Isolation and characterization of a mutant of L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nucleoside transport

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.     

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.     

A microfibril-generating factor from the cellulase of Trichoderma reesei

A combination of ionic strength reduction and diafiltration of Trichoderma reesei cellulate complex through a hollow fiber apparatus of 5000 molecular weight (MW) cutoff and subsequent passage of filtrate over a Spherogel-TSK 3000-SW column provided extracts that had the ability to generate microfibrils in filter paper and to disrupt filter paper and corn leaf tissue. Milligram quantities of material obtained from these extracts released small amounts of soluble carbohydrate from filter paper, required ferric iron for increased activity, and contained amino acids. Short fiber formation and disruption of filter paper during interaction with these extracts was enhanced by prior acid treatment and eliminated by prior base treatment. The amount of soluble carbohydrate hydrolyzed in 24 h from filter paper by whole cellulase complex was not changed by first disrupting the substrate with the extracts.     

Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H(2)SO(4) and H(3)PO(4)) such that the cooking liquor pH 相似文献   

Aspirin enhances evaporation in hydrated and dehydrated rats

The effect of acetysalicylic acid (aspirin) on thermoregulation in a warm environment was studied in hydrated and dehydrated adult rats to test the hypothesis that dehydration hyperthermia can be modified by an antipyretic drug. Metabolic rate (MR), evaporative water loss (EWL), and deep body temperature (Tb) were measured during 2 h of exposure to an ambient temperature of 36 degrees C after the rats had received an oral pellet of aspirin (100 mg.kg-1) or placebo. The dehydrated placebo group had a higher Tb and lower EWL than the hydrated placebo group. Aspirin increased MR and EWL in both hydrated and dehydrated animals. Aspirin did not affect Tb in hydrated rats, but reduced Tb by 0.2 degree C in dehydrated rats during the heat exposure. The elevation in EWL appears to be a thermoregulatory response to increased heat production in both hydrated and dehydrated animals after aspirin treatment. The possibility that aspirin may act in dehydrated animals to restore central thermosensitivity toward hydrated levels needs to be tested further.     

Synthesis and evaluation of a series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines

A series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines (3'-alkyl-3'-deoxythymidines) has been prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pent-2- enono-1,4-lactone ((S)-5-[(tert-butyldiphenylsilyl)oxymethyl]-2(5H)- furanone) by Michael addition of the appropriate organocopper reagent, followed by reduction of the lactone, acetylation of the resulting hemiacetal, and trimethylsilyl triflate-catalyzed coupling with 2,4-di-O-(trimethylsilyl)thymine. The protected nucleosides were desilylated by using tetrabutylammonium fluoride to give anomeric mixtures of the free nucleosides. The unsubstituted 2',3'-dideoxynucleoside analog was similarly prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pentono- 1,4-lactone ((S)5-[(tert-butyldiphenylsilyl)-oxymethyl]-dihydro-2(3H)-fu r anone).     

The effect of salts in fermenting mashes on the microbiological assay ofl-lysine

During a study of the effects of high concentrations of NaCl, NaNO₃ and KC1 on the production of lysine bySaccharomyces cerevisiae during the fermentation of glucose (Richmond 1980) it was learned that amounts of salt greater than 0.6 M in the microbiological assay sample could change the apparent concentration of lysine in the sample. This could be corrected by adding to the lysine assay broth sufficient salt to match the concentration in the sample tube and then developing a revised standard curve. The additional salt, in turn, required that the microbiological assay time be lengthened.     

A deletion map of the human Y chromosome based on DNA hybridization.

The genomes of 27 individuals (19 XX males, two XX hermaphrodites, and six persons with microscopically detectable anomalies of the Y chromosome) were analyzed by hybridization for the presence or absence of 23 Y-specific DNA restriction fragments. Y-specific DNA was detected in 12 of the XX males and in all six individuals with microscopic anomalies. The results are consistent with each of these individuals carrying a single contiguous portion of the Y chromosome; that is, the results suggest a deletion map of the Y chromosome, in which each of the 23 Y-specific restriction fragments tested can be assigned to one of seven intervals. We have established the polarity of this map with respect to the long and short arms of the Y chromosome. On the short arm, there is a large cluster of sequences homologous to the X chromosome. The testis determinant(s) map to one of the intervals on the short arm.     

Vicilin genes ofPisum

Summary We describe four genomic clones of pea 7S storage protein gene, one of which corresponds to convicilin, and the others to vicilin. Hybridization studies exploiting these clones, and previously identified cDNA clones, have enabled us to define six different loci. Three of these loci have been mapped to positions on chromosome 7.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.