Genome Engineering of Human Urine-Derived Stem Cells to Express Lactoferrin and Deoxyribonuclease.

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on transposon modification.

Functional analysis of the catalytic triad of the hAT-family transposase TcBuster.

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid.

Percentage of canals filled in apical cross sections - an in vitro study of seven obturation techniques.

Aim: To compare the apical density of several obturation techniques when used in palatal roots of extracted maxillary molars.

Methodology: Seventy extracted molars were randomly divided into seven groups with 10 teeth each. The palatal root canals were instrumented to size 60 MAF, coated with Kerr's Pulp Canal Sealer, and obturated using one of seven techniques.

Glucose metabolism in proliferating epithelial cells from the rat colon.

1. The effects of fasting and fasting followed by refeeding on the activities of the oxidative pentose pathway (OPP) and the tricarboxylic acid cycle (TCA) in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]glucose and [6-14C]glucose, respectively. 2.

Pyruvate sparing by butyrate and propionate in proliferating colonic epithelium.

1. The effects of fasting and fasting followed by refeeding on the relative activities of the pyruvate dehydrogenase (PDH) complex and the tricarboxylic acid (TCA) cycle in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]pyruvate and [3-14C]pyruvate, respectively. 2.

Zinc deficiency is associated with suppression of colonocyte proliferation in the distal large bowel of rats.

As zinc status may influence susceptibility to colon cancer, we examined the effect of dietary zinc deficiency on the proliferation of epithelial cells (colonocytes) in the large bowel of rats. When compared to feed-restricted rats, those with zinc deficiency showed a significant reduction in proliferation in the distal colon as assessed by accumulated metaphase arrest and crypt cell production rates in vivo. Zinc deficiency had no apparent effect on thymidine kinase activity in colonocytes but was accompanied by minor changes in fecal mass and fecal pH.

Proliferative activity in the proximal and distal colon of the rat after fasting and refeeding.

Several methods were used to assess proliferation of colonocytes in the proximal and distal colon of the rat after fasting and refeeding. Those applied in vivo included metaphase arrest, labelling with bromodeoxyuridine and uptake of tritiated thymidine. The latter two techniques were also applied after isolation of colonocytes in vitro.

The application of microcalorimetry to the assessment of metabolic efficiency in isolated rat hepatocytes.

1. Heat output by suspensions of isolated rat hepatocytes was determined by using a modified batch-type microcalorimeter. 2.

Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide--adenine dinucleotide couple acting on adenosine 3':5'-cyclic monophosphate phosphodiesterase.

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2.

An effect of extracellular redox state on the glucagon-stimulated glucose release by rat hepatocytes and perfused liver.

The effect of changes in the extracellular redox-state on glucagon-stimulated glucose release by intact isolated rat hepatocytes and the perfused liver was examined. For hepatocytes from the fed rat an increase in pyruvate, ammonium ion or oxygen concentration or a decrease in the lactate/pyruvate or sorbitol/fructose ratios decreased the ability of 1 microM-glucagon to stimulate glucose release without significantly altering the control rate. These changes coincided with a decrease in the lactate/pyruvate ratio of the cell suspension.

Amino acid nutrition of the fetal lamb.

With three exceptions amino acids were retained by the fetal lamb from the maternal circulation in proportions appropriate for the synthesis of whole blody protein. Only glutamate, aspartate and serine, amino acids involved in the synthesis of nucleic acids, seemed to be produced by the fetus itself.

Glucose and acetate metabolism in sheep at rest and during exercise.

Total entry rate of blood glucose and the rate of irreversible loss of blood acetate and its oxidation have been examined in sheep at rest and while walking on a horizontal treadmill at 5 km/h for 2 h. Sheep were given their daily ration of 1000 g chaff in 24 eaual portions at hourly intervals and received multiple intravenous injections of [2-3H]glucose and intravenous infusions of [1-14C]acetate and NaH14CO3. At rest the total entry rate of blood glucose was 0-44 +/- 0-03 mmol/min (values given as mean +/- s.

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact.

Utilization of oxidizable substrates by the sheep hind limb: effects of starvation and exercise.

Measurements of substrate uptake by the sheep hind limb show a pattern similar to human and other monogastric animals. Thus free fatty acids (FFA) are the principal substrates at rest and during exercise while beta-hydroxybutyrate and acetoacetate are major nutrients in starved animals. The hind limb has arteriovenous differences for glucose and lactate which indicate that glucose supplies about 27% of the fuel of respiration during exercise, but the hind limb in resting, fed, and starved animals returns essentially all of the glucose carbon to the blood in the form of lactate.

Amino acid uptake and output by the sheep hind limb.

Arteriovenous differences across the hind limb of fed sheep show release of alanine, glutamine, and tyrosine and uptake of serine, glutamate, and possibly lysine. In starved animals there is a net output of most amino acids, although the amount of alanine released, 26 nmol/ml blood, is much lower than reported for human muscle and is less than the cumulative release of valine, leucine, and isoleucine. Since it has been argued that the carbon of alanine is derived from glucose and the nitrogen from the deamination of branched chain amino acids, we suggest that either nutrient availability is limiting alanine output or else sheep muscle has an impaired ability to degrade the branched chain amino acids.

Utilization of amino acids by the isolated perfused sheep liver.

To test Elwyn's suggestion [1970], that high concentrations of amino acids supplied to the liver from the hepatic artery do not stimulate protein synthesis, substrates containing amino acids have been infused into either the hepatic artery or portal vein of isolated sheep livers. The livers received highly oxygenated blood from the hepatic artery and partly deoxygenated blood from the portal vein. There were no significant differences in amino acid uptake (81 +/- 4% of input), urea output (69 +/- 6% of uptake) or 'protein synthesis' as assessed by N accumulation in the liver.

Production and utilization of acetate in mammals.

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.

Effects of carbohydrate availability on lipogenesis in sheep.

1. Lipogenesis in sheep liver and adipose tissue was investigated by incorporation studies in vitro with radioactive glucose and acetate and by assays of key enzymes. 2.