Simultaneous saccharification and fermentation of cellulose: effect of ethanol on enzymatic saccharification of cellulose

It was confirmed that simultaneous saccharification and fermentation are effective for accelerating enzymatic saccharification of cellulose. In this work, the effects of ethanol on the saccharification of tissue paper by Trichoderma cellulase (Meicelase CEPB) have been investigated. The following results were obtained. (1) Saccharification was inhibited by at least 0.2M ethanol. (2) Less than 4M ethanol did not affect the enzymatic activities of beta-glucosidase and endoglucanase (C(x)) at all. The thermal stability of endoglucanase was not also varied by ethanol. (3) It is suggested that ethanol depresses the adsorption of exoglucanase on cellulose. (4) The rate expression of saccharification of cellulose in the presense of ethanol is proposed. (5) The inhibititory effect of ethanol was found to become more significant in the later stages of the reaction than just the initial stage.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Distribution of Glycinebetaine in Old and Young Leaf Blades of Salt-Stressed Barley Plants

In barley plants exposed to stepwise salt-stress (up to 200mM NaCl), sodium and chloride ions accumulated preferentiallyin old rather than in young leaf blades. Furthermore, the levelof glycinebetaine in young leaf blades was approximately threetimes that in old leaf blades. ³Present address: Department of Biochemistry, Faculty of Science,Kasesart University, Bangkok, 10900 Thailand     

Synechococcus sp. PCC7942 Transformed with Escherichia coli bet Genes Produces Glycine Betaine from Choline and Acquires Resistance to Salt Stress

Synechococcus sp. PCC7942, a fresh water cyanobacterium, was transformed by a shuttle plasmid that contains a 9-kb fragment encoding the Escherichia coli bet gene cluster, i.e. betA (choline dehydrogenase), betB (betaine aldehyde dehydrogenase), betI (a putative regulatory protein), and betT (the choline transport system). The expression of these genes was demonstrated in the cyanobacterial cells (bet-containing cells) by northern blot analysis, as well as by the detection of glycine betaine by 1H nuclear magnetic resonance in cells supplemented with choline. Endogenous choline was not detected in either control or bet-containing cells. Both control and bet-containing cyanobacterial cells were found to import choline in an energy-dependent process, although this import was restricted only to bet-containing cells in conditions of salt stress. Glycine betaine was found to accumulate to a concentration of 45 mM in bet-containing cyanobacterial cells, and this resulted in a stabilization of the photosynthetic activities of photosystems I and II, higher phycobilisome contents, and general protective effects against salt stress when compared to control cells. The growth of bet-containing cells was much faster in the presence of 0.375 M NaCl than that of control cells, indicating that the transformant acquired resistance to salt stress.     

Refractoriness at peripheral and pituitary receptors in general and pituitary types of thyroid hormone resistance.

Patients with the general type (patient #1 and #2) and the selective pituitary type (#3) of thyroid hormone refractoriness (TR) were studied to clarify defects at peripheral and pituitary receptors. Products of T3 and TSH (n = 63) were calculated when T3 was above the normal limit (T3 > 1.8 ng/ml, 2.8 nmol/l) as one of the indices of pituitary resistance. Means of T3 (ng/ml) x TSH (mU/l) of patient #1 (mean; 40.8), #2 (15.0) and #3 (8.6) were significantly greater than patients with Graves' disease (2.1), suggesting pituitary refractoriness in the 3 patients. The products of patient #1 and #2 were also significantly larger than patient #3, demonstrating that the pituitary insensitivity in the latter (#3) was less than the former patients. Means of serum cholesterol in patients #1 and #2 were higher than patient #3 and patients with Graves' disease. Products of T3 (ng/ml) and cholesterol (mg/ml) (n = 28) in the patient #1 (541.9) and #2 (461.0) were significantly greater than the patient #3 (292.8) and the patients with Graves' disease (275.3). The results demonstrate generalized refractoriness in the patient #1 and #2 and selective pituitary resistance in the patient #3. It is suggested that our patient with the pituitary type (#3) had less severely affected receptors at the pituitary than our two patients with the general type. These results are consistent with the previous hypothesis that the pituitary type of TR is a partial form of this disease.     

Glutaraldehyde pretreatment blocks temperature-induced high-affinity [3H]tryptamine binding

The effect of glutaraldehyde (and Azure A) on temperature-sensitive high-affinity [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. In the 0.01-0.1% concentration range, the glutaraldehyde pretreatment preferentially inhibited only the above-mentioned portion of the binding, whereas the posttreatment of this reagent had no effect. On the other hand, in cases of pretreatment or posttreatment, a concentration of glutaraldehyde as high as 0.1% was inactive on the basal [3H]ligand binding capacity of the membranes (i.e., temperature-independent binding). Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with glutaraldehyde (0.05%) conformed to a straight line, as did a similar plot of temperature-independent binding. And, it was interesting to find that the binding parameters (KD and Bmax values) of both samples corresponded closely to each other. On the contrary, in all concentrations, Azure A affected nonspecifically both the temperature-dependent and the independent [3H]tryptamine binding to the same degree, regardless of whether or not there was pretreatment or posttreatment. All these observations clearly demonstrate that an appropriate concentration (0.01-0.1%) of glutaraldehyde pretreatment specifically blocks the temperature-induced allosteric modifications of high-affinity [3H]tryptamine binding sites.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

A novel inhibitor of ceramide trafficking from the endoplasmic reticulum to the site of sphingomyelin synthesis

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3R)-HPA-12, which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3R)-HPA-12 did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3R)-HPA-12 inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3R)-HPA-12 inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.     

Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.