Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth,respiration and protein biosynthesis

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O₂ consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.Abbreviations DNP dinitrophenol - PG propylgallate - SHAM salicylhydroxamic acid     

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.     

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     

Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.     

Canopy transpiration and water fluxes in the xylem of the trunk of Larix and Picea trees — a comparison of xylem flow,porometer and cuvette measurements

Summary Leaf gas exchange, transpiration, water potential and xylem water flow measurements were used in order to investigate the daily water balance of intact, naturally growing, adult Larix and Picea trees without major injury. The total daily water use of the tree was very similar when measured as xylem water flow at breast height or at the trunk top below the shade branches, or as canopy transpiration by a porometer or gas exchange chamber at different crown positions. The average canopy transpiration is about 12% lower than the transpiration of a single twig in the sun crown of Larix and Picea. Despite the similarity in daily total water flows there are larger differences in the actual daily course. Transpiration started 2 to 3 h earlier than the xylem water flow and decreased at noon before the maximum xylem water flow was reached, and stopped in the evening 2 to 3 h earlier than the water flow though the stem. The daily course of the xylem water flow was very similar at the trunk base and top below the lowest branches with shade needles. The difference in water efflux from the crown via transpiration and the water influx from the trunk is caused by the use of stored water. The specific capacitance of the crown wood was estimated to be 4.7 x 10^-8 and 6.3 x 10^-8 kg kg^-1 Pa^-1 and the total amount of available water storage was 17.8 and 8.7 kg, which is 24% and 14% of the total daily transpiration in Larix and Picea respectively. Very little water was used from the main tree trunk. With increasing transpiration and use of stored water from wood in the crown, the water potential in the foliage decreases. Plant water status recovers with the decrease of transpiration and the refilling of the water storage sites. The liquid flow conductance in the trunk was 0.45 x 10^-9 and 0.36 x 10^-9 mol m^-2s^-1 Pa^-1 in Larix and Picea respectively. The role of stomata and their control by environmental and internal plant factors is discussed.