A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Pyruvate uptake induced by a pH jump in mesophyll chloroplasts of maize and sorghum, NADP-malic enzyme type C4 species

A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP-malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD-malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient-collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP-malic enzyme type C4 species is primarily driven by the proton gradient across the envelope.     

Multivariable analysis of serum 3,5,3'-L-triiodothyronine concentration in patients of diabetes mellitus by blood glucose level and body weight

Serum thyroid hormone concentrations, 1-thyroxine (T4), free T4 and 3,5,3'-l-triiodothyronine (T3) were measured in 213 patients of diabetes and analyzed their correlation with metabolic parameters, hyperglycemia and body weight. Haemoglobin A1 (HbA1) was used as an index of hyperglycemia. Body weight was expressed by relative body weight (body weight/standard weight). Among the thyroid hormones, only T3 had significant correlation with HbA1 and body weight (r = -0.476, P less than 0.01 and r = 0.369, P less than 0.01, respectively). Multivariable analysis of serum T3 by HbA1 and relative body weight gave the following regression equation. Serum T3 (ng/dl) = 108 + 0.362 x relative body weight (%) - 3.88 x HbA 1 (%). Though relative body weight had inverse correlation with HbA1, the contribution of the two metabolic parameters to the serum T3 was independent from each other. Our results confirm the previous reports that low T3 in diabetes correlates with severity of hyperglycemia and we report for the first time that serum T3 of diabetic patients has positive correlation with body weight, probably due to still available carbohydrate in spite of disturbances in the metabolism.     

Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.     

Estimation of Genetic Variability in Natural Populations of DROSOPHILA SIMULANS by Two-Dimensional and Starch Gel Electrophoresis

Genic variation in natural populations of Drosophila simulans was surveyed using allozymic and two-dimensional electrophoretic techniques. Consistent with some previous reports, allozymic heterozygosity appeared lower than in the sibling species D. melanogaster (0.07 vs. 0.16). No variation was detected by two-dimensional electrophoresis of 19 lines scored for 70 abundant proteins. This is consistent with reported reductions in estimates of genic heterozygosity by two-dimensional electrophoresis in D. melanogaster, Mus musculus, and man. Although the amount of intraspecific variation detected in abundant proteins was lower than that detected for allozymes in D. simulans and D. melanogaster, the genetic distances between the sibling species calculated from the two data sets are not significantly different (0.35 and 0.20). The allozyme and two-dimensional electrophoresis data confirmed the impression from other measures of genetic variation (mitochondrial DNA restriction maps and inversion polymorphisms) that D. simulans is substantially less variable than D. melanogaster.     

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.     

A contractile ring and cortical changes found in the dividing Tetrahymena pyriformis

A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with siender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of microfilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments.The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing ceil, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epipiasmic layer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved in cytokinesis of Tetrahymena.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.