Modification of the Recalde method for the isolation of human monocytes

A modification of the method for monocyte isolation reported by Recalde (1984. J. Immunol. Methods. 69: 71-77) is described. Application of the modified method to 36 consecutive healthy adult donors gave a monocyte purity of 73 +/- 8% monocytes with less than 1% polymorphonuclear leukocytes and a yield of 3.44 +/- 0.93 x 10(5) monocytes/ml blood. While the monocyte purity of the modified Recalde method was lower than that obtained by elutriation (method BB in Fogelman et al. 1981. J. Lipid Res. 22: 1131-1141) in 15 donors (71 +/- 10% vs. 83 +/- 6%) the monocyte yield and the viability of the cells before and after culture were similar in both methods. The modified Recalde method does not require the expensive or complicated equipment required for elutriation and permits the isolation of human monocytes for culture in autologous serum from multiple donors in a single day.     

Relative contribution of humoral and metastatic factors to the pathogenesis of hypercalcaemia in malignancy.

Some relations between metastatic bone disease and calcium homoeostasis were determined in a consecutive series of 81 patients with solid malignant tumours attending for radionuclide bone scans. Biochemical evaluation showed that bone resorption from metastatic disease was generally not enough to account for hypercalcaemia. While skeletal metastases were present in about half of the patients who developed hypercalcaemia, biochemical indices of bone resorption in these subjects were greatly increased and disproportionate to the extent of metastatic disease detected by the bone scans. Furthermore, a reduced renal phosphate threshold and increased tubular calcium reabsorption were generally observed in hypercalcaemic patients when compared with their normocalcaemic counterparts. These findings suggest that in most cases malignancy associated hypercalcaemia may be caused by the release of a humoral factor by tumour tissue which exhibits "parathyroid-hormone-like" activity with regard to bone resorption, renal phosphate threshold, and renal calcium handling. It may be postulated that this putative humoral mediator predisposes to hypercalcaemia both by stimulating generalised osteolysis and in most cases also by impairing the renal excretion of the resultant increase in filtered calcium load. While hypercalcaemia may arise as a result of metastatic bone disease alone, these data indicate that this may be the exception rather than the rule. Hence the term "metastatic hypercalcaemia" should probably be reserved for patients with extensive skeletal tumour disease in whom biochemical evaluation fails to yield evidence of an underlying humorally mediated cause.     

Improved methods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A method for solubilizing HMG-CoA reductase is described that reproducibly yielded approximately 190% of the activity assayed in rat liver microsomes. Optimal solubilization occurred when microsomal membranes were frozen at a fixed concentration, thawed, homogenized in a buffer containing 50% glycerol, and incubated at 37 degrees C for 60 minutes. A rapid spectrophotometric assay of the reductase has been developed and the optimal conditions defined. Using this assay, the kinetics were determined for HMG-CoA reductase purified to a specific activity of 17,400 nmol NADPH oxidized per minute per mg protein.     

Localization of monocyte chemotactic protein-1 gene (SCYA2) to human chromosome 17q11.2-q21.1

Monocyte chemotactic protein-1 (MCP-1) is a member of the small inducible gene (SIG) family. It has been shown to play a role in the recruitment of monocytes to sites of injury and infection. By analysis of a panel of somatic cell hybrids, we have localized the MCP-1 gene, designated SCYA2, to human chromosome 17. In situ hybridization confirmed this assignment and further localized the gene to 17q11.2-q21.1.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.