Evaluation of an isotope ratio method for measuring biliary cholesterol secretion

We have evaluated an isotope ratio method for measuring biliary cholesterol secretion. Secretion was measured in eight nonhuman primates by analysis of radioactivity of feces and plasma 4 weeks after intravenous administration of a single dose of [3H]cholesterol. For the test, [14C]cholesterol was fed in known amounts daily for 10 days. The ratio of isotopes in feces (14C/3H) was equivalent to the ratio of total radioactivity that entered the intestine from diet and bile. Assuming biliary cholesterol specific activity equals plasma cholesterol specific activity, the mass of cholesterol secreted daily in the bile could be calculated. In paired experiments in four animals we were able to directly compare biliary secretion by the new method with mass measurement by intraduodenal intubation (Grundy, S. M., and A. L. Metzger. 1972. Gastroenterology. 52: 2100-1216). The two methods correlated well in these four animals (r = 0.97). We further noted that bile secretion by the new method (eight animals) and by the mass method (four animals) correlated well with body weight (r = 0.94 for weight vs secretion by the isotope ratio method; r = 0.97 for weight vs secretion by the mass ratio method). Ranges of body weight and secretion for the eight animals were 4.2-13.1 kg and 3.6-11.4 mg/hr, respectively. The slopes of the two regression lines for body weight vs. secretion measured by the two methods did not differ significantly from one another (F(1.8) = 1.42; 0.25 less than P less than 0.50).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

Summary The 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNA^Ala and tRNA^Ile, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.     

Kinetics of gas diffusion in mammalian cell culture systems. I. Experimental

It has been demonstrated experimentally that the thickness of fluid overlay in conventional tissue culture systems limits the oxygen available to mammalian cells growing as a submerged monolayer. A rocker culture system is described which circumvents critical problems associated with thin film culture while permitting nearly unlimited access of oxygen to the cell monolayer. Good growth of primary hepatic cells as isolated sheets has been obtained.     

Studies of low density lipoprotein molecular weight in human beings with coronary artery disease

Low density lipoprotein molecular weight (LDL MW) correlates positively with coronary artery disease in cholesterol-fed nonhuman primates. To evaluate this in human beings with coronary artery disease (CAD) we measured LDL MW in 93 volunteers undergoing coronary angiography (47 controls and 46 CAD patients). LDL MW of CAD patients was less than that of controls (patients, 2.79 +/- 0.17 g/mumol; controls, 2.93 +/- 0.19 g/mumol; P less than 0.001). However, LDL MW decreased as plasma triglyceride increased and concentrations of triglyceride were greater in CAD patients than in controls. Since decreased LDL MW is likely to result, in part, from increased plasma triglyceride concentrations, we attempted to determine the effect of triglyceride on the relation of LDL MW to CAD in this study. After covariance adjustment for triglyceride, there was no LDL MW difference between CAD patients and controls. Because LDL heterogeneity has been identified in other studies and was apparent on inspection of agarose column profiles of LDL of these volunteers, we sought differences in the profiles that might distinguish coronary disease cases from controls. No differences could be found. In addition, we used density gradient ultracentrifugation to characterize LDL in more detail in a subset of volunteers who had a wide range of plasma triglyceride concentrations (50 mg/dl to 900 mg/dl). LDL mean hydrated density was inversely related to LDL MW and increased as triglyceride increased. The increase in peak density was reflected in an increase in percent of total protein in LDL found to have d greater than 1.045 g/ml and a decrease in protein in LDL of d 1.035-1.040 g/ml. These interrelationships were not apparently influenced by coronary artery status.     

Synthesis and characterization of singly modified (carboxyferrocenyl)cytochrome c derivatives.

Carbodiimide-activated coupling chemistry has been used to covalently attach 1,1'-dicarboxyferrocene (dcFc) to the epsilon-amine of surface lysine residues of horse heart cytochrome c. Conditions have been found that optimize the production of singly modified (dcFc)cytochrome c derivatives and the presence of one free carboxylate per modification site allows separation and purification of about 10 of these derivatives by cation-exchange chromatography. Reversed-phase HPLC tryptic peptide mapping techniques have been used to identify the attachment sites of eight pure (dcFc)cytochrome c derivatives (at lysines 7, 8, 13, 25, 60, 72, 73, and 100). Through-space distances from these lysines to the nearest heme edge span the 6-16 A range and these derivatives should prove useful in exploring the distance dependence of long-range intramolecular electron transfer in cytochrome c.     

Thymic epithelium. I. Lymphoid-free organ cultures grafted in syngeneic intact mice

Low temperature organ culture of 14 day gestational age mouse thymic lobes leads to the development of a lymphoid-free epithelial matrix. Morphologically, these explants exhibit two distinct epithelial components, which may be indicative of the dual embryologic origin (ectodermal/endodermal) of the thymic epithelium. When such explants are grafted into syngeneic intact recipients, the compound matrix is repopulated by lymphohematopoietic precursors that give rise to an intragraft lymphocyte population. The graft shows additional morphologic development parallel to normal thymus ontogeny. Studies in congenic mice when using the TIa alloantigen as a marker of derivation demonstrate the host origin of these lymphocytes. Cytotoxic assays for the presence of cell surface Thy-1.2, TIa, Lyt-1.2, and Lyt-2.2 reveal that graft lymphocytes express a phenotypic profile and developmental progression that is typical of normal thymocytes. Furthermore, mitogen and mixed leukocyte culture assays show that intragraft lymphopoiesis leads to the generation of a mature population. In addition to the lymphocytes, host-derived Ia+ adherent cells can also be isolated from these thymic epithelial grafts. We conclude that low temperature organ culture-derived thymic epithelium appears to retain those properties of the thymic microenvironment that permit lymphohematopoietic colonization and support thymocyte differentiation.