Simultaneous saccharification and fermentation of cellulose: Effect of ethanol and cellulases on particular stages

The effects of ethanol and Trichoderma reesei cellulase on the saccharification and fermentation processes as well as the tolerance of the cellulase complex for ethanol have been investigated. The studies were conducted with respect to their usefulness in the process of simulataneous saccharification and fermentation of cellulose to ethanol. The following results were obtained. (1) Fermentative activity of Kluyveromyces fragilis yeasts was gradually depressed with increasing intial ethanol concentrations and temperature of fermentation between 35–46°C. (2) Crude cellulase preparation introduced to the culture broth to a final enzyme activity of 0.5 to 2.0 FPU/ml had not distinct effect on the biomass production, ethanol yield, and glucose uptake by yeasts in 48 h fermentation at 43°C. On the other hand, only a negligible decrease in the cellulase complex activity was observed during fermentation process. (3) Saccharification of wheat straw was inhibited by at least 1% w/v ethanol. (4) The enzymes of the cellulase system showed a high stability to exposure to ethanol for 48 h at 43°C.     

Novel inhibitors of enkephalin-degrading enzymes. II: N5'-substituted-4-thioxohydantoic acids as aminopeptidase inhibitors

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.     

The mutagenic potential of high flash aromatic naphtha

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent — high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow.There was no evidence that high-flash aromatic naphtha was either a gene or chromosomal mutagen. Thus it is unlikely to be a genotoxic carcinogen.Abbreviations Brdu 5-Bromo-2-deoxyuridine - C9 Aromatic species with 9 carbons (i.e., ethyl toluene and trimethyl benzene isomers) - CE Cloning efficiency - CHO Chinese hamster embryo - CP Cyclophosphamide - DMSO Dimethyl sulfoxide - HGPRT Hypoxanthine-guanine phosphoribosyl transferase - HVAC Heating, Ventilation, Air Conditioning - 3MC 3 Methylcholanthrene - MMC Mitomycin C - MMS Methyl methanesulfonate - S9 S9 Mammalian microsomal enzyme activation mixture - SCE Sister chromatid exchange     

Cloning and characterization of a tandemly repeated DNA sequence in the crane family (Gruidae)

A tandemly repeated DNA sequence possessing a unique PstI site has been characterized in several species of the crane family. The "Pst family" comprises at least 8800 monomer units 187 base pairs (bp) in length and constitutes 0.14% of the genome of the sarus crane (Grus antigone). The array is located in the centromeric heterochromatin of chromosome 2 in the two species where in situ hybridizations of a cloned monomer to metaphase chromosome spreads were carried out. DNA sequence comparisons between five monomer units from G. antigone revealed a high degree of homology between four of the individual repeats, while the fifth was somewhat divergent. The G + C content deduced from the DNA sequence makes it likely that the Pst family constitutes part of a density satellite seen in profiles of crane DNA centrifuged to equilibrium in CsCl. The common occurrence of tandem arrays such as the Pst family, with repeat lengths close to 200 bp, leads us to an hypothesis implicating nucleosomes in the evolution of such families.     

The human glucocerebrosidase gene and pseudogene: structure and evolution

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.     

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of hyperlipidemic serum on lipid peroxidation, synthesis of prostacyclin and thromboxane by cultured endothelial cells: protective effect of antioxidants

An increased lipid peroxides and a decreased production of prostacyclin have been shown in advanced atherosclerotic lesions and plasma. Our purpose was to determine whether the similar findings could be observed in cultured endothelial cells, and whether antioxidants could protect the cell against peroxide injury. In these experiments we have used bovine aortic endothelial cells in culture to address the issue of hyperlipidemia-induced arterial damage. Results of the present study showed that different concentration of hyperlipidemic sera from atherogenic rabbits induced a time- and dose-dependent alteration in the production of prostacyclin and levels of lipid peroxides in endothelial cells. Endothelial cells incubated with hyperlipidemic serum increased prostacyclin generation significantly during the initial stages and then continuously decreased. When endothelial cells were incubated for 36 h, TXA2 generation was also impaired and at the same time the cellular lipid peroxides content increased. There was a positive correlation between the concentration of hyperlipidemic serum and lipid peroxides and an inverse correlation with prostacyclin synthesis. The medium supplemented with antioxidant selenium or vitamin E showed a significant decrease in lipid peroxides and an increase in prostacyclin synthesis. These results suggest that both hyperlipidemic serum and lipid peroxides injury endothelial cells and inactivate prostacyclin synthetase, resulting in a decrease of prostacyclin production, while antioxidants have a protective effect. We conclude that the increase in lipid peroxides in association with hyperlipidemia results in alteration of prostacyclin synthesis that may play an important role in the pathogenesis of atherosclerosis.     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Effect of Nitrogen Fertilization and Growth Suppression on Pitch Canker Development on Loblolly Pine Seedlings

One-yr-old loblolly pine seedlings of two half-sib families, grown in sand, were fertilized three times per week with nutrient solution containing 20 μg/ml (low) or 80 μg/ml (high) nitrogen. Nitrogen concentration in the nutrient solution was either constant throughout the experiment, or interehanged after the inoculation of stems or shoots with Fusarium subglutinans, 55 days after initiation of fertilization. Growth was suppressed by a weekly excision of shoots branching from the stem apex. Either high nitrogen nutrition or shoot excision generally enhanced canker elongation on stem inoculated plants; the combination of both was extremely conducive for disease development. With intact plants of family 8–68, interchange of pre-inoculation low nitrogen nutrition with high nitrogen after inoculation enhanced canker elongation and rate of wilt. Nitrogen content varied in wood, bark and needles, as well as with time intervals, but was consistently in accordance with nitrogen level in the nutrient solution. In shoot excised plants, nitrogen content was higher than in the respective treatment without shoot excision. The higher nitrogen nutrient accelerated disease development on inoculated shoots, compared to low nitrogen, on both pine families. With respective treatments, stem cankers were larger and rates of shoots exhibiting lesions or wilt were higher on plants of family 8–68 than on 8–61. It is postulated that the disease enhancing effect associated with higher nitrogen content in stem tissues results from an increased nitrogen availability to the pathogen.     

Estrogens induce malpighian metaplasia of the endocervical junction in female rats immunohistochemistry study

Immature female Wistar rats were treated with 1 mg of estradiol benzoate for 6 days. The injections were started on the 20th day of age; the animals were autopsied every 3 days after the last injection until the age of 45 days. Islets of hyperplastic cells and metaplasia area were seen in the endocervix in the majority of the animals autopsied. We have the expression of cytokeratin polypeptides in reserve cells, in areas exhibiting reserve cell hyperplasia and squamous metaplasia, using a panel of monoclonal cytokeratin antibodies. The reserve cells were positive for antibodies directed against stratified squamous epithelia, type cytokeratins No. 5, 13 and 17. In addition, hyperplastic cells revealed the presence of cytokeratins No. 7, 8, 18 and 19, specific for simple epithelia, but in a variable manner. The Squamous metaplasia cells exhibited cytokeratins No. 13, 18 and 19, but only weakly reactive. Our observations indicate that estrogen-induced endocervix metaplasia results from a transformation of reserve cells towards an epidermoid type epithelium. Hyperplasia would be the intermediate step in the mechanism of induced cervical metaplasia. This transformation is accompanied by the loss of cytoplasmic keratin proteins and the acquisition of new high molecular weight keratin proteins, specific for stratified squamous epithelia. The basal or reserve cells of the cervix can proliferate to produce regions of squamous cell metaplasia. It appears to be a direct effect of estrogen stimulation. Immunohistochemical staining for different molecular weight keratin proteins may be helpful in the evaluation of reserve cell differentiation.