Presence Of Low Concentrations Research Articles

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.

Direct activation by okadaic acid of the contractile elements in the smooth muscle of guinea-pig taenia coli.

Okadaic acid isolated from black sponge (Halichondria okadai), at the concentration of 10 mumol/l, caused contraction in saponin-treated skinned smooth muscle of guinea-pig taenia coli in the absence of Ca2+. In the presence of low concentration (0.3 mumol/l) of Ca2+, okadaic acid induced a greater contraction than in the absence of Ca2+. Okadaic acid potentiated the contractions induced by Ca2+ and pCa2+-tension curve was shifted to the left as well as upward by 1 mumol/l okadaic acid. Native actomyosin preparation (myosin B) containing calmodulinmyosin light chain kinase system and phosphatase was obtained from taenia coli. Okadaic acid (10 mumol/l) increased the actomyosin Mg2+-ATPase activity in the presence or absence of Ca2+. Okadaic acid (1-100 mumol/l) had no effect on calmodulin activity as monitored by Ca2+-calmodulin activated cyclic nucleotide phosphodiesterase activity and the (Ca2+ + Mg2+)-ATPase activity or erythrocyte membranes. These results suggest that okadaic acid directly activates contractile elements of smooth muscle.

Combined effects of interferon and mutagens on survival of mouse L929 cells

We studied the effect of mouse interferon (IFN) alpha/beta on the sensitivity of L929 cells to cytotoxic activity of ultraviolet (UV) irradiation or ethyl methanesulfonate (EMS) administration. Exposure of L929 cells to IFN for 48 h before mutagen treatment markedly enhanced their sensitivity to UV, but only slightly to EMS. Incubation of L929 cells after UV-irradiation for 24 h in the presence of low concentrations of IFN (15 units/ml) increased the cellular recovery, while with graded concentrations of IFN the protective activity was inhibited (60 units/ml) and after 250 units/ml the ability of IFN treated cells to recover was found to decrease. Incubation of cells after EMS treatment for 24 h with low (15 units/ml) or moderate (60 units/ml) IFN concentrations markedly reduced the cellular recovery. It has been shown that the extent and the direction of IFN activity on cellular sensitivity to mutagens was influenced by the type of mutagen, IFN-concentration and length of its action on the cell and upon application of IFN before or after mutagen treatment.

Ascorbic acid transport in mammalian kidney.

Ascorbic acid is known to circulate free in the plasma of several species and is therefore filtered in the kidney; reabsorption subsequently takes place and prevents urinary loss. However, no specific mechanism of renal ascorbic acid transport has previously been presented. In the present study, rat and guinea pig kidney were incubated as slices or as isolated tubules in vitro in the presence of low concentrations of [14C]ascorbic acid. The kidneys of both species handle ascorbic acid similarly. Ascorbic acid accumulates in the renal tissue to a concentration three to four times that present in the bathing media. Recently absorbed ascorbic acid diffuses freely from the kidney and is predominantly nonmetabolized during absorption. Uptake is reduced following replacement of bathing solution sodium by lithium or cesium, or when incubation is performed in the presence of metabolic inhibitors or at low temperatures. The results indicate that ascorbic acid is reabsorbed in the kidney by a sodium-dependent active transport mechanism that operates by concentrating ascorbic acid in the cellular fluid. Renal slices and tubules both appear to transport ascorbic acid and galactose across the brush-border membrane; this indicates that the tubular lumens in these preparations are not collapsed or sealed off.

Substrate-ligand interactions with acetylcholinesterase and energetics of binding

1. 1. Ester hydrolysis by acetylcholinesterase (from electric eel, Electrophorus electricus) increased in the presence of low concentrations ( ca 10 −7 M) of edrophonium, propidium, d-tubocurarine, gallamine, decamethonium or bis- N-methylacridinium, and decreased at higher concentrations. 2. 2. The overall sensitivity of the substrates to inhibition by the ligands was acetylcholine (Ach) > phenylacetate (PA) > indophenylacetate (IPA). 3. 3. Complete (saturable) inhibition was unattainable with edrophonium or gallamine for IPA and, to a lesser degree, with propidium for PA. 4. 4. Reaction heats between the enzyme and the ligands (edrophonium, propidium and decamethonium) measured directly in a microcalorimeter revealed binding sites that are different from each other in their interaction energetics.

Permanganate peroxidation of cyclohexene. III. Hydroxide ion and salt effect studies

Good yields of cis-1,2-cyclohexanediol were formed by the reaction of cyclohexene and aqueous potassium permanganate under turbulent stirring conditions only in the presence of low concentrations of sodium hydroxide. Larger amounts had no further benefit or were deleterious. The lack of sensitivity to low concentrations of hydroxide ion indicated that its benefit is to inhibit acid promoted oxidations. There was no indication of its participation in the mechanism of glycol formation. The addition of the neutral salts, sodium sulfate or sodium nitrate, caused a decrease in glycol yields presumably due to a positive salt effect on a secondary negative ion – negative ion reaction, which formed other oxidation products. Stoichiometric amounts of hydroxide and potassium ions were not isolated as reaction products due to interaction with the hydrated manganese dioxide in a 1:2 ratio. The mechanisms in acidic and basic solutions appear to be quite different. A mechanism for basic solutions is suggested.

INFLUENCE OF THE ACETATE MEDIUM ON THE EXTRACTIONOF Co(II) BYDI-2ETHYLHEXYL PHOSPHORIC ACID

The distribution of Co(II) between aqueous sodium nitrate medium or acetate-buffered medium and kerosene solutions of di-2-ethylhexyl phosphoric acid has been studied. Under these conditions it has been possible to observe that the ions existing in the aqueous medium play a significant role in the extraction of metal. Thus, the main species extracted from nitrate medium is Co(DEHP)2(HDEHP)2, whereas in the presence of low concentrations of acetate a dimerized species (CoAc)2 (DEHP) 2 (HDEHP)4 is extracted, but (Co2Ac3DEHP) (HDEHP)3 at high acetate concentrations.

Differential effect of anionic and cationic drugs on the synaptosome-associated acetylcholinesterase activity of dog brain.

The evoked effects of the negatively charged drugs phenobarbital and barbituric acid, the positively charged imipramine, perphenazine and trifluoperazine, and the neutral primidone, on the synaptosome-associated acetylcholinesterase activity were studied. A marked increase in the enzyme activity was exhibited in the presence of low concentrations (up to 3 mM) of phenobarbital, barbituric acid and primidone. Higher concentrations (up to 10 mM), however, led to a progressive inhibition of the enzyme activity. However, the activity of the enzyme was not affected by imipramine, but it was decreased by perphenazine and trifluoperazine. Arrhenius plots of acetylcholinesterase activity exhibited a break point at 23.4 degrees C for the untreated (control) synaptosomes, which was shifted to around 16 degrees C in the synaptosomes treated with the charged drugs. The allosteric inhibition by F- of acetylcholinesterase was studied in control synaptosomes and in those treated with the charged drugs. Changes in the Hill coefficients in combination with changes in Arrhenius activation energy produced by the charged drugs would be expected if it is assumed that charged drugs 'fluidize' the synaptosomal plasma membranes.

Oxidative metabolism associated with phagocytosis in Acanthamoeba castellanii.

When amebae were incubated with latex beads, cyanide-insensitive oxygen consumption increased nearly two-fold. This cyanide-insensitive respiration was inhibited by salicylhydroxamate. Furthermore, cell fractionation studies revealed a localization for a portion of the NAD(P)H oxidase activity in phagolysosomes. The presence of low concentrations of divalent metal during fractionation resulted in an increased yield of oxidative activity in the phagolysosome fraction. In addition, the phagolysosome membrane was enriched about two-fold in a b-type cytochrome. These results show that oxidative metabolism in amebae has some striking similarities to the respiratory burst oxidase of neutrophils.

Mating behavior, serum testosterone and semen characteristics in vasectomized and short scrotum rams

Ten similar rams were either vasectomized (VAS) or altered by securing the testes near the abdomen using elastrator bands (short scrotum, SS). Semen characteristics (electroejaculation) were similar (P>0.10) between groups before treatment and ejaculate volumes remained relatively constant (P>0.10) throughout the trial. Remaining sperm cells were nonmotile and 95% abnormal by 6 days after vasectomy. By 12 days after surgery, ejaculates contained no cells. Two SS rams had detectable sperm numbers at 12 days after treatment, but 95% were abnormal and all but 1% were nonmotile. Ejaculates collected 3 months after surgery were similar between treatments, in that all existing cells were nonmotile and abnormal. Partial spermatogenic function was regained in SS rams 15 months after treatment as indicated by the presence of low concentrations of motile sperm. Three and 15 months after treatment, sexual activity was estimated by exposing rams to a group of four estrous ewes. During the 15-minute exposure, VAS males exhibited more (P<0.10) nonmating sexual approaches than did SS animals during the first year. However, nonmating approaches and incomplete and completed matings were similar (P>0.10) between VAS and SS rams during the second year. No treatment differences in serum testosterone were observed before, during or after either libido trial. Shortening the scrotum of 6-month-old ram lambs has little effect on libido or testosterone when compared with VAS rams. However, the SS method of alteration does not result in complete sterility as indicated by the presence of motile spermatozoa in ejaculates 15 months after SS treatment.

Effect of feed and water deprivation or force-feeding on plasma prolactin concentration in turkey hens.

Plasma concentrations of prolactin (Prl), glucose, corticosterone, and D(-)-3-hydroxybutyrate (DBHB) were compared in nonlaying, nonincubating turkey hens subjected to feed and/or water deprivation. Neither Prl nor corticosterone concentrations were significantly (P greater than 0.05) altered by any of the treatments, whereas fasting significantly (P less than 0.05) reduced the concentration of glucose and increased the concentration of DBHB. Plasma levels of Prl in incubating hens were significantly (P less than 0.05) reduced by nest deprivation either in the absence of feed and water or when the hens were force-fed the normal intake for a laying hen. After 48 h of nest deprivation, the hens resumed nesting within 5 min of being returned to the pen although the plasma levels of Prl were low. Neither nest attentiveness nor the concentration of Prl were affected by force-feeding the hens while they were incubating eggs. The concentration of glucose increased in response to force-feeding or nest deprivation, whereas the concentration of corticosterone was increased only by force-feeding. These results suggest that Prl may not be involved in the striking changes in both intermediary and water metabolism which occur during incubation in the turkey hen. Furthermore, since incubation behavior can occur in the presence of low concentrations of Prl, elevated levels of Prl during broodiness appear to be maintained by a stimulus associated with the nest itself or some other aspect(s) of the environment.

Mobilization of 45Ca from insulin-producing RINm5F cells attached to microcarriers.

A technique was designed allowing studies of 45Ca efflux from insulin-releasing cells of a clonal cell line (RINm5F) attached to fibronectin-coated plastic beads. Supporting the existence of Ca2+-Ca2+ and Na+-Ca2+ exchange mechanisms, the efflux of 45Ca in a Ca2+-deficient perifusion medium was increased after introduction of Ca2+ and decreased after removal of Na+. The washout of radioactivity was similar to that from pancreatic islets in also being inhibited by glucose in the presence of low concentrations of extracellular Ca2+. During perifusion with a medium containing 2.56 mM Ca2+, increasing the K+ concentration resulted in an increased 45Ca efflux, a process counteracted by D-600. Also, exposure to tolbutamide resulted in a distinct stimulatory peak. Glucose was a poor stimulator of 45Ca efflux, suggesting that this sugar lacks the ability to depolarize the RINm5F cells sufficiently to induce normal opening of the voltage-dependent channels in the plasma membrane. Such a defect might well explain why the RINm5F cells do not respond to glucose with stimulation of insulin release.

Induction of rat alpha 2-macroglobulin in vivo and in hepatocyte primary cultures: synergistic action of glucocorticoids and a Kupffer cell-derived factor.

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.

Binding ofEscherichia coliK-12 to cellulose

Escherichia coli K-12 populations containing up to 3 · 109 bacteria were reversibly retained by columns containing fibrous cellulose. Using chromatographic retention as an assay of attachment to cellulose, the factors affecting bacterial binding were investigated. Minimal binding was observed in the absence of ions or at high pH. Optimal binding was observed in the presence of low concentrations of divalent cations at any pH or at pH below 4 in the absence of divalent cations. Factors found not to significantly affect bacterial retention by cellulose included the culture conditions of the bacteria, the presence of surface appendages, the presence of various sugars during chromatography or variations in elution temperature between 0 and 70°C. These results suggest that mechanisms not dependent on appendages contribute to the adhesion of a bacterium to an inert surface.

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains ofEscherichia coli

When grown on glucose in K+-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD+ and NADP+, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H-pyrrolo(2,3-f)quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phosphoenolpyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.

Potentiation by purines of the growth-inhibitory effects of sulphonamides on Escherichia coli K12 and the location of the gene which mediates this effect.

The increased toxicity of sulphonamides for Escherichia coli in the presence of low concentrations (50-100 microM) of purines or purine nucleosides has been confirmed and investigated further. The potentiating effect of a purine was dependent upon the activity of the appropriate phosphoribosyl transferase: a gpt mutant strain was not potentiated by guanine but remained fully sensitive to the addition of adenine. Mutants resistant to the potentiating effect of all purines have been isolated and partially characterized. The site of these mutations has been located in the region between oriC and asnA at minute 83 on the E. coli chromosome map. It is suggested that this locus be temporarily designated psp (potentiation of sulphonamides by purines) because these mutants have unaltered sensitivities to sulphonamides acting alone. Mutations in PurA, purR and folB did not affect the potentiation of sulphonamides by purines. Hypoxanthine-insensitive strains harbouring lambda asn20 were as sensitive as the wild-type to the potentiating effect. This result suggests that these lysogens are heterozygous for psp and that the wild-type allele is dominant. It is probable that psp is a regulatory gene, affecting some rate-limiting step in the biosynthesis of methionine.

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme.

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.

Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors.

An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.

Reconstruction of Photosynthetic, Cyclic Electron Transport System from Photoreaction Unit, Ubiquinone-10 Protein, Cytochrome c2 and Polar Lipids Purified from Rhodospirillum rubrum1

It was previously reported that in chromatophores of Rhodospirillum rubrum, reaction center, which consists of three kinds of protein (Mm, about 78K), is a small fragment of a large protein complex (PRU; photoreaction unit), which contains six other kinds of protein including light-harvesting bacteriochlorophyll protein, has Mm of about 700K and is free of phospholipid [J. Biochem. 86, 1211-1224 (1979); 94, 1815-1826 (1983(]. In the present study, the photosynthetic, cyclic electron transport system sensitive to antimycin A was effectively reconstructed by incubating 60 nM PRU (which contained 1 mol of reaction center and 2 mol of ubiquinone-10 per mol) with 300 nM each of oxidized ubiquinone-10 protein, reduced cytochrome c2 and lipoamino acid (which were all purified from Rhodospirillum rubrum) in the presence of low concentrations of cholate and deoxycholate (pH 8.0). In the light, the cytochrome was oxidized while the quinone was reduced. The oxidation and reduction each progressed rapidly at first, then slowly, reaching maxima (steady states) 1-2 min after the light had been turned on. At the steady states, 30% of the cytochrome was oxidized while 11% of the total quinone was reduced. When the light was turned off, the original oxidation-reduction states of the cytochrome and quinone were restored at rapid rates initially then at slow rates. Antimycin A stimulated the slow rates in the light-on state and depressed them in the light-off state, but did not influence the fast rates. Ubiquinone-10 protein was required for the antibiotic-sensitive, slow oxidation reactions. This indicates that the slow rates were due to cyclic electron transport. Cytochrome c2 was tightly bound to PRU at a molar ratio of 1:1. This cytochrome as well as the quinone bound to PRU was responsible for the fast rates. PRU had other sites able to bind cytochrome c2 and ubiquinone-10 protein with Km of 0.4 and 0.1 microM, respectively. Of the polar lipids tested, lipoamino acid was the most effective for reconstruction, and its effect was maximal at 300 nM, which is far below its critical micelle concentration.

Does the large T protein of polyoma virus regulate the expression of the cellular myc gene?

Upon expression of the large T protein of polyoma virus from genes encoding only this protein (plt genes: intron-less gene in pPyLT1 plasmid) (Tyndall et al., 1981), hrt mutant NG18 (Schaffhausen et al., 1978)), changes were observed in the behaviour in culture of rodent embryo fibroblast cells in primary cultures (REF) (Rassoulzadegan et al., 1982, 1983): the cells grew in culture for an unlimited number of generations (“immortalization”) and grew in the presence of low concentrations of serum (below 1% newborn calf serum). They maintained, however, the extended morphology, the low saturation density and the anchorage dependency characteristic of the normal fibroblast. Unlike the original REF- cells, these “immortalized” lines could be transformed by transfer of the polyoma virus gene encoding only the middle T protein (pmt gene: plasmid pPyMT1, Treisman et al., 1981), thus demonstrating a two-step process in tumorigenic transformation. A similar two-step mechanism was suggested by Land et al. (1983) for the myc and ras families of cellular oncogenes: expression either of the polyoma plt or of v-myc and rearranged c-myc genes allowed transformation of REF cells with activated forms of the ras genes. These results suggest that both genes exert similar function (s) at an early step of the transformation of REF cells in culture. This class of oncogenes also includes the E1a genes of adenovirus 2, required for transformation by the E1b genes of the same virus and which can also cooperate with a ras oncogene in REF transformation (Van den Elsen et al., 1982, Ruley, 1983).