Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (c) 1996 John Wiley & Sons, Inc.     

Growth kinetics of Lactobacillus acidophilus under ohmic heating

Lactobacillus acidophilus OSU133 was inoculated into MRS broth in a fermenter vessel and incubated at 30, 35, or 45 degrees C with agitation. Incubation temperatures were attained by conventional or ohmic heating. An electrical current at low (15 V) or high (40 V) voltage was used to heat the culture directly during fermentations under ohmic heating. The growth parameters (lag period, minimum generation time, and maximum growth) and changes in pH were determined during fermentation. Metabolic activities (consumption of glucose and production of lactic acid and bacteriocin) were determined during fermentation at 35 degrees C under both heating methods. Lag period for L. acidophilus was affected appreciably by the method of heating, but the magnitude of these changes depended on the fermentation temperature. When fermentation was done at 30 degrees C, lag period decreased by 94% under low-voltage ohmic, compared with conventional, heating methods. Ohmic heating did not change the generation time significantly and caused slight, but significant (p < 0.01) decrease in maximum growth. Therefore, the electric current enhances the early stages, but it inhibits the late stages of growth. Ohmic, compared with conventional, heating resulted in higher final pH and lower bacteriocin activity in the fermented medium. However, ohmic heating at 35 degrees C had minimal effect on glucose utilization and lactic acid production by L. acidophilus. Results show that measurement of the electric current when ohmic heating is done at a constant voltage may be used in monitoring such fermentations. In conclusion, ohmic heating is potentially useful in certain applications related to fermented foods. (c) 1996 John Wiley & Sons, Inc.     

The effect of rebamipide on gastric xanthine oxidase activity and type conversion in ethanol-treated rats

Rebamipide, a novel antipeptic ulcer drug, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic acid, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intraperitoneal administration of rebamipide at 60 mg/kg body weight reduced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of rebamipide on alcohol-induced xanthine oxidase-type conversion and enzyme activity. It was also observed that the activity of xanthine oxidase was significantly inhibited by administration of rebamipide at 60 mg/kg body weight, leading to a significant reduction of lipid peroxide content in alcohol-treated rats. The results suggest that alcohol-induced gastric mucosal lesions might be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and the protective effect of rebamipide on gastric mucosal lesions would result from its ability to protect against oxidative stress on gastric mucosal lesions of alcohol-treated rats.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Dendrochronological analysis of the canopy history of two Ohio old-growth forests

This study examined the temporal patterns of establishment, suppression, and release of major tree species in two old-growth Ohio forest remnants as a means to determine the past disturbance history of these forests. Increment cores were taken from a total of 154 trees from two well-drained, upland plots and two poorly-drained, bottomland plots in each of the two forested areas. Acer saccharum and Fagus grandifolia exhibited multiple episodes of suppression and release prior to becoming canopy trees, and could tolerate suppressions as long as 84 years. In contrast, Quercus macrocarpa, Q. muehlenbergii, Prunus serotina, and Acer saccharinum rerely exhibited any tolerance to suppression and appeared to have entered the canopy after single disturbances had opened large areas of canopy. There was clear synchrony in the temporal pattern of establishment and final release from suppression among trees from bottomland plots scattered throughout the stands, indicating that relatively large disturbances were important in these poorly-drained areas. In contrast, there was little synchrony among trees from well-drained upland plots, except in a single instance where selective cutting of Quercus trees opened the canopy. Thus, the canopy of upland site was likely subjected only to small disturbances resulting from the death of one or a few trees. At the whole of forest level, there was evidence of episodic recruitment of canopy trees in both forests. Establishment of Fraxinus spp. and Quercus spp. were particularly episodic, and few Fraxinus or Quercus trees alive today established during the last century. These data suggest that large disturbances have affected canopy dynamics of both upland and bottomland areas prior to 1900 and in bottomland forests through this century. In contrast, disturbances in upland areas during this century have been restricted to small, treefall-generated canopy gaps.     

Decreasing the distance between the two conserved sequence elements of histone pre-messenger RNA interferes with 3' processing in vitro.

Histone mRNA 3' end formation requires the presence of two cis-acting conserved sequence elements: a stem-loop structure upstream from the site of cleavage and a purine-rich region downstream from the site of cleavage called the histone downstream element (HDE). Possible interactions between these two elements and their respective binding factors were investigated by a series of deletions (1-7 nt) in the region between the two. The efficiency of processing decreased as the stem-loop and the HDE were moved closer together. In contrast with the documented ability of the U7 snRNP to direct cleavage at a fixed distance from the HDE in insertion mutants (Scharl & Steitz, 1994), all deletion substrates for which processing was observed were cleaved at or 1-nt upstream from the wild-type site. The reason for the inability of the system to cleave closer to the stem-loop remains unclear, but the removal of stem-loop binding protein(s) (SLBP) did not activate upstream cleavage events. Thus, although the processing machinery measures the distance between the cleavage site and the HDE of mammalian histone pre-mRNAs, there is a barrier limiting how far upstream cleavage can occur. These data allow a reevaluation of the sites of 3' end processing in known histone pre-mRNAs.