The radiation response of asynchronous cells at low dose: evidence of substructure

Flow cytometry and cell sorting techniques have been used together with repeated measurement in an attempt to define better the radiation survival response of asynchronously dividing Chinese hamster V79-171 cells under aerobic and hypoxic conditions. Although the first two decades of cell inactivation have been examined, particular attention has been given to the low-dose range of a few grays, as used in individual radiation therapy treatments. A single linear-quadratic dose-response function was consistently unable to fit both the low-dose and high-dose data satisfactorily, suggesting a two-component response. Separate fitting of the low-dose and high-dose portions of the response yielded alpha and beta values which differed significantly (P = 0.001 to 0.002). The data are consistent with the hypothesis that the observed substructure simply reflects the presence of subpopulations of sensitive (G1-, G2-phase) and resistant (late S-phase) cells, which are resolved in these measurements. These results may have significance for certain situations in radiation therapy and in biophysical modeling of the radiation response.     

Male sterility and restored fertility in annual mercuries, relations with sex differentiation

The paper summarizes the researches conducted on male sterility in Mercurialis annua. Totally sterile individuals are very scarce in the dioecious species showing as the other Mercuries, unisexual flowers devoid of rudiments of the opposite sex. From one sterile male mutant, a ‘sterile series’ was conducted and genetics was studied. Sterile, semisterile, restored fertile male lines were constructed as well as female lines containing the inducer gene of male sterility, both fertility restorers and the sensitive cytoplasm. Morphology and ontogeny of these isogenic lines were presented. Male sterile anthers (empty) present a splitted tapetum and an abnormal meiotic end. Restored fertile male lines were normal. The relative abundance of auxin and cytokinins was studied. A specific cytokinin pathway measured as a background in fertile lines, the cis-oxidized pathway characterised the ‘sterile series’. Restoration of normal meiosis and tapetum appeared for the highest quantities of cis-zeatin (669 ng instead of 192 ng/100 g fresh weight in totally sterile). Auxin quantities were abundant compared with the normal males. Gene expression in the ‘sterile series’ was also compared with the fertile lines. t-RNAs specific for normal females were expressed in the male ‘sterile series’. Hybridization kinetics and in vitro translations pf poly(A)⁺RNAs demonstrate specific sequences for each line. Comparisons between identical organs (normal fertile male/restored fertile male or normal female/female of the ‘sterile series’) exhibited nearly 10% differences. The results suggest that for stamen development, a cascade of regulators probably exists: sex genes acting on the induction of stamen or pistil, then genes for sterility/restoration of fertility acting in anthers. Fertility-sterility regulators control the synthesis of a specific cytokinin pathway. The new hormonal signals are linked to several specific genes expressed in the floral morphology characterizing each line of the ‘sterile series’.     

Microtubule disruption interferes with the structural and functional integrity of the apical pole in primary cultures of rat hepatocytes.

The effect of microtubule disruption on the development and maintenance of cell polarity was studied in rat hepatocytes cultured as primary monolayers in the presence of colchicine or nocodazole. Addition of colchicine immediately after plating did not inhibit the generation of bile canaliculi (the apical pole) after 1 day of culture, as judged by electron microscopic examination, and did not allow penetration of Ruthenium Red through the tight junctions. However, the bile canaliculi developed in the presence of colchicine or nocodazole were not fully normal since they were not able to concentrate fluorescein diacetate in their lumina, and did not enrich with proteins of the apical plasma membrane domain, as control cells did. When the drugs were added after 1 or 2 days of culture, the new bile canaliculi appeared to be unaffected when examined by electron microscopy, but many of them did not concentrate fluorescein and were not enriched with apical membrane proteins within 4 to 24 h after drug addition. Whenever the drugs were added, the proteins that would normally concentrate on the membrane of the bile canaliculi accumulated intracellularly in endocytic vesicles after 2 to 4 h of drug treatment, and in vacuoles resembling lysosomes when the drugs were maintained for 24 h or more. These results show that microtubule disruption does not inhibit the structural reconstitution of bile canaliculi, but impairs their normal function and the transport of proteins of the apical plasma membrane domain.     

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.     

Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum

Summary An amylolytic lactic acid bacterium identified as Lactobacillus plantarum was isolated from cassava roots (Manihot esculenta var. Ngansa) during reting. The amylolytic enzyme synthesized was an extracellular -amylase with an optimum pH of 5.0 and an optimum temperature of 55° C. Cultured on starch, the strain displayed a growth rate of 0.43 h^–1, a biomass yield of 0.19 g·g^–1 and a lactate yield of 0.81 g·g^–1. The growth kinetics were similar on starch and glucose. Sufficient enzyme was synthesized and starch hydrolysis was not a limiting factor for growth. Biosynthesis of the enzyme was observed when the glucose concentration was less than 6.7 g·l^–1 and reached up to 4 IU·ml^–1 at the end of the fermentation. Offprint requests to: M. Raimbault     

Biphasic nature of the binding of cationic amphipaths with artificial and biological membranes

We have studied the interaction with liposomes and red cell membrane of various cationic amphipaths, chlorpromazine, methochlorpromazine, imipramine and propranolol. At low concentrations the interaction is a partition of the molecule between the lipid hydrophobic phase and the aqueous medium. The extent of the partition is dependent on the membrane composition or physical properties, on the incubation conditions (pH, ions) and on the amphipath used. After a given amount of amphipath has entered in the membrane, a new type of interaction appears which leads to an apparent saturable association. This association, which probably involves the anionic groups of the membrane components, might result from structural or/and electrical membrane perturbations induced by the presence of drug molecules between the phospholipids. Thus the interaction of a molecule of cationic amphipath with a membrane varies according to the amount of drug present.     

Effects of harmaline on ion transport and oxygen consumption by the bovine tracheal epithelium

The alkaloid harmaline is known to affect various membrane transport systems. This study examines the action of the drug on the short-circuit current (I0) and on the oxidative metabolism (Jr) in the tracheal epithelium of the cow. In this tissue I0 corresponds to the sum of two active transports: Na+ is absorbed and Cl- is secreted by a process based on the activity of the Na+ pump. A well defined relationship has been previously demonstrated between these active transports and the rate of O2 consumption (Schoenenweid et al., 1984 b). Low concentrations of harmaline (10(-6) to 5.10(-6) M) induced a small stimulation of I0. In contrast, larger concentrations (between 5.10(-5) and 10(-3) M) yielded a dose-related inhibition of I0, with an apparent concentration yielding 50% of maximal effect of 7.1.10(-4) M and maximal effect approaching 100%. The action was fully reversible after removal of the drug. The measurements of the fluxes of 22Na and 36Cl revealed that harmaline at a concentration of 8.10(-4) M, which decreased the I0 by 74 +/- 1% (n = 23), diminished both Na+ and Cl- transports, by 81 and 52%, respectively. The time course of I0 decay following the administration of harmaline was made of three components, with half-times of 0.34, 2.2 and 15.2 min. The time course was not appreciably modified when Cl- secretion was abolished with furosemide. Although harmaline, 10(-3)M, inhibited markedly I0, it did not modify Jr significantly. In contrast, when K+ in the incubation solution was omitted, both Ji and Jr were lowered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.     

Positional Distribution of Acyl and Alk-1-enyl Groups in Grey and White Matter Ethanolamine and Choline Phosphoglycerides of a Marsupial, the Koala (Phascolarctos cinereus)

The major phosphoglycerides in grey and white matter from the brain of the koala have been separated and examined. The major polyunsaturated fatty acids present in both the diacyl- and alk-1-enyl acylglycerophosphorylethanolamines from grey matter were 22:6 omega 3, 20:4 omega 6, and 22:4 omega 6. In both grey and white matter, 22:6 omega 3 and 20:4 omega 6 were concentrated in the 2-position of diacylglycerophosphorylethanolamines and 22:4 omega 6 in the 2-position of alk-1-enylacylglycerophosphorylethanolamines; polyunsaturated fatty acid levels were higher in diacylglycerophosphorylethanolamines. Ethanolamine phosphoglyceride fractions from grey matter were enriched in polyunsaturated fatty acids compared with those from white matter. The acyl groups 18:0, 18:1, and 16:0 and their alk-1-enyl analogues were prominent in grey and white matter ethanolamine phosphoglycerides; 18:1 was dominant in white matter alk-1-enylacylglycerophosphorylethanolamines. The plasmalogen composition of ethanolamine phosphoglycerides was 55% in grey matter and 76% in white matter. Choline phosphoglycerides contained negligible plasmalogen and low polyunsaturated fatty acid levels. Diacylglycerophosphorylcholine was characterized by high levels of 16:0 and 18:1. Similar acyl group distributions were estimated in the 1-position in both grey and white matter, 16:0 being present at greater than 50%. The presence of the molecular species 18:0/22:6 omega 3 was indicated in grey matter diacylglycerophosphorylethanolamine, 18:1/18:1 in white matter alk-1-enylcylglycerophosphorylethanolamine, and 16:0/18:1 in white matter diacylglycerophosphorylcholine.