Vitamin E and organic selenium for broilers from 22 to 42 days old: performance and carcass traits.

This study was conducted to evaluate the effect of vitamin E and selenium on performance, viability, productive efficiency, and yields of carcass, major cuts, and organs of broilers from 22 to 42 days submitted to cyclic-heat stress. The experimental design was randomized blocks, in a 2 × 3 factorial arrangement with two levels of selenium (0.1 and 0.3 mg/kg) and three levels of vitamin E (300, 400, and 500 mg/kg), plus a control treatment. Animals were submitted to a natural condition of high cyclic temperature. Organic selenium levels of 0.1 and 0.3 mg/kg associated with 300, 400, and 500 mg/kg of vitamin E were tested. The level of vitamin E did not affect the performance or production efficiency of broilers in the period from 22 to 33 days and 22 to 42 days. However, the selenium inclusion level of 0.3 mg/kg improved the viability in both phases. The yields of carcass, major cuts, intestine, and heart were not influenced by the levels of selenium and vitamin E, whereas abdominal fat for the selenium level 0.1 mg/kg decreased linearly with the inclusion in vitamin E.

Cystic fibrosis hetero- and homozygosity is associated with inhibition of breast cancer growth.

Cystic fibrosis (CF) is the most common lethal recessive genetic disease of the Caucasian population. Although reports of cancer frequency in CF have emphasized an elevated observed-to-expected ratio of 6.5 for digestive tract cancers, these studies also show a significantly decreased observed-to-expected ratio for other malignancies including breast cancer. The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATP channel. We found that heterozygous and homozygous CFTR knockout mice had elevated blood ATP concentrations. Elevated extracellular ATP is known to inhibit tumor growth in vivo and in vitro. Using double mutant mice created by F2 generation crosses of CFTR knockout and nude mice, we observed reduced breast tumor implantability in CFTR homozygous nude animals. Decreased tumor growth rate was observed in both CFTR heterozygous and homozygous nude animals. Extracellular ATP reduced human breast tumor cell growth rate in vitro, and a breast tumor transfected with human CFTR that had high extracellular ATP concentrations in vitro correspondingly had a slower growth rate in vivo. The results suggest that both CFTR heterozygosity and homozygosity suppress breast cancer growth and that elevated extracellular ATP can account for this phenomenon.

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Transporting renal epithelium: culture in hormonally defined serum-free medium.

A hormonally defined medium was used to isolate a homogeneous epithelioid cell population from canine kidney. Monolayers of these cells form domes, an indication of active ion transport, and this process is inhibited by ouabain. This technique allows the isolation of primary cultures of renal epithelial cells, free of fibroblasts, for the characterization of biochemical and physiological properties related to renal function.

Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis.

Posttranscriptional modulation of gene expression in cultured rat hepatocytes.

The maintenance of high levels of two liver-specific mRNAs in cultured hepatocytes was achieved in a serum-free hormonally defined cell culture medium. However, this maintenance of liver-specific mRNA levels did not correlate with the level of transcription of the genes but was apparently due to increased stabilization of the tissue-specific mRNAs. The mRNA stabilization did not occur in serum-supplemented medium. In both defined and serum-supplemented medium, actin and tubulin mRNAs were also greatly increased, in both cases predominantly if not entirely due to increased mRNA stability.

Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines.

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Mineralocorticoid-induced membrane proteins in MDCK cells.

The Madin-Darby canine kidney (MDCK) cell line, which exhibits properties indicative of a distal tubule origin, evidently binds and responds to mineralocorticoid hormones. We investigated the effects of aldosterone and deoxycorticosterone on protein synthesis in MDCK cells grown either in medium supplemented with serum or in a hormonally defined, serum-free medium. Aldosterone induced the synthesis of at least 2 membrane proteins with molecular weights of 35000 and 14000. The MDCK line may prove a useful model system for examining the mechanism of mineralocorticoid-regulated sodium transport and, in particular, the identification and study of hormone-induced proteins in a homogeneous cell population.

Development of salivary gland cell lines for studies of signaling and physiology.

We developed and characterized an immortalized rat parotid cell line to use in salivary gland studies. The cells were immortalized by retroviral transduction of SV40 large T antigen into isolated parotid cells. Using immunocytochemical techniques, we found that the immortalized epithelial cells were ductal, rather than acinar, in nature. Cells were grown under coculture conditions with lethally irradiated NIH3T3 cells. One cell line, which was designated RPG1/SV40 cells (for rat parotid gland 1/SV40 transformant), was selected for characterization. These cells formed a sheet epithelium with tight junctions and a measurable transepithelial resistance. RPG1/SV40 cells responded to muscarinic receptor (carbachol) and/or P2 purinoceptor (ATP and UTP) stimuli with increases in the following: (1) intracellular free-calcium concentration ([Ca2+]i); (2) the short-circuit current (ISC) across the epithelium; (3) the tyrosine phosphorylation of PKC delta; and (4) MAP kinase activity. Thus, the cells appear to be useful for a wide range of studies involving physiology, biochemistry, and signal transduction approaches.

Cystic fibrosis transmembrane conductance regulator protein expression in brain.

The cystic fibrosis transmembrane conductance regulator protein (CFTR) has been identified in bovine brain clathrin-coated vesicles, rat brain and a human neuroblastoma cell line using affinity-purified polyclonal peptide antibodies against CFTR. Immunocytochemical staining of multiple dendrites and soma of neurons of the diencephalon, midbrain, pons and medulla oblongata, has also been demonstrated. Whole cell lysates and membranes derived from rat brain, neuroblastoma cells and bovine brain clathrin-coated vesicles express the mature 150-165 kDa and 130 kDa unglycosylated forms of CFTR. The localization of CFTR to brain regions controlling homeostasis and energy expenditure may relate to the pathogenesis of non-pulmonary manifestations of cystic fibrosis. CFTR expression in neurons and coated vesicles suggests a possible effect on neuropeptide vesicle trafficking by mutant CFTR.

Synthesis and degradation of eicosanoids in primary rat hepatocyte cultures.

Arachidonic acid metabolites may play an important role in liver physiology, yet hepatocyte prostaglandin synthesis has not been characterized extensively. We used RIA to study production and clearance of several eicosanoids in confluent primary cultures of rat hepatocytes in serum-free, hormonally-defined medium. Under basal, unstimulated conditions 6-keto-PGF1 alpha (spontaneous breakdown product of prostacyclin) and 13,14-dihydro-15-keto-PGE (DHK-PGE, a metabolite of PGE) accumulated in the culture medium. Hepatocytes cleared 6-keto-PGF1 alpha, thromboxane B2, and DHK-PGE from the medium. Production of eicosanoids by primary cultures appeared resistant to indomethacin and several other cyclooxygenase inhibitors. This apparent resistance to indomethacin was not caused by rapid metabolism of indomethacin, by failure of the drug to enter hepatocytes, or by insensitivity of hepatocyte cyclooxygenase to the drug. Metabolism of PGE to DHK-PGE may be saturated under in vitro conditions. Hepatocytes can synthesize significant amounts of eicosanoids, although they are probably less active in this regard than are non-parenchymal cells.

Controlled drug release from polymeric delivery devices V: Hydroxy group effects on drug release kinetics and thermodynamics.

The effects of progesterone hydroxylation on silicone matrix drug release kinetics and thermodynamics were investigated. Hydroxylation at positions 11, 17, and/or 21 substantially reduced progesterone release. The magnitude of this reduction depended on the number and position of the hydroxy groups and could be attributed to decreased polymer matrix diffusivity (Dm) and polymer solubility (Cp). Thermodynamically, hydroxy group addition to positions 11 and/or 21 reduced the activation energy for matrix diffusion (Ed,m) but increased the solvation energy for dissolution in silicone polymer (delta HT,m)). Adding an hydroxy group to position 17 increased the Ed,m but decreased the delta HT,m. The overall (Ed,m) + delta H(T,m)) values were relatively constant and independent of hydroxylation.

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes.

When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate or N-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight of 60,000 to 70,000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with 35S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin E and organic selenium for broilers from 22 to 42 days old: performance and carcass traits

ABSTRACT This study was conducted to evaluate the effect of vitamin E and selenium on performance, viability, productive efficiency, and yields of carcass, major cuts, and organs of broilers from 22 to 42 days submitted to cyclic-heat stress. The experimental design was randomized blocks, in a 2 × 3 factorial arrangement with two levels of selenium (0.1 and 0.3 mg/kg) and three levels of vitamin E (300, 400, and 500 mg/kg), plus a control treatment. Animals were submitted to a natural condition of high cyclic temperature. Organic selenium levels of 0.1 and 0.3 mg/kg associated with 300, 400, and 500 mg/kg of vitamin E were tested. The level of vitamin E did not affect the performance or production efficiency of broilers in the period from 22 to 33 days and 22 to 42 days. However, the selenium inclusion level of 0.3 mg/kg improved the viability in both phases. The yields of carcass, major cuts, intestine, and heart were not influenced by the levels of selenium and vitamin E, whereas abdominal fat for the selenium level 0.1 mg/kg decreased linearly with the inclusion in vitamin E.

Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba.

Acanthamoeba keratitis is a vision-threatening corneal infection. The mannose-binding protein of Acanthamoeba is thought to mediate adhesion of parasites to host cells. We characterized the amoeba lectin with respect to its carbohydrate binding properties and the role in amoeba-induced cytopathic effect (CPE). Sugar inhibition assays revealed that the amoeba lectin has the highest affinity for alpha-Man and Man(alpha1-3)Man units. In vitro cytopathic assays indicated that mannose-based saccharides which inhibit amoeba adhesion to corneal epithelial cells were also potent inhibitors of amoeba-induced CPE. Another major finding was that N-acetyl-D-glucosamine (GlcNAc) which does not inhibit adhesion of amoeba to host cells is also an inhibitor of amoeba-induced CPE. The Acanthamoebae are thought to produce CPE by secreting cytotoxic proteinases. By zymography, one metalloproteinase and three serine proteinases were detected in the conditioned media obtained after incubating amoebae with the host cells. The addition of free alpha-Man and GlcNAc to the co-culture media inhibited the secretion of the metalloproteinase and serine proteinases, respectively. In summary, we have shown that the lectin-mediated adhesion of the Acanthamoeba to host cells is a prerequisite for the amoeba-induced cytolysis of target cells and have implicated a contact-dependent metalloproteinase in the cytopathogenic mechanisms of Acanthamoeba.

Effect of haemodialysis on immune response.

Uraemia depresses immune response by altering cellular reactivity to mitogenic and antigenic stimulation. One might expect that amelioration of uraemia by dialysis would improve immune responses. We have investigated the effect of haemodialysis on in-vitro parameters of cellular immunity. Our data suggest that haemodialysis leads to loss of a factor or factors from both uraemic and normal plasma essential for DNA synthesis. Our data do not suggest that measurements of celluar immunity are useful in monitoring adequacy of haemodialysis in chronic uraemic patients.

A comparison of protein synthetic patterns of MDCK cells grown in serum and hormonally defined serum-free medium.

The development of hormonally defined serum-free media (HDM) for the culture of mammalian cells has allowed the study of specific cellular functions in a totally defined environment. Recently, several reports have indicated that there are differences in the basic cellular physiology of cells cultured in HDM when compared to cells cultured by the more traditional method of using serum-supplemented culture media (SM). We report here that there are significant changes in the protein synthetic pattern in MDCK cells grown in HDM. There were no changes exhibited during the first passage in HDM, but following 10 passages in HDM there was an increased isotope ratio of 1) plasma membrane proteins with molecular weights of 12,000, 36,000 and 68,000 and 2) endoplasmic reticulum proteins with molecular weights of 12,000 and 37,000. Additionally, the incorporation of methionine and uridine were significantly increased in cells cultured in HDM for 10 passages. At present, we believe that these changes in the protein synthetic patterns are due at least partially to increased protein synthesis as indicated by monosome/polysome ratios. Therefore, though the use of HDM offers a completely defined system for studying cellular function, results obtained using HDM must be interpreted with caution when comparing them to previous studies that have used SM.

Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit.

Familial hypercholesterolemia is an inherited disease in humans that is caused by a defect in the receptor for low density lipoproteins (LDLR). The existence of an animal model for this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, makes it an attractive candidate for developing new therapies that involve gene transfer into liver. As a first step toward the development of these therapies, we report the use of retrovirus-mediated gene transfer to correct the genetic defect in hepatocytes isolated from WHHL rabbits. A series of retroviral vectors that express the gene for human LDLR were constructed, each differing in the transcriptional elements used to drive LDLR expression. Helper-free amphotropic virus stocks representing each construct were then used to infect primary cultures of hepatocytes that were isolated from newborn WHHL rabbits. The efficiency of transduction, as measured by Southern analysis of integrated proviral sequences, ranged from 20% to 100%. Expression of human LDLR was analyzed by blot hybridization analysis of total cellular RNA and by biochemical and in situ analyses of transduced cultures for receptor function. The vector in which the expression of LDLR was driven by the viral long terminal repeat sequence produced the greatest quantity of LDLR RNA and protein in WHHL hepatocytes; LDLR activity approached normal levels in these cultures.

Regulation of cytokine secretion by cystic fibrosis airway epithelial cells.

Chronic airway inflammation is an important feature of cystic fibrosis (CF), markedly influencing morbidity and mortality. We wanted to assess the contribution of the respiratory epithelium in the mediation of local inflammatory events, and, more particularly, its regulating role through cytokine secretion. We have studied the regulation of interleukin-6 and 8 (IL-6 and IL-8) production by the SV40 transformed airway epithelial cell line JME/CF15 (homozygous for the deletion of Phe 508). We show that unstimulated JME/CF15 cells secrete IL-6 and IL-8. Neutrophil chemotactic activity (NCA) is detected in supernatants. The secretion of IL-6 and IL-8 is increased following stimulation of the JME/CF15 cells by IL-1 beta and neutrophil elastase. Lipopolysaccharide and granulocyte macrophage colony stimulating factor (GM-CSF) have no effect on secretion of IL-6 or IL-8. Neutrophil elastase inactivates recombinant human IL-6 at 37 degrees C in vitro, but has no effect at 4 degrees C, suggesting a proteolytic effect of elastase on IL-6. IL-8 activity remains preserved, even after prolonged exposure to elastase. Our data suggest that the airway epithelium may play an active role in the mediation of neutrophil chemotaxis. Local production of IL-8 in response to elastase and IL-1 beta, together with the inactivation of the anti-inflammatory protein IL-6, may result in a significant upregulation of airway inflammation in cystic fibrosis.

An in vitro model of infection of human biliary epithelial cells by Cryptosporidium parvum.

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.