Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

Effect of particle size of the calcium source on performance of broiler chicks fed diets with different calcium and phosphorus levels.

Broiler chicks were fed diets differing in calcium (Ca) level, source, and particle size. The basal diet fed in most experiments contained .62% total phosphorus and .49% Ca from sources other than the Ca sources being compared. Performance was not affected by Ca source in diets with .9% Ca. In diets with 1.5% Ca, gain and bone ash were significantly lower with a powdered, USP grade calcium carbonate than with limestone particles passing through a 1,000-mu screen and retained by a 417-mu screen. Crushing these particles to allow them to pass through a 74-mu screen caused them to behave like the powdered calcium carbonate. Particles of this limestone had to at least pass through a 147-mu screen to cause a greater reduction in gain; the finest particles tested (passed a 74-mu screen) reduced performance most. When graded levels of fine and medium particles were fed, the higher levels reduced gain, and less of the fine particles was required to produce a given reduction. In a high Ca-low P diet, fine particles of the Ca source were more deleterious than medium. Apparently, chicks can shunt excess Ca in the form of medium particles (150 to 1,000 mu) through the digestive system better than they can the more reactive, fine particles.

Comparison of DL-methionine and its sodium salt in water solution in broiler starter diets with two choline levels.

Graded levels of DL-methionine or a water solution of its sodium salt were added at equivalent levels to a broiler diet based on corn, soybean, and poultry by-product meals. Methionine addition to the basal diet produced only a small and statistically nonsignificant increase in gain. Gain/feed ratio was significantly increased by either methionine source; the two forms did not differ in value. Increasing the choline chloride added to the diet from 300 to 800 mg/kg did not improve performance nor reduce the value of the added methionine. The sulfur amino acid requirement of the broiler chicks was less than the .93% given by the National Research Council (1977).

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT-PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION.

Enriched mitochondrial fractions isolated from durum wheat seedlings via differential centrifugation exhibited classical cyanide- or antimycin A-insensitive O(2) uptake which was inhibited by either salicylhydroxamic acid or propyl gallate. Further purification of this fraction using Percoll density gradients resulted in two discrete bands which were essentially homogeneous mitochondrial populations, as verified by electron microscopy. Respiratory O(2) uptake in these two fractions was completely inhibited by cyanide or antimycin A. Addition of linoleic acid to a third-step gradient band, which was shown to contain virtually no mitochondria, resulted in demonstrable cyanide-insensitive O(2) uptake. This O(2) consumption was completely inhibited by propyl gallate or salicylhydroxamic acid, two known lipoxygenase inhibitors. In contrast, addition of linoleic acid to the two purified mitochondrial fractions did not stimulate O(2) uptake. These data indicate that lipoxygenase oxygenation, the enzyme physically separable from the mitochondria, is responsible for the cyanide-insensitive component of O(2) uptake that was observed in subcellular fractions isolated from etiolated wheat seedlings.

Effects of protectants on the activity of wheat mitochondria.

A mitochondrial fraction was isolated from durum wheat seedlings via differential centrifugation using three different tissue grinding buffers. One buffer contained bovine serum albumin (BSA), a protein proven effective in protecting mitochondria from damage during isolation. The other two buffers contained either polyethyleneglycol of M.W. 20,000D (PEG-20), a phenolic binding compound or PEG-20 plus XAD-2, a non-ionic adsorbent material. Organelles isolated with the BSA exhibited an average ADP/O ratio of 2.3 and an average respiratory control ratio of 5.1, substantially higher than those obtained with the other media. Addition of the XAD-2 to a buffer containing PEG-20 resulted in an increase in the respiratory control ratio to an average value of 3.7 vs 2.7 for organelles isolated with PEG-20 alone without causing a concomitant rise in the ADP/O ratio which remained an average of 1.9. Protein values for the mitochondrial fraction isolated with BSA ranged from 2.5 to 3 times greater than those obtained from the PEG-20 preparations. This difference in organelle protein is discussed in relation to the accurate measurement of mitochondrial respiration.

Factors affecting efficacy of methionine hydroxy analogue for chicks fed amino acid diets.

Chicks were fed mixtures of methionine, cystine, and the calcium salt of the hydroxy analogue of methionine (MHA) in a diet based on a mixture of amino acids. Rate of gain with the basic amino acid diet containing a mixture of methionine and cystine was about 90% of the rate noted with a practical type diet. Efficacy of MHA depended on its level in the diet and on the levels of methionine and cystine fed with it. It was least effective when fed as the only sulfur amino acid or when fed with cystine. When fed with methionine it had intermediate value and was most efficacious when fed with a mixture of methionine and cystine. Essentially it was fully effective when it provided 25% of the sulfur amino acids with the remainder as equal parts of methionine and cystine. Replacing part of the cystine in a mixture of cystine and MHA with methionine resulted in a marked improvement in performance with L-methionine being slightly more effective than D-methionine.

Factors affecting efficacy of methionine hydroxy analogue for chicks fed practical diets.

The relative values of the calcium salt of the hydroxy analogue of methionine (MHA) and L-methionine were studied in chick feeding tests with five practical type diets deficient in the sulfur amino acids. Performance was improved with the addition of either substance to each of the five diets. Methionine was slightly more effective at low levels of addition to the four diets primarily deficient in methionine. The two supplements were equally effective in one diet based on corn, soybean, and meat and bone meals, a diet with a wider methionine/cystine ratio. Both the practical and highly purified amino acid diets thus showed similar differences in efficacy of the two products as the methionine/cystine ratio of the diet changed. Methionine also was more effective than MHA in overcoming the deleterious effects produced by the addition of ethionine to a practical type diet.

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT-PURIFIED MITOCHONDRIA.

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O(2) uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A-insensitive O(2) uptake in either gradient band. Antimycin A-insensitive O(2) consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O(2) uptake. This O(2) consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O(2) consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development.

Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation.

The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 mug/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.It was suggested that the resistance mechanism in P. arundinacea involves an induction of cathodic isoperoxidases in challenged tissue. These peroxidases may function in the biosynthesis of lignin at the site of attempted penetration.

Replacing dietary methionine and cystine in chick diets with sulfate or other sulfur compounds.

A purified diet deficient in the sulfur amino acids and sulfate was fed to chicks in tests designed to compare the effectiveness of several sulfur compounds in sparing the sulfur amino acids. The addition of a mixture of methionine and cystine to this diet increased growth rate and feed efficiency more than any of the other additions. Sulfate improved performance as much as any inorganic sulfur source tested. Taurine, sulfite, thiosulfate, and sulfur also had value. When graded levels of sulfate were added to diets with sulfur amino acid levels about 0.2 and 0.3% below the level required for optimum performance, the weight gain and feed efficiency response curves were curvilinear. Most of the response to sulfate was produced with a level of 200 p.p.m., but 500 to 660 p.p.m. was needed to produce the maximum sparing effect. The sulfur amino acid requirement was found to be about 0.075% less with 0.1% sulfate in the diet than without it when growth rate was the criterion. When feed efficiency was the criterion, the requirement was reduced about 0.05% by sulfate.