylacyl glycerophospholipids of human platelets.
The phospholipids of human platelets were isolated and analysed for their constituent 1-0-alkyl- and 1-0-(1'-alkenyl)glycerol species. The serine and inositol phospholipids contained only small amounts (0.5-1.6%) of both alkylacyl and alkenylacyl species; the ethanolamine phospholipids contained little alkylacyl (1.7%) but much more alkenylacyl (45.3%) species, while the choline phospholipids contained 4.5% and 1.4% of these compounds, respectively. Because choline phospholipids amount to 41% of the total phospholipids of human platelets, it appears that they could provide a sufficient supply of alkylacyl glycerophosphocholine for the generation of the alkylacetyl analog (platelet activating factor) by a deacylation-acetylation pathway.
Blood Platelets↗
Inhibitory activity of polyamines on phospholipase C from human platelets.
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Blood Platelets↗
Changes in the natural abundance 13C NMR spectra of intact frog muscle upon storage and caffeine contracture.
The carbons of phospholipids have limited mobility in fresh, resting, gastrocnemius frog muscle, but a population of phospholipids gains considerable mobility upon storage in the muscle or in contracture induced by caffeine. In parallel with the appearance of sharp phospholipid resonances, lactic acid also appears in the 13C NMR spectra. There is a correlation between the mobility of phospholipids and the depletion of phosphocreatine and ATP in muscle.
Animals↗
Preparation of phosphatidyl[2-3H]inositol from yeast grown in medium containing myo[2-3H]inositol.
Phosphatidyl[2-3H]inositol was prepared from Saccharomyces cerevisiae (YSC-2), grown in synthetic medium containing myo[2-3H]inositol. Over 44 microCi (or 81%) of the radiolabeled inositol was taken up by the organism, with 34 microCi incorporated into phosphatidylinositol. Upon purification by silicic acid pressure liquid chromatography (MPLC), a final yield of 24 to 26 microCi of phosphatidyl[2-3H]inositol with a specific radioactivity of 40 X 10(3) dpm/nmole was obtained. The purified phosphatidyl[2-3H]inositol was found to be a suitable for phospholipase C from human platelets.
Inositol↗