The effects of technology use in feedlot production systems on feedlot performance and carcass characteristics.

The objectives of this study were to examine the effects of feedlot production systems with and without the use of a ß-adrenergic agonist compared to an all-natural production program on feedlot performance and carcass characteristics. Crossbred beef steers ( = 336; initial BW = 379 ± 8 kg) were randomized to 1 of 3 treatments in a randomized complete block design (RCBD; 14 steers/pen; 8 pens/treatment). Treatments consisted of an all-natural treatment (NAT), a conventional treatment (CONV), and a conventional treatment with a ß-agonist (CONV-Z). All treatments were fed the same basal diet with NAT cattle receiving no growth promoting technologies. The CONV and CONV-Z cattle were implanted with 40 mg of estradiol and 200 mg of trenbolone acetate (TBA) on d 0 and were fed 33 and 9 mg/kg of monensin and tylosin daily, respectively. The CONV-Z cattle were fed zilpaterol hydrochloride (ZH) at 6.76 mg/kg (90% DM basis) for the last 20 days on feed (DOF) There was no effect of treatment on DMI ( = 0.83); however, CONV-Z steers gained 3.8% faster (1.64 vs. 1.58 kg/d; < 0.01) and were 5.3% more efficient (0.160 vs. 0.152; < 0.01) than CONV steers, and CONV steers gained 32.8% faster (1.58 vs. 1.19 kg/d; < 0.01) and were 26.7% more efficient (0.152 vs. 0.120; < 0.01) than NAT steers. There was a 35.7% improvement in estimated carcass gain (1.29 vs. 0.95 kg/d; < 0.01) and a 32.6% improvement in carcass efficiency (0.126 vs. 0.095; < 0.01) for CONV-Z steers compared to NAT steers. Hot carcass weight was increased by 8 kg for CONV-Z steers compared to CONV steers (394 vs. 386 kg; = 0.05) and 46 kg compared to NAT steers (394 vs. 348 kg; < 0.01). Longissimus muscle area was increased by 3.6 cm for CONV-Z steers compared to CONV steers (92.29 vs. 88.67 cm; = 0.02) and 12.1 cm for CONV-Z steers compared to NAT steers (92.29 vs. 80.16 cm; < 0.01), resulting in a 9.6% unit increase in USDA yield grade (YG) 1 (15.14 vs. 5.52%; < 0.05) and a 21.6% unit reduction in USDA YG 3 for CONV-Z steers compared to CONV steers (30.70 vs. 52.32%; < 0.05). The CONV-Z steers had a lower marbling score compared to the other treatments (432; 0.01), resulting in an 11.7% unit increase (20.70 vs. 9.03%; < 0.05) in USDA Select carcasses compared to CONV steers. The results of this experiment show that CONV-Z and CONV production results in a significant improvement in feedlot performance and USDA YG compared to NAT.

Neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease.

RATIONALE: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. OBJECTIVE: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. METHODS AND RESULTS: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)-null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C-null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C-null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C(-/-) ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. CONCLUSIONS: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system.

The effect of UVB irradiation on ferritin subunit synthesis, ferritin assembly and Fe metabolism in cultured canine lens epithelial cells.

Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue-specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc-induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

Transferrin in after-cataract and as a survival factor for lens epithelium.

The Fe-transport protein, transferrin (Tf), is synthesized and secreted by whole lenses and cultured lens epithelial cells. Because of Tf's central role in cell growth and proliferation, its participation in lens cell proliferation following cataract extraction was explored using a rabbit model of after-cataract. Varying amounts of the central anterior lens capsule were removed (0, 35, or 80%) following extraction of the lens. The Tf content of and secretion by after-cataract lens capsular sacs containing regenerated lens tissue was determined ex vivo at 0, 3, 5, 7 and 9 weeks post-surgery. In all cases Tf content of and secretion by the lens sacs was higher than that of their contralateral controls (whole lenses). Tf secretion was up to 5-fold higher and metabolic labeling studies indicated secretion of newly synthesized Tf. The sacs contained up to 10 times the concentration of Tf as the control lenses. Human lens after-cataract capsular bags also secreted Tf. The function of Tf as a survival factor was tested on cultured lens epithelial cells. Cells cultured in serum-free medium had a survival rate of only 20-34% if the medium was changed each day. If the medium was never changed during this period, the survival rate was 43-52%, suggesting secretion of essential growth factors by these cells. Addition of 200 microg ml-1 Tf to the medium during each daily change increased survival to levels attained when the medium was not changed. Addition of Tf antibodies to the culture medium during each daily change decreased cell survival to 14%. Apparently Tf acts as a survival factor for lens epithelia and its synthesis is up-regulated in after-cataract lens sacs. These factors suggest that Tf may play an important role in the pathogenesis of lens epithelial cell proliferation and after-cataract formation following cataract surgery.

Molecular basis of the brindled mouse mutant (Mo(br)): a murine model of Menkes disease.

The brindled mouse mutant (Mo(br)) is the closest animal model of the human genetic copper deficiency, Menkes disease, which is presumed to be due to a mutation at the X-linked mottled locus (Mo). The mutant mice are hypopigmented and die at around 15 days after birth, but can be saved by treatment with copper before the 10th postnatal day. Menkes disease has been shown to be due to mutations of the gene ATP7A which encodes P-type ATPase (referred to here as MNK). MNK is likely to function in copper efflux from cells, but the full range of its biological activity is not fully understood. The nature of the mutation in the brindled mouse is of importance in our understanding of the role of MNK and for devising treatment strategies for Menkes disease. Here we show that the brindled mouse has a deletion of two amino acids in a highly conserved, but functionally uncharacterized, region of Mnk. Comparison with the Ca ATPases suggests this region may be involved in conformational changes associated with the E1/E2 transition fundamental to the action of P-type ATPases. We also describe the first Western blot data for Mnk in tissues, and these show normal levels of Mnk in mutant and brindled kidneys but none in liver. In the kidney, immunohistochemistry demonstrated Mnk in the proximal and distal tubules, the distribution is identical in mutant and normal. This distribution is consistent with Mnk being involved in copper resorption from the urine.

Decreased carbonic anhydrase III levels in the liver of the mouse mutant 'toxic milk' (tx) due to copper accumulation.

The mouse mutant 'toxic milk' (tx) is characterized by marked hepatic accumulation of copper, similar to that found in patients with the genetic disorder of copper transport, Wilson disease. In addition, lactating tx females produce copper-deficient milk. To characterize further the biochemical basis of this defect, Western blots of tissue extracts from normal and tx mice were probed with various heavy-metal radioisotopes (63Ni. 65Zn and 64Cu). A 30 kDa Ni/Zn-binding polypeptide was found to be markedly decreased in the livers of the tx mice. This protein was isolated from normal adult mice using a procedure based on Ni-chelation chromatography. The amino acid sequences of two CNBr peptides were identical with portions of the mouse skeletal muscle carbonic anhydrase III (CAIII) sequence. Two other peptides sequenced had closely related sequences to that of CAIII, but with two differences in 45 amino acids. These two peptides may be derived from a novel CAIII isoform, which we term CAIIIB to distinguish it from the published form, CAIIIA. We isolated a cDNA clone corresponding to CAIIIA and used this to show that CAIIIA mRNA was also decreased in the mutant liver, but not in muscle. Copper loading of normal mice also decreased hepatic CAIIIA mRNA, suggesting that the decrease in CAIII mRNA in the tx mouse liver is a secondary consequence of the high copper levels in the liver.

Expression of Menkes disease gene in mammary carcinoma cells.

Two P-type ATPases, MNK and WND were recently shown to be defective in the human disorders of copper transport, Menkes disease and Wilson disease respectively. These proteins are important in copper homeostasis but their full physiological function has not been established. This study uses the human breast carcinoma line, PMC42, to investigate copper transport in the mammary gland. Northern blot analysis indicated that both MNK and WND mRNA are expressed in these cells. Western blot analysis with an MNK-specific antibody demonstrated a band of approx. 178 kDa, close to the expected size of 163 kDa. Treatment of PMC42 cells with lactational hormones (oestrogen and progesterone for 3 days followed by dexamethasone, insulin and prolactin for a further 3 days) did not produce an obvious increase in MNK expression as measured by Northern and Western blots. By using indirect immunofluorescence with the MNK antibody, the intracellular distribution of MNK was found to be predominantly perinuclear, consistent with Golgi localization. Punctate staining was also seen in a smaller proportion of cells, suggesting that some MNK is associated with endosomes. Treatment of PMC42 cells with lactational hormones increased the intensity of the perinuclear and punctate fluorescence. Exposure of cells to 100 mM copper resulted in the dispersion of the fluorescence towards the periphery of the cell. The results suggest a role for MNK in the secretion of copper into milk and that PMC42 cells are a valuable model for investigating the detailed cellular function of MNK and WND.

Iron uptake by cultured lens epithelial cells.

BACKGROUND: Transferrin and Fe concentrations increase in the intraocular fluids in pathological conditions and the lens accumulates Fe during ocular inflammation. Tissues take up Fe from transferrin by two mechanisms, receptor-medicated endocytosis of diferric transferrin and a process occurring at the cell membrane which may be mediated by an oxido-reductase. However, Fe metabolism, transport and storage have not been previously investigated in the lens. This study was designed to characterize the uptake of Fe from transferrin by lens epithelial cells in culture. METHODS: Primary, secondary and tertiary cultures of canine lens epithelial cells and cultures obtained from cataractous lenses were studied. Uptake of 59Fe from transferrin by these cultured cells was measured. Transferrin receptor populations were determined in receptor-binding assays. RESULTS: There was a distinct relationship between the amount of Fe-transferrin added and the amount of Fe taken up, which was linear for the primary cultures but significantly reduced for the secondary, tertiary and cataract cultures (252 +/- 21, 169 +/- 14, 153 +/- 14 and 96 +/- 2 ng Fe/mg protein, respectively). Transferring receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free medium for 1 week. CONCLUSIONS: The reduction of Fe uptake by the subcultured and cataract cell lines probably reflects a decrease in transferrin receptor expression and in the activity of an alternative pathway for Fe transferrin uptake occurring over time. This reduced Fe uptake may result from long-term exposure to relatively high Fe concentration in the media. A reduction in the expression of the transferrin receptor after incubation with high concentrations of Fe supports this conclusion.

Regulation of ferritin levels in cultured lens epithelial cells.

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.

Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice.

The murine homologue of the Menkes disease gene (MNK) was isolated from cDNA libraries, using human cDNA clones as probes, and by PCR. The predicted amino acid sequence shows a high level of identity (89.9%) with the human protein, and the predicted functional domains in the human protein are present. Using probes to the mouse Mnk gene, we found that the mottled dappled mutation was caused by alteration in the Mnk locus and lack of expression of Mnk RNA. Tissues of the blotchy mouse contained two larger sizes of MNK mRNA demonstrating a likely defect in RNA splicing. Thus, the mottled locus is homologous to the human MNK locus and dappled and blotchy are allelic mutations in this gene.

Isolation of a partial candidate gene for Menkes disease by positional cloning.

Menkes disease is an X-linked recessive disorder of copper metabolism resulting in death in early infancy. The gene has been mapped to band Xq13 based, in part, on a translocation breakpoint in a female with the disease, which was found to lie within 300 kilobases (kb) of the PGK-1 locus, allowing the isolation of a YAC clone spanning the breakpoint. Phage subclones from the breakpoint region were isolated and used to screen cDNA libraries. cDNA clones were found which detect an 8 kb transcript from normal individuals but show diminished or absent hybridization in Menkes disease patients. Partial sequence of the cDNA shows a unique open reading frame containing putative metal binding motifs which have been found in heavy metal resistance genes in bacteria. This gene is a strong candidate for the Menkes disease gene.

Red-cell metabolism in patients with iron deficiency.

18 components of metabolism were determined in the red cells of iron-deficient patients and data were expressed per 10(12) red cells to avoid the complicating effects of hypochromia and microcytosis. Glucose consumption, AMP and ATP, glycolytic intermediates except 2,3-bisphosphoglycerate (2,3-DPG) and phosphoenolpyruvate (PEP), red-cell Na+ and the net passive leakage of Na+ and K+ at 4 degrees C were all normal. Creatine, 6-phospho-D-gluconate: NADP oxidoreductase (6PGD) activity and fresh red-cell K+ were raised, suggestive of a young cell population. However, ATP: D-fructose-6-phosphate 1-phosphotransferase (PFK) activity and ADP were low. An elevated 2,3-DPG level was attributable to the anaemia present but the somewhat raised PEP level is unexplained. It is concluded that red cells in iron deficiency show some characteristics of a young cell population; in other respects they appear normal, but in containing a low PFK activity they are abnormal.

Rapid correction of red-cell nucleotide abnormalities following successful renal transplantation.

Erythrocyte levels of 5' mono-, di- and triphosphates of adenosine, inosine and guanosine, together with NAD+ (H) and NADP+ (H) were measured before and serially after renal transplantation. The high levels of ATP and guanosine triphosphate (GTP) fell to normal within 3 d of achieving a normal plasma creatinine. The decline in ATP and GTP levels paralleled the fall in plasma creatinine. Acute graft rejection was accompanied by a rise in ATP and GTP, both of which returned rapidly to normal with resolution of the rejection episode. Radiolabelling of red cells confirmed that the metabolism of the existing red-cell population was corrected so that observed changes in nucleotide concentrations were not due to a new cohort of cells.

Identification of two molecular-mass forms of phenylalanine hydroxylase that segregate independently in rats. Specific association of each form with certain rat strains.

The nature of the different molecular-mass forms of phenylalanine hydroxylase in rat livers was examined by immunoprecipitation of the enzyme from crude liver extracts that had been radiolabelled by reductive methylation. The two forms of the enzyme were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and detected by fluorography. Segregation of the two forms of the enzyme was demonstrated in Sprague-Dawley rats, as would be expected if the two forms were products of allelic genes. In addition, hooded and albino Wistar rat livers contained only the slower-migrating form and Lewis rat livers contained only the faster-migrating form, and hence we suggest that the forms be referred to as W (for Wistar) and L (for Lewis). Peptide mapping showed that the W and L forms are closely related, and the difference between them appears to reside at one or other end of the polypeptide chain. The kidney contained the same forms as the liver in one-tenth the quantity, providing further evidence that the same phenylalanine hydroxylase gene is expressed in liver and kidney.

beta-hydroxyisobutyryl coenzyme A deacylase deficiency: a defect in valine metabolism associated with physical malformations.

An infant, born to parents who were first cousins had multiple physical malformations. An associated biochemical abnormality was suggested by the urinary excretion of cysteine and cysteamine conjugates of methacrylic acid. The coenzyme A (CoA) ester of this compound is an intermediate in the pathway of valine oxidation. Subsequent investigation revealed a deficiency of beta-hydroxyisobutyryl-CoA deacylase, an enzyme unique to valine metabolism. The enzyme defect results in accumulation of methacrylyl-CoA, a highly reactive compound, which readily undergoes addition reactions with free sulfhydryl groups. Tissue damage due to reactions between methacrylyl-CoA and important sulfhydryl-containing enzymes and cofactors may account for the teratogenic effects seen in this patient.

Viscosity and iron deficiency in treated polycythaemia.

An in vitro study of the effect of red-cell changes due to iron deficiency on whole-blood viscosity has been made on samples from patients with polycythaemia treated by venesection. When the PCV of the samples was adjusted to a standard value of 0.45, whole-blood viscosity was unrelated to decreasing MCH. In contrast, when samples were adjusted to a standard Hb concentration of 14 g/dl, whole-blood viscosity rose exponentially with decreasing MCH. This increase in whole-blood viscosity was shown to be a function of the increasing PCV that accompanies the falling MCH at this standard Hb value. It is suggested that in polycythaemia not due to hypoxia, hyperviscosity can be satisfactorily corrected by venesection controlled by monitoring the PCV alone. Other consideration may apply in hypoxic polycythaemia and these are discussed.

Leucocyte ATP and renal failure.

1. We have measured adenosine 5'-triphosphate (ATP) levels in isolated leucocyte suspensions from patients with renal failure. 2. Leucocyte ATP is reduced in these patients, unlike erythrocyte ATP which is known to be elevated in uraemia. 3. The relevance of reduced leucocyte ATP to the abnormalities of cation transport and cell function which occur in uraemia is discussed.

Anaerobic glycolysis in normal human erythrocytes incubated in vitro with sodium salicylate.

1. Some effects of sodium salicylate upon anaerobic glycolysis have been studied in normal human erythrocytes incubated for up to 6 h at 37 degrees C in autologous sera. 2. Both glucose consumption and lactate production were stimulated by concentrations of salicylate up to 60 mmol/l but at the highest concentration used (90 mmol/l) an initial stimulus was followed by inhibition of glycolysis. 3. Losses occurred of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-phosphate(AMP)at higher concentrations of salicylate and there was a concomitant increase of inorganic phosphate. 4. Other phosphate esters underwent concentration changes at higher concentrations of salicylate that reflected inadequate concentrations of ATP for glycolysis. 5. The rates of sodium efflux from, and potassium influx into, erythrocytes were unaffected by the presence of salicylate at concentrations sufficient to stimulate glycolysis.