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PubMed2006

Diagnostic significance of urinary thiodiglycolic acid as a possible tool for studying the role of vitamins B12 and folates in the metabolism of thiolic substances.

We have found that the determination of thiodiglycolic acid (TDGA) in urine may help to characterize metabolic imbalance of substances participating in methionine synthesis, which leads to hyperhomocystinuria. From the metabolic scheme, based on a proper combination of known facts, we attempted to theoretically explain and to demonstrate the possibilities of TDGA formation via different ways of homocysteine transformation. This scheme was used in evaluating the results obtained by testing urine of a woman suffering from impaired function of methionine synthase reductase (CblE type of homocystinuria). The amount of TDGA excreted in her morning urine was very sensitive to the changes in her treatment based upon a combination of N5-formyl tetrahydrofolate, betaine and vitamin B12. Vitamin B12 given in the evening either alone or together with betaine increased the TDGA excretion in the morning urine up to ten times. On the other hand, in the absence of vitamin B12, betaine in combination with N5-formyl tetrahydrofolate hindered the appearance of TDGA in the morning urine. Generally, the determination of TDGA in urine of an appropriately pretreated patient may indicate the degree of success of the treatment.

Adult↗
PubMedDate not supplied

[The antioxidant activity of S-(4-quinazolyl)mercaptoacetic acid benzylidene hydrazides in vitro in the "nitrosation stress" model].

The "nitrozation stress" causes modification of the antioxidant enzymes leading to a decrease in their activity. A decrease in this activity and the resulting increase in the production of OH* and NO2* radicals via ONOO* decomposition leads to a significant increase in the extent of free-radical peroxidation during the "nitrozation stress". These factors are unfavorable from the standpoint of oxidative phosphorylation, which is manifested by inhibited activity of the oxidative enzymes. S-(4-Quinazolyl)mercaptoacetic acid benzylidene hydrazides exhibit a high antiradical activity related to the presence of a mercapto group in the quinazoline fragment. The activity depends on the position of this group on and the number of substituents in the benzylidene fragment. The compounds studied reduce the extent of damage in the brain homogenates of rats under conditions of nitrozation stress. This is manifested by inhibition of the free-radical peroxidation and an increase in the activity of antioxidant and prooxidant enzymes.

Animals↗
PubMed1984

The role of immunomodulators in the production of lipid mediators by macrophages (M phi).

There is evidence indicating that bioreactive lipid mediators, PAF-acether (platelet-activating factor: 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and arachidonic acid (AA) metabolites, are formed upon deacylation of ether lipids. M phi obtained from mice treated with the sterile irritant thioglycolate exhibited an impaired formation of PAF-acether, leucotrienes (LT) C4 and prostaglandins (PG). In order to assess whether the impaired formation in lipid mediators is a general feature of M phi found at inflammatory sites, we have compared the capacity of resident (R-M phi) and immunostimulant-activated M phi to release, upon a zymosan challenge, PAF-acether and cyclooxygenase and lipoxygenase products. Activated M phi was obtained from mice injected intraperitoneally with non-viable C74 streptococci (St-M phi), bacilli Calmette-Guerin (BCG-M phi) or trehalose dimycolate (TDM-M phi), a defined immunostimulant isolated from Mycobacterium tuberculosis. All populations were capable of releasing PAF-acether. However, the amount of cell-associated PAF-acether was reduced by 75-90% in activated M phi populations as compared to R-M phi. Although the acetyltransferase level was comparable, acetyl-CoA supplementation restored the formation of PAF-acether by activated M phi to control (R-M phi) level. The amount of 14C-AA metabolites released by St-M phi was much lower compared with TDM-M phi or R-M phi, as was the amount of LTC4 detected as SRS contractile activity after HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Purification of murine macrophage cytotoxin (MCT).

Macrophage cytotoxin (MCT) can be induced from peritoneal exudate macrophage monolayers (PEMM) obtained from thioglycollate-treated mice, by exposure of PEMM to lipopolysaccharide (LPS) or to double-stranded polyinosinic: poly-cytidylic acid (poly-l:poly-C). MCT is highly labile even upon storage at 4 degrees C, and is irreversibly denatured by isolectricfocusing, polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS), or by exposure to ethylene glycol. alpha-MCT [150,000 daltons (d)] has been highly purified (2,000- to 5,000-fold) from serum-free, PEMM supernatants by a scheme of concentration, molecular sieving on Ultrogel AcA 44, negative hydrophobic affinity chromatography on benzyl-agarose, and ion exchange chromatography on aminoethyl-agarose. The scheme results in high yield of MCT, in part because of the rapidity with which the labile toxin is manipulated due to the tandemization of the chromatographic steps.

Animals↗
PubMed2006

VEGF gene expression is regulated post-transcriptionally in macrophages.

The macrophage is critical to the innate immune response and contributes to human diseases, including inflammatory arthritis and plaque formation in atherosclerosis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that is produced by macrophages. To study the regulation of VEGF production in macrophages we show that stimulation of monocyte-macrophage-like RAW-264.7 cells by lipopolysaccharide (LPS) increases expression of VEGF mRNA and protein. Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation. To study post-transcriptional regulation of the VEGF gene the 3'-untranslated region (3' UTR) was introduced into the 3' UTR of the luciferase gene in a reporter construct. In both RAW-264.7 cells and thioglycollate-elicited macrophages, the 3' UTR sequence dramatically reduces reporter expression. Treatment with activators of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3' UTR reporters. Finally, mapping studies of the 3' UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW-264.7 cells, but does not affect reporter activation with LPS. Together these results demonstrate that a post-transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells.

Alternative Splicing↗
PubMed1983

Differences in cytotoxic effects of activated murine peritoneal macrophages and J774 monocytic cells on metastatic variants of B16 melanoma.

The cytotoxic effects of activated peritoneal macrophages and the J774 reticulum cell sarcoma cell line on B16 melanoma cells of differing metastatic potential were investigated in vitro. The melanoma target cells were sublines of low (B16-F1) or high (B16-F10) lung colonization potential as well as a subline of high (B16-B14b) brain colonization ability. Thioglycolate-elicited peritoneal macrophages from syngeneic C57BL/6 mice and J774 cells were activated in vitro with polyinosinic-polycytidylic acid (poly I:C) and used as effector cells. Macrophage-mediated cytolysis was determined by means of 24- to 72-hour radioactivity release assays with [125I]5-iodo-2'-deoxyuridine-labeled melanoma cells; the results indicated that the more metastatic sublines B16-F10 and B16-B14b were less susceptible to cytolysis by activated macrophages and J774 cells than was the poorly metastatic B16-F1 subline. The poly I:C-activated effector cells also released soluble cytotoxin(s), which resulted in melanoma cell lysis and growth inhibition. Cytotoxin-mediated melanoma cell cytolysis was determined by counting the number of viable mitomycin-treated target cells remaining after a 40-hour incubation period in the absence or presence of various concentrations of cell-released factors, and cytotoxin-mediated cytostasis was performed with the use of the same procedures without mitomycin pretreatment of targets. The factors released from both activated macrophages and J774 cells were more effective against the poorly metastatic B16-F1 cells than against the highly metastatic B16-F10 or B16-F14b cells. In addition, the activity of the factors from both activated effector cells was inhibited by fetal bovine serum. The J774 cells and the activated peritoneal macrophages demonstrated similar activities against B16 melanoma variants, indicating that the J774 cell line may be suitable as a model for the study of macrophage cytotoxicity. The results suggested that the potential of the highly metastatic melanoma cells to implant, survive, and grow at secondary sites may be due, in part, to their ability to circumvent host antitumor mechanisms.

Animals↗
PubMed1999

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.

Amino Acid Sequence↗
PubMed2002

Optimizing sporulation of Clostridium perfringens.

Many sporulation media have been developed for Clostridium perfringens, but none stimulates sporulation for all strains. The aim of our experiments was to develop a sporulation method using Duncan and Strong (DS) medium, which supports sporulation of a wide variety of strains. Different inoculation levels were tested, and the effects of sporulation-promoting substances and acid shock were evaluated. Furthermore, DS medium was compared with other sporulation media. Highest spore numbers in DS medium were obtained with a 10% 24-h fluid thioglycollate broth inoculum (5.0 x 10(5)/ml). Addition of theophylline and replacement of starch by raffinose increased spore yields for some strains, but most strains were not affected (average increases in log N/ml of 0.2 and 0.3, respectively). One strain was enhanced by the addition of bile, but other strains were strongly inhibited (average decrease in log N/ml of 2.5); agar did not influence sporulation. Neither short-time acid exposure nor addition of culture supernatant fluids of well-sporulating strains resulted in higher spore numbers in DS medium. None of the tested methods enhanced sporulation in general; only strain-dependent effects were obtained. Peptone bile theophylline medium was the most promising sporulation medium tested; peptone bile theophylline starch medium yielded highest spore numbers (2.5 x 10(5)/ml), but some strains failed to sporulate. In conclusion, adding theophylline to DS medium may optimize sporulation of C. perfringens, but peptone bile theophylline medium with or without starch is most suitable.

Clostridium perfringens↗
PubMed2000

Effects of metabolic inhibitors on ingestive behavior and physiology in preweanling rat pups.

We have previously demonstrated that administration of 2-Mercaptoacetate (MA) stimulates independent intake after 1 h in 12 and 15-day-old rat pups, but not younger pups. MA also produces decreases in beta-HBA levels, consistent with the development of a role of altered fatty acid oxidation in modulating independent ingestion in rat pups by 12 days of age. The present experiments extended investigations of the role of changes in energy utilization in young rats by investigating the duration of the effects of altered fatty acid oxidation and the effects of combined blockade of fatty acid oxidation and glucose utilization. Pups were tested at 9, 12 or 15 days of age 3 or 6 h following administration of a dose of 0, 11.4, 22.8, 45.6 or 91.2 mg/kg MA. In pups aged 12 and 15 days, moderate doses of MA stimulated intake 3 h, but not 6 h, following administration. Administration of the highest dose of MA produced significant decreases in beta-HBA levels in pups at all ages when tested after 3 h, but not after 6 h. In the second set of experiments, behavioral and physiological responses to administration of MA (0, 11.4 or 22.8 mg/kg) combined with 2-Deoxyglucose (2-DG: 0, 100 or 200 mg/kg) were investigated in pups aged 6, 9, 12 or 15 days of age. The results demonstrated that while administration of 2-DG produced physiological responses, intake was not stimulated at any age by 2-DG alone or in combination with MA. In fact, in 12 and 15-day-old pups, administration of 2-DG blocked the stimulatory effects of administration of MA. Therefore, while altered utilization of glucose does not appear to be an effective stimulus for increased independent ingestion in pups at this age, altered fatty acid oxidation may be an early metabolic modulator of intake.

Age Factors↗
PubMed1975

4,4'-Dinitrodiphenyldisulfides of different charge type as probes for the electrostatic environment of sulfhydryl groups.

5,5'-Dithiobis-(2-nitro-N-trimethylbenzyl ammonium iodide) (I) and 5,5'-dithiobis-(2-nitro-N-2'-hydroxyethyl benzamide) (II) were synthesized as positively charged and neutral analogs of Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) (III). Their reaction rates with a variety of thiols with different charge showed that sulfhydryl groups with no charge react about 25 times more rapidly with I than with III. A positive charge removed three single bonds from the sulfhydryl group decreases this ratio to about 3.5 to 1 while a negative charge within three single bonds increases it to 120 to 1. The reactivity of II was much higher than that of III but smaller than that of I. Comparison of the rates at 15 and 25 degrees C gave activation enthalpies (10.6-17.4 kcal/mol) that did not depend in a clear-cut way on the charge of the reacting species. Measurements at different salt concentrations showed an enhancement of the differences between I, II, and III at low ionic strength and a leveling effect of added salt.

Binding Sites↗
PubMed1983

Reactive oxygen production, arachidonate metabolism and cyclic AMP in macrophages.

Chemiluminescence was used as an indicator of the production of reactive oxygen species by thioglycollate-elicited rat peritoneal macrophages stimulated by A23187. This action of the ionophore was inhibited by bromophenacyl bromide and nordihydroguaiaretic acid, inhibitors of the phospholipase A2 and lipoxygenase enzymes, respectively. The cyclo-oxygenase inhibitors, indomethacin and aspirin, did not diminish the light output. Preincubation of the cells with the 8-bromo- or dibutyryl analogues of cyclic AMP or with the cyclic AMP-phosphodiesterase inhibitors theophylline and RO-20-1724, or with PGE2, inhibited the A23187-evoked chemiluminescence. The results suggest that he lipoxygenase pathway of arachidonic acid metabolism may make a significant contribution to reactive oxygen production. This process may be modulated, and its duration limited, by cyclic AMP-mediated actions of prostaglandins, which are products of the cyclo-oxygenation of arachidonate.

8-Bromo Cyclic Adenosine Monophosphate↗