Progression of Epiphytic Microflora in Wheat and Alfalfa Silages as Observed by Scanning Electron Microscopy

Wheat and alfalfa silages were examined by scanning electron microscopy and standard methods of microbial enumeration. Epiphytic microflora were present at levels of 10⁶ to 10⁸/g in the fresh-cut plants. This flora was initially observed microscopically primarily on the surfaces. After 4 days of fermentation, lactic acid bacteria were observed on the surface in high concentrations near open stomata and throughout the interior mesophyll air sac spaces. At 4 days, populations on interior surfaces were restricted to the exterior surfaces of the air sacs. After 8 days the mesophyllic cells showed marked deterioration, and bacteria were observed on their inner surfaces. At 32 days, the end of the fermentation, vascular bundles and epidermal cells remained intact whereas stomata and mesophyllic cells were collapsed and often contained microorganisms. It is concluded that the interior of the leaves offers substantial nutritional and environmental advantages to epiphytic flora and is an important if not major deterioration site in fermented products. Since little deterioration of exterior surfaces was observed, these sites may play a minor role in supplying nutrients for microbial growth.     

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.     

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.     

Actions of LH/hCG on accumulation and catabolism of progestins in cultured rat granulosa cells

Immature hypophysectomized rats were treated with estradiol-17 beta and follicle-stimulating hormone. Granulosa cells were isolated and incubated for 24 h with or without varying doses of ovine luteinizing hormone (NIMADD-oLH-24) or human chorionic gonadotropin (NIADDK CR 125) and accumulations of progesterone and 20 alpha-hydroxy-4-pregnen-3-one were determined. The cells were reincubated for 3 h with [4-14C]progesterone (0.5 nmol/mL) and the radiolabelled metabolites were separated and quantified. Both LH (0.04-1.0 ug/mL) and hCG (0.04-1.0 ug/mL) enhanced the accumulation of endogenous progesterone (by up to 300 and 150%, respectively) and 20 alpha-hydroxy-4-pregnen-3-one (by up to 90 and 85%, respectively) producing dose-dependent increases of the ratio of progesterone to 20 alpha-hydroxy-4-pregnen-3-one (by up to 125 and 70%, respectively). Studies of the metabolism of [1-14C] progesterone have demonstrated that both LH and hCG led to a dose-dependent decrease of the utilization of radiolabelled progesterone (down to 64 and 70%, respectively, of the control value). This effect was associated with an LH- and hCG-dependent inhibition of 20 alpha-hydroxysteroid dehydrogenase activity (down to 60 and 70%, respectively, of the control value) but had no significant effect on 5 alpha-reductase. The present results indicate that LH and hCG stimulate accumulation of progesterone at least in part by decreasing the 20 alpha-hydroxysteroid dehydrogenase activity.     

Reconciling nutritional geometry with classical dietary restriction: Effects of nutrient intake,not calories,on survival and reproduction

Dietary restriction (DR) is one of the main experimental paradigms to investigate the mechanisms that determine lifespan and aging. Yet, the exact nutritional parameters responsible for DR remain unclear. Recently, the advent of the geometric framework of nutrition (GF) has refocussed interest from calories to dietary macronutrients. However, GF experiments focus on invertebrates, with the importance of macronutrients in vertebrates still widely debated. This has led to the suggestion of a fundamental difference in the mode of action of DR between vertebrates and invertebrates, questioning the suggestion of an evolutionarily conserved mechanism. The use of dietary dilution rather than restriction in GF studies makes comparison with traditional DR studies difficult. Here, using a novel nonmodel vertebrate system (the stickleback fish, Gasterosteus aculeatus), we test the effect of macronutrient versus calorie intake on key fitness‐related traits, both using the GF and avoiding dietary dilution. We find that the intake of macronutrients rather than calories determines both mortality risk and reproduction. Male mortality risk was lowest on intermediate lipid intakes, and female risk was generally reduced by low protein intakes. The effect of macronutrient intake on reproduction was similar between the sexes, with high protein intakes maximizing reproduction. Our results provide, to our knowledge, the first evidence that macronutrient, not caloric, intake predicts changes in mortality and reproduction in the absence of dietary dilution. This supports the suggestion of evolutionary conservation in the effect of diet on lifespan, but via variation in macronutrient intake rather than calories.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH

Hydrogen was produced by an ethanol-acetate fermentation at pH of 5.0 +/- 0.2 and HRT of 3 days. The yield of hydrogen was 100-200 ml g Glu(-1) with a hydrogen content of 25-40%. This fluctuation in the hydrogen yield was attributed to the formation of propionate and the activity of hydrogen utilizing methanogens. The change in the operational pH for the inhibition of this methanogenic activity induced a change in the main fermentation pathway. In this study, the main products were butyrate, ethanol and propionate, in the pH ranges 4.0-4.5, 4.5-5.0 and 5.0-6.0, respectively. However, the activity of all the microorganisms was inhibited below pH 4.0. Therefore, pH 4.0 was regarded as the operational limit for the anaerobic bio-hydrogen production process. These results indicate that the pH plays an important role in determining the type of anaerobic fermentation pathway in anaerobic bio-hydrogen processes.     

Possible Role of the Glycogen Synthase Kinase-3 Signaling Pathway in Trimethyltin-Induced Hippocampal Neurodegeneration in Mice

Trimethyltin (TMT) is an organotin compound with potent neurotoxic effects characterized by neuronal destruction in selective regions, including the hippocampus. Glycogen synthase kinase-3 (GSK-3) regulates many cellular processes, and is implicated in several neurodegenerative disorders. In this study, we evaluated the therapeutic effect of lithium, a selective GSK-3 inhibitor, on the hippocampus of adult C57BL/6 mice with TMT treatment (2.6 mg/kg, intraperitoneal [i.p.]) and on cultured hippocampal neurons (12 days in vitro) with TMT treatment (5 µM). Lithium (50 mg/kg, i.p., 0 and 24 h after TMT injection) significantly attenuated TMT-induced hippocampal cell degeneration, seizure, and memory deficits in mice. In cultured hippocampal neurons, lithium treatment (0–10 mM; 1 h before TMT application) significantly reduced TMT-induced cytotoxicity in a dose-dependent manner. Additionally, the dynamic changes in GSK-3/β-catenin signaling were observed in the mouse hippocampus and cultured hippocampal neurons after TMT treatment with or without lithium. Therefore, lithium inhibited the detrimental effects of TMT on the hippocampal neurons in vivo and in vitro, suggesting involvement of the GSK-3/β-catenin signaling pathway in TMT-induced hippocampal cell degeneration and dysfunction.