Evaluation of near-infrared reflectance spectroscopy (NIRS) techniques for total and phytate phosphorus of common poultry feed ingredients.

The purpose of this study was to determine the feasibility of estimating the total and phytate P content of common poultry feed ingredients by near-infrared reflectance spectroscopy (NIRS). Samples of 8 plant-origin feedstuffs were collected from poultry producers in the USA and Canada during the summer of 2009: corn (133), soybean meal (114), corn distillers dried grains with solubles (DDGS; 89), bakery by-product meal (95), wheat (22), wheat middlings (31), canola meal (21), and wheat shorts (15). The samples were assayed by standard wet chemical techniques for total and phytate P contents. There was considerable variation found in most of the ingredient components. The average values for the laboratory determinations versus NIRS predictions were all within 0.030 for total phosphorus and 0.012 for phytate P. For phytate P, the magnitude of the standard errors of the predictions ranged from 0.009% for soybean meal to 0.012% for canola meal. These values may be sufficiently precise for nutritionists to use the NIRS predictions to estimate how much of the P in their ingredients is not available to the birds. For total P, the magnitude of the standard errors of the predictions ranged from 0.027% for corn DDGS to 0.142% for wheat middlings. In general, total P predictions by NIRS were not generally sufficiently precise for most nutritionists to use in feed formulation. Decision making may be quite easy in using NIRS estimates for the phytate P content of bakery by-product meal [R(2) = 0.89 for predicted = f (determined)] but not for the total P content of soybean meal (R(2) = 0.03). It is concluded that precise estimates of phytate P through NIRS should allow nutritionists for more efficient formulate and mix feed, lowering feed costs and reducing the amount of residual polluting phosphorus in poultry excreta.

Phytate and other nutrient components of feed ingredients for poultry.

Samples of feed ingredients were collected from poultry feed mills in the United States and Canada: corn (133), soybean meal (114), corn distillers dried grains with solubles (DDGS; 89), bakery by-product meal (95), wheat (22), wheat middlings (31), canola meal (21), and wheat shorts (15). The samples were assayed by standard wet chemical techniques for CP, fat, neutral detergent fiber (NDF), acid detergent fiber, calcium, phosphorus, phytate phosphorus, and ash. There was considerable variation found in most of the ingredient components. Forty-two of the 64 CV were above 10.0%. The calcium contents of the ingredients were the most variable, followed by the fat contents. The CP contents were the least variable. There were some fairly consistent relationships observed across samples; in general, acid detergent fiber and NDF were positively correlated, as were ash and mineral levels. Crude protein and fiber levels were positively related, except for wheat shorts, but the relationships were not strong. Phytate P was found to be positively related to ash and total P, as expected, except for corn DDGS. The fat content of corn was found to be negatively related to the NDF content. Significant (P < 0.004) linear regressions were found between phytate P and total P for corn, soybean meal, bakery by-product meal, wheat, wheat middlings, and wheat shorts. The average nonphytate P content of the ingredients was 49.8%, ranging from 38.8% for wheat middlings to 73.2% for DDGS. The phytate P content of wheat and wheat by-products could be predicted from their proximate compositions, with coefficients of determination in excess of 0.740. Predictions for the other ingredients were not as good.

Lycopene incorporation into egg yolk and effects on laying hen immune function.

Carotenoids are partially responsible for the colors of plants and when consumed by humans and animals are deposited into tissues (e.g., skin and egg yolk in laying hens) and may have health benefits. Because carotenoids are more available when consumed from egg yolk sources than vegetables, this research examined the ability of the laying hen to deposit dietary lycopene, a carotenoid that imparts red color in tomatoes, into the egg yolk and to investigate effects on immune function. All birds were housed in commercial cages, had ad libitum access to water, and were fed 100 g/bird per day. Experiment 1 consisted of 4 dietary concentrations of lycopene (0, 65, 257, and 650 mg of lycopene/kg of diet). High-performance liquid chromatography analysis confirmed that dietary lycopene was incorporated into egg yolks. Experiment 2 was a completely randomized design, with 3 concentrations of lycopene (0, 420, and 840 mg of lycopene/kg of diet) and 6 concentrations of alpha-tocopherol (0, 84, 164, 200, 284, and 364 mg of alpha-tocopherol/kg of diet). Egg yolk lycopene (P < 0.05) and vitamin E (P < 0.05) were increased with increasing dietary concentrations, whereas lutein and zeaxanthin concentrations remained constant. Immune responses (inflammatory, cutaneous basophil hypersensitivity, 1 degrees and 2 degrees antibody response) were induced but were not affected by dietary lycopene or vitamin E. These data indicate that lycopene can be incorporated into egg yolks, and at these dietary concentrations, alpha-tocopherol and lycopene may not affect the immune system of the laying hen.

Role of specific interactions between protein kinase C and triphenylethylenes in inhibition of the enzyme.

The tumor promoter receptor protein kinase C (PKC) has been implicated as a key enzyme in cellular growth regulation. It is, therefore, believed that specific PKC inhibitors may include effective antiproliferative agents. Previously, we have shown that the antiestrogen tamoxifen and related triphenylethylenes are potent inhibitors of PKC. Although the mechanism of inhibition of PKC by triphenylethylenes clearly involves nonspecific interactions between the antiestrogens and the lipid cofactor of PKC, we recently demonstrated that PKC itself has specific triphenylethylene-binding sites, suggesting that the inhibitory mechanism also involves specific drug-protein interactions. In this report, we characterize the direct interactions between PKC and triphenylethylenes and demonstrate their relevance to the inhibitory action of triphenylethylenes against PKC. We show (a) that the triphenylethylene-binding sites of PKC are located in the catalytic domain of the enzyme, (b) that MgATP (i.e., 10 mM MgCl2 plus 1 mM ATP) competes with the triphenylethylenes for binding sites on PKC, and (c) that triphenylethylenes are competitive inhibitors of PKC with respect to MgATP. Taken together, these data provide strong evidence that triphenylethylenes can inhibit PKC by binding directly to the ATP-binding region of the active site of the enzyme. The specific interactions between triphenylethylenes and PKC characterized here may provide a rationale for developing more specific PKC inhibitors.

Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue.

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), is a critical enzyme in the regulation of cell growth. In this report, we demonstrate elevated expression of PKC activity in surgical specimens of eight of nine spontaneous human breast tumors, as compared with the expression of PKC activity in normal breast tissue obtained from the same patients. The mean PKC specific activity in histologically normal breast tissue was 166 +/- 63 pmol 32P/min/mg, whereas the mean PKC specific activity in the breast tumors was 460 +/- 182 pmol 32P/min/mg (P = 0.0003; Student's t test). The low interpatient variability among the PKC levels observed in the histologically normal breast tissue specimens and the significant elevation of PKC levels observed in the tumors indicate that elevated expression of PKC activity in breast tissue is a potential marker for malignant disease in the breast.

Level of protein kinase C activity correlates directly with resistance to adriamycin in murine fibrosarcoma cells.

In this report, we demonstrate a direct correlation between protein kinase C (PKC) activity and adriamycin (ADR) resistance in mouse fibrosarcoma cells. PKC activity was measured in four murine UV-2237M fibrosarcoma cell lines that differed in the degrees to which they expressed resistance to ADR, which is an inhibitor of PKC. A comparison of the four cell lines revealed a positive correlation between the level of PKC activity and resistance to ADR. Incubation of the cells with the PKC inhibitor H-7 produced a partial reversal of ADR resistance. Taken together, these results suggest a role for PKC in the mechanism of ADR resistance.

Distinct patterns of phorbol ester-induced downregulation of protein kinase C activity in adriamycin-selected multidrug resistant and parental murine fibrosarcoma cells.

Specific activators of protein kinase C (PKC), including the phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), can reduce the chemosensitivities of a variety of mammalian tumor cell lines and their cytotoxic drug-selected multidrug resistant (MDR) variants to MDR-linked drugs, thus implicating PKC in the MDR phenotype. Previously, we reported that the adriamycin-selected MDR murine fibrosarcoma cell line UV-2237M-ADRR has approximately twice as much PKC activity as the parental UV-2237M line. In this report, we show that the level of [3H]phorbol-12,13-dibutyrate specific binding activity was elevated 3.5-fold in the MDR cells, thus establishing that phorbol-ester responsive PKC is overexpressed in the MDR line. Phorbol esters mediate downregulation of PKC by stimulating proteolysis of the enzyme, without altering the rate of PKC synthesis. We report that the kinetics of TPA-induced downregulation of PKC activity differ markedly in parental and MDR UV-2237M cells, providing evidence that the overexpression of phorbol-ester responsive PKC in adriamycin-selected MDR UV-2237M-ADRR cells results, at least in part, from a reduced rate of PKC degradation in the cells.

Inhibition of protein kinase C by a synthetic peptide corresponding to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 envelope glycoprotein.

This report describes the inhibition of protein kinase C (PKC) by a synthetic peptide corresponding to a viral sequence expressed in mammalian cells. The peptide corresponds to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp41), and it inhibits Ca(2+)- and phosphatidylserine (PS)-dependent phosphorylation of synthetic peptide substrates and histone by purified PKC with IC50 values ranging from 9 to 32 microM. Although previously described pKC-inhibitory synthetic peptides corresponding to sequences expressed in mammalian cells are also effective against the phosphorylation of synthetic peptide substrates, they fail to affect PKC-catalysed phosphorylation of potent protein substrates such as histone. This may limit their usefulness as inhibitors of PKC-catalysed protein phosphorylation in cellular systems. PKC activation is a major contributing factor in multidrug resistance (MDR) in cancer. Our observation that the synthetic peptide gp41(828-848) inhibits pKC-catalysed phosphorylation of a protein substrate suggests the potential value of expressing the viral sequence gp41(828-848) in cancer cells as a novel in vitro model system of MDR reversal.

Inhibition of protein kinase C by the 12-O-tetradecanoylphorbol-13-acetate antagonist glycyrrhetic acid.

Glycyrrhetic acid is an anti-inflammatory agent isolated from licorice root that inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion in mouse skin. Although it has been established that glycyrrhetic acid inhibits a number of events induced by the phorbol ester tumor promoter TPA in cultured cells, its mechanisms of action has remained obscure. In this report, we demonstrate that glycyrrhetic acid inhibits the Ca2+-and phospholipid-dependent phosphotransferase activity of protein kinase C (PKC), the phorbol ester tumor promoter receptor. Therefore, inhibition of PKC may play a role in the anti-promoting activity of glycyrrhetic acid.

Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site.

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which has been implicated as a key enzyme in the regulation of cellular growth. The naphthalenesulfonamide W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] is representative of a number of cationic amphiphilic inhibitors of PKC which appear to inhibit PKC by interacting with the acidic phospholipid cofactor of the enzyme, according to kinetic studies. In a previous report, we demonstrated that PKC binds directly to W7 when the naphthalenesulfonamide is immobilized on agarose. In the present report, we have defined the mechanism of the binding of PKC to W7-agarose, and its relevance to the inhibitory mechanism of the naphthalenesulfonamide. We demonstrate that PKC bound W7-agarose through the catalytic domain of the enzyme. An active catalytic fragment of PKC was generated by limited proteolysis, and we found that this fragment bound W7-agarose and coeluted with intact PKC upon the addition of Triton X-100. W7 inhibited PKC activity by two different mechanisms. As previously reported, W7 inhibited PKC by interacting with the phospholipid cofactor of the enzyme (IC50 = 260 microM). However, at higher concentrations of W7, we found that this naphthalenesulfonamide inhibited PKC by serving as a competitive inhibitor with respect to the substrate ATP, according to a kinetic analysis of the inhibition of the active catalytic fragment of PKC by W7. W7 inhibited the active catalytic fragment of PKC as well as PKC-catalyzed phosphorylation of protamine sulfate, a reaction which is independent of Ca2+ and phospholipid, with similar potencies. Consistent with the kinetic evidence that W7 serves as a competitive inhibitor of PKC with respect to ATP, we found that, in the presence of 10 mM MgCl2, 1 mM ATP was sufficient to elute PKC from W7-agarose. Thus, naphthalenesulfonamide PKC inhibitors may include both agents which primarily function by interacting with the phospholipid cofactor of the enzyme and agents which primarily serve as active site inhibitors of PKC.

Inhibition of protein kinase C by melittin: antagonism of binding interactions between melittin and the catalytic domain by active-site binding of MgATP.

Melittin inhibits the lipid cofactor-independent activity of protein kinase C (PKC) by directly binding to the catalytic domain in a MgATP-sensitive manner. The catalytic domains of certain PKC isozymes have a consensus sequence for a second nucleotide binding site outside their active site regions. In this report, we show that PKC isozymes containing the second nucleotide binding site motif (alpha, beta) and an isozyme lacking the motif (epsilon) all have MgATP-sensitive binding interactions with melittin. Our results support a mechanism of PKC inhibition by melittin in which active-site binding of MgATP antagonizes binding interactions between PKC and melittin.

Evidence that protein kinase C-alpha activation is a critical event in phorbol ester-induced multiple drug resistance in human colon cancer cells.

We previously designed and characterized an in vitro model of the intrinsic drug resistance of human colon cancer. The human colonic epithelium is chronically exposed to endogenous protein kinase C (PKC) stimulatory factors, and our model demonstrated that activation of PKC induces resistance to multiple anticancer drugs in the metastatic human colon cancer cell line KM12L4a. PKC is an isozyme family with ten members, eight of which are phorbol ester-responsive. In this report, we show that thymeleatoxin (Tx), a daphnane tumor promoter that selectively activates the phorbol ester-responsive isozymes cPKC-alpha, -beta 1, -beta 2, and -gamma, was just as effective in inducing drug resistance in KM12L4a cells as phorbol dibutyrate, a potent activator of all phorbol ester-responsive PKC isozymes. The induction of resistance by Tx was associated with a reduction in cytotoxic drug accumulation in KM12L4a cells. We demonstrated by immunoblot analysis and hydroxylapatite chromatography that KM12L4a cells express active cPKC-alpha but not cPKC-beta 1, -beta 2, or gamma. Our results provide strong evidence that phorbol-ester activation of cPKC-alpha is sufficient for the induction of resistance observed in KM12L4a cells. The possibility that endogenous PKC activators may induce intrinsic drug resistance in clinical colon cancer by an analogous mechanism is strongly suggested by our detection of active cPKC-alpha in surgical specimens of human colon carcinomas.

Transient enhancement of multidrug resistance by the bile acid deoxycholate in murine fibrosarcoma cells in vitro.

Recent studies have implicated protein kinase C (PKC) activation in drug resistance in vitro. PKC can be activated directly by phorbol-ester tumor promoters as well as by the bile acid deoxycholate. In this report, we demonstrate that deoxycholate, at concentrations that are chronically present in the lumen of the colon in vivo, mimicked phorbol-ester tumor promoters by protecting Adriamycin (ADR)-sensitive and multidrug-resistant (MDR) murine fibrosarcoma UV-2237M cells from ADR cytotoxicity. Deoxycholate also enhanced the resistance of the MDR cell line UV-2237M-ADRR to the cytotoxic effects of vincristine and vinblastine. In contrast to cytotoxic drug-selected MDR phenotypes, deoxycholate-induced drug resistance was transient and required continuous exposure to the bile acid. The protein kinase inhibitor H7 completely reversed the protection against ADR cytotoxicity conferred on UV-2237M-ADRR cells by deoxycholate, providing evidence that deoxycholate exerts its protective effects by a mechanism that involves stimulation of protein phosphorylation and not merely by detergent effects on membrane permeability. PKC consists of a family of at least seven isozymes with distinct modes of activation and substrate specificities. We previously reported that MDR UV-2237M cell lines contain higher levels of PKC activity than the parental ADR-sensitive UV-2237M cell line (O'Brian et al., FEBS Lett 246: 78-82, 1989). The present report shows that PKC-III is a major PKC isozyme in ADR-sensitive and MDR UV-2237M cell lines. Thus, the resistance to ADR induced by the phorbol esters in UV-2237M cell lines provides strong evidence that PKC-III activation confers protection against ADR on ADR-sensitive and MDR UV-2237M cell lines. Furthermore, since deoxycholate is an endogenous molecule in the colonic epithelium, our finding that physiological concentrations of deoxycholate can render cells more resistant to chemotherapeutic drugs in vitro may have implications for the biology and therapy of intestinal cancers.

N-myristyl-Lys-Arg-Thr-Leu-Arg: a novel protein kinase C inhibitor.

In view of the critical role that the Ca2+- and phospholipid-dependent enzyme protein kinase C (PKC) plays in mediating proliferative responses to a number of growth factors, hormones, and tumor promoters, it is thought that selective PKC inhibitors may provide a new class of antiproliferative drugs. Established PKC inhibitors include three major classes of agents: agents that compete with the substrate ATP, agents that compete with the protein substrate, and agents that both compete with ATP and interact with the cofactor phosphatidylserine (PS). In this report, we have characterized the interactions between PKC and N-myristyl-Lys-Arg-Thr-Leu-Arg, a myristylated analogue of a synthetic peptide substrate of PKC. We determined that the myristylated peptide was a novel PKC inhibitor that interacted with PS as well as competed with the protein substrate of PKC. The inhibitory activity of the peptide was conferred by myristylation. We found that the myristylated peptide antagonized Ca2+- and PS-activated PKC with an IC50 of 75 microns, whereas the nonmyristylated peptide lacked this inhibitory activity. A fully active, Ca2+- and PS-independent catalytic fragment of PKC can be generated by limited proteolysis. Although the myristylated peptide was a very poor PKC substrate, this peptide inhibited the catalytic fragment of PKC by apparent competition with the phosphoacceptor substrate histone IIIS with an IC50 of 200 microM, whereas the nonmyristylated peptide showed no inhibitory activity against the catalytic fragment. Thus, the myristylated peptide may serve as a model for the development of selective PKC inhibitors, because its inhibitory mechanism exploits the substrate specificity of PKC, as well as the novel regulation of the enzyme. Furthermore, since endogenous PKC substrates include acylated proteins, the observations that we report here concerning a myristylated synthetic peptide suggest that acylation of proteins may be important in the regulation of PKC activity in vivo.

Differential non-redox inhibitory effects of glutathione against protein kinase C isozyme family members.

Glutathione (GSH) and the GSH metabolic precursor N-acetylcysteine (NAC) are potent antioxidants that have clear potential either as cancer chemopreventive agents or as lead compounds for new cancer chemopreventive agents. The potential efficacy of GSH and NAC in clinical cancer chemoprevention is suggested by their antagonism of tumor promotion in animal models. Protein kinase C (PKC) is an isozyme family that plays a critical role in phorbol ester-mediated tumor promotion. We recently found that GSH and NAC exert direct inhibitory effects against a purified PKC isozyme mixture by a mechanism that did not involve their antioxidant properties. In this report, we characterize non-redox inhibitory effects of glutathiones on PKC isozymes that have been shown to produce partially or fully transformed phenotypes in mammalian cells. We show that GSH, NAC, and oxidized GSH analogs exert potent inhibition of the isozyme cPKC-ç and are somewhat less effective against cPKC- 1. In contrast, the oncogenic isozyme nPKC-â was unaffected by NAC, and it was inhibited by GSH and oxidized GSH analogs very modestly. Our results suggest that the potential impact of non-redox GSH/NAC-mediated PKC inhibition on cellular responses to tumor promoters and indeed, on cell growth regulation in general, may depend upon the pattern of PKC isozyme expression in the cells.

Stable expression of a cDNA encoding rat brain protein kinase C-beta I confers a multidrug-resistant phenotype on rat fibroblasts.

Protein kinase C (PKC) is a Ca++- and phospholipid-dependent protein kinase that plays an important role in signal transduction pathways that regulate cell growth. Tumor cells selected for a multidrug resistant (MDR) phenotype often express elevated levels of PKC activity. To directly test whether PCK overexpression can produce an MDR phenotype, we studied rat embryo fibroblasts that were infected with the full-length cDNA clone RP58 encoding the beta I form of rat brain PKC. The PKC-beta I gene recipient R6-PKC3 cells are stable, overproduce PKC, and express an elevated level of PKC activity. R6-PKC3 cells exhibited significant resistance to adriamycin, actinomycin D, vinblastine, and vincristine but not to 5-fluorouracil. Intracellular accumulation of adriamycin, vinblastine, and vincristine was decreased in the R6-PKC3 cells, but this was not associated with an altered level of P-glycoprotein expression. Moreover, the reduction in drug accumulation appeared to be a consequence of a decreased rate of drug uptake. The data indicate that overexpression of PKC in rat fibroblasts produces an MDR phenotype without altering P-glycoprotein expression.

Immunological effects of tumor vaccines: III. Influenza virus oncolysates inhibit the TPA induced activation of peripheral blood mononuclear cells.

This paper investigates the effects of tumor vaccines on T cell proliferation induced by 12-0 tetradecanoylphorbol-13-acetate (TPA). Viral oncolysate (VO) tumor vaccines containing inactivated influenza virus A significantly inhibited TPA-induced T cell proliferation. In contrast, a control tumor vaccine (CO) that contained the same cellular components as VO but lacked influenza virus did not affect the TPA-induced proliferation. These effects were also observed with peripheral blood mononuclear cells (PBMC) from ovarian cancer patients, although VO and CO each induced significant and similar levels of proliferation in these cells in the absence of TPA. Protein kinase C (PKC) is a pivotal enzyme in signal transduction pathways that control cell proliferation, and TPA is a specific activator of PKC. VO and CO showed differential effects on the inhibition of purified protein kinase C (PKC). These studies demonstrate the antagonistic effects of different tumor vaccines on T lymphocyte proliferation and suggest that influenza virus A or virus-modified cellular components may interfere with signal transduction in the immune cells of the recipient of the tumor vaccine.

Vitamin B12 nutriture of chickens fed raw soybean meal.

Experiments were conducted to investigate a possible interference by raw soybean meal (RSM) with B12 nutriture of chickens. In Experiment 1, day-old chicks were fed B12-free isonitrogenous and equienergetic diets containing 0 or 40% RSM to determine if RSM accelerated storage losses of B12. After 42 days, RSM decreased growth (P less than .01), decreased hepatic (P less than .01) and blood (P less than .05) concentrations of glutathione (GSH), and increased (P less than .01) pancreas and liver weights. However, statistically significant differences due to treatment were not detected in hepatic B12 concentration, indicating that RSM does not enhance B12 turnover in chicks. To ascertain the effect of RSM on B12 absorption, 9 micrograms B12/kg diet was added to diets containing 0 or 40% RSM and fed to chicks to 42 days of age. Raw soybean meal depressed growth (P less than .001) and hepatic GSH (P less than .01) and increased (P less than .001) pancreas weights. The RSM had no effect on hepatic B12 concentration, suggesting that RSM trypsin inhibition does not impair B12 absorption in chicks. A third experiment was designed to test the hypothesis that vitamin B12 stimulates egg production or feed intake of hens fed a diet with 27% RSM. Vitamin B12-depleted hens were fed RSM or heat-treated soybean meal (HSM) diets containing equivalent amounts of soybean protein, oil, and hulls. Hens received either 26 micrograms B12/kg BW per os or 13 micrograms B12/kg BW intramuscularly twice within a 30-day period.(ABSTRACT TRUNCATED AT 250 WORDS)

Ability of cimetidine to increase intestinal pH of chicks.

Cimetidine was tested for its ability to increase the pH of intestinal contents of chicks. In Experiment 1, 18 mg cimetidine injected i.m. per kilogram body weight (BW) increased (P less than .05) proventricular pH within 1 hr, but proventricular pH began to decrease 4 hr postinjection. The pH of the contents of the duodenum was increased (P less than .05) 4 hr postinjection, whereas the pH of the contents of the jejunum was unaffected. In Experiment 2, 36 mg cimetidine injected i.m. per kilogram BW increased (P less than .05) the pH of the contents in the proventriculus and duodenum, but did not alter the pH in the jejunum and ileum. These results show that cimetidine can be used for experimental purposes to increase the pH of the proventricular and duodenal contents in chickens.