The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation.

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.02. A progressive and significant (P <.0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.1, +1 hour: 1.3 +/- 0.9, +2 hours: 4.1 +/- 0.9), peaking at +3 hours (10 +/- 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. 2000;96:554-559)

Cloning of a gene that is rearranged in patients with choroideraemia.

Choroideraemia (tapetochoroidal dystrophy, TCD), a common form of X-linked blindness, is characterized by progressive dystrophy of the choroid, retinal pigment epithelium and retina. Previous studies have assigned the TCD gene to a small segment of the Xq21 band. By making use of reverse genetics strategies we have isolated eight overlapping complementary DNA clones from the same chromosomal region. The corresponding gene is expressed in retina, choroid and retinal pigment epithelium. The cDNAs encompass an open reading frame of 948 base pairs that is structurally altered in eight TCD patients with deletions, and in a female patient with a balanced translocation involving Xq21. These findings provide strong evidence that we have cloned the gene underlying choroideraemia. Elucidation of its function should provide new insights into the molecular mechanisms responsible for this disorder and other hereditary retinopathies.

Effect of exercise on lipid metabolism of rats fed high carbohydrate diets.

Two experiments were conducted to determine whether high carbohydrate diets or exercise would have a greater influence on certain parameters of lipid metabolism. Male Fischer rats were used in both experiments, separated into exercise and sedentary groups, and fed either a high sucrose (63%) or a high starch (63%) diet. There were no differences in body weight or food consumption between the two diets. Exercise resulted in a highly significant increase in food consumption in both experiments. Rats fed sucrose had a higher serum cholesterol value than rats fed starch. Diet did not influence serum triglycerides but the rats on excercise had significantly lower serum triglycerides than the sedentary rats. Liver weight was significantly larger in rats fed sucrose. Sucrose caused an increase in the activity of both glucose-6-phosphate dehydrogenase and malic enzyme activities in liver tissue, whereas exercise caused an increase in the activity of these enzymes in adipose tissue.