The bioconversion of wood hydrolyzates to butanol and butanediol

Steam-exploded aspenwood chips were acid hydrolysed to their component sugars. Near theoretical solvent yields were achieved in both the acetone-butanol-ethanol (ABE) fermentation and 2,3-butanediol fermentation of these liberated sugars. When Clostridium acetobutylicum was grown on wood hydrolysates, final butanol yields of 9.0 g/L (0.26 g of butanol per g of sugar consumed) were obtained. When Klebsiella pneumoniae was grown on the wood hydrolysates, final butanediol concentrations exceeded 20 g/L, resulting in a bioconversion efficiency approaching 0.5 g of butanediol per g of sugar utilised.     

Suitability of aspenwood biologically delignified with Pheblia tremellosus for fermentation to ethanol or butanediol

Summary Enzymatic conversion of lignocellulosic material to fuels and chemicals depends on a initial pretreatment to render the cellulose more susceptible to enzymatic attack. Biological delignification of aspenwood with the fungus Phlebia tremellosus was compared to steaming as a pretreatment method.The biologically delignified aspenwood (BDA) had a high pentosan content and did not contain inhibitors of enzymatic hydrolysis or subsequent fermentation. In contrast, the steamed aspenwood required a water extraction step to remove the inhibitory material and this step also removed most of the pentosan. The yield of treated material was 90% from biological delignification and 70% from steaming.The cellulose in the BDA was less accessible to the cellulase enzymes than the steamed aspenwood. Combined hydrolysis and fermentation with Saccharomyces cerevisiae gave a lower yield of ethanol from BDA than from the steamed aspenwood, but the yields based on the weight of substrate before pretreatment were comparable. Combined hydrolysis and fermentation with Klebsiella pneumoniae gave higher yields of butanediol from BDA than from steamed aspenwood, because Klebsiella can ferment the xylose which was present in the biologically treated aspenwood. Trichoderma harzianum produced lower levels of cellulase enzymes when grown on BDA than when grown on steamed aspenwood and this was related to the xylan found in the biologically treated material.Abbreviations BDA biologically delignified aspenwood - SEA-WI steam-exploded, water-extracted aspenwood - AI-SEA-WI acid-impregnated, steam-exploded, water-extracted aspenwood - CHF combined hydrolysis and fermentation - FP filter paper     

Estimation of fungal growth on lignocellulosic substrates using an enzyme-linked immunosorbent assay

Summary The feasibility of using an enzyme-linked immunosorbent assay (ELISA) for quantifying the growth ofThielavia terrestris on cellulosic substrates was studied. WhenT. terrestris was grown, using glucose as the substrate, the ELISA readings correlated well with the mycelial dry weight values. When lignocellulosic substrates were used, the ELISA appeared to slightly overestimate fungal growth. The ELISA values indicated that the fungi grew more slowly on steam-treated aspenwood than on commercial sources of cellulose (Avicel).     

Butanol production from cellulosic substrates by sequential co-culture ofClostridium thermocellum andC.acetobutylicum

A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to fuels and chemicals. Growth ofClostridium acetobutylicum on solka floc (or a mixture of solka floc and aspenwood xylan), in co-culture withC.thermocellum, resulted in the efficient utilization of all the hydrolysis products derived from the lignocellulosic substrates. This co-culture approach resulted in a 1.7–2.6 fold increase in the total fermentation products formed. The majority of the fermentation products were acids and not solvents, however the solventogenesis step could be induced by the addition of butyric acid to the fermentation medium.     

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum

Summary A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.     

Identification of Mycoplasma Binding Proteins Utilizing A 100 Kilodalton Lung Fibroblast Receptor

Abstract

A 100 kilodalton glycoprotein receptor for Mycoplasma pneumoniae has been isolated from MRC-5 human lung fibroblasts. This receptor, as well as antireceptor serum, were both capable of inhibiting the attachment of ¹⁴C-labelled M. pneumoniae to MRC-5 fibroblasts. The receptor was also capable of inhibiting the attachment of C-labelled M. gallisepticum and M. genitalium, but not M. pulmonis, to MRC-5 fibroblasts. This indicates that a common sequence may exist in these binding proteins of M. pneumoniae, M. genitalium, and M. gallisepticum, This receptor and anti-receptor serum were utilized to probe M. pneumoniae, M. genitalium, and M. gallisepticum for their corresponding binding proteins. A 32 kilodalton protein in M. pneumoniae, a 90 kilodalton protein in M. genitalium and a 139 kilodalton protein in M. gallisepticum were recognized.     

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site

Human blood coagulation factor X has two N-linked oligosaccharides at Asn³⁹ and Asn⁴⁹ residues and two O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn³⁹ and Asn⁴⁹ residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac-26 residue. In addition, the structures of O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues were suggested to be disialylated Gal/3GalNAc sequences by their component analyses.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose - HPLC high-performance liquid chromatography - NDV Newcastle disease virus - Neu5Ac 5-N-acetylneuraminic acid - ODS octadecylsilyl - PA pyridylamino - RVV-X Russell's viper venom factor X activator - TBS Tris-buffered saline - XAP factor X activation peptide.     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

The antagonism by anti-inflammatory analgesics of prostaglandin F2α-induced contractions of human and rabbit myometrium in vitro

The anti-inflammatory analgesic drugs, aspirin, indomethacin, phenylbutazone, mefenamic acid, ibuprofen and flurbiprofen are shown to inhibit in a dose-dependent manner the force of contraction of isolated human pregnant myometrial strips which have been stimulated to contract by adding prostaglandin (PG) F_2α to the tissue bath. These drugs and also flufenamic acid and salicin show a similar antagonism of the action of PGF_2α with isolated rabbit non-pregnant myometrium. The ratio of the inhibitory concentration in vitro to the maximum plasma level after a normal dose in vivo suggests that phenylbutazone and possibly ibuprofen may be capable of inhibiting human uterine contractions in vivo. Patients who were treated with aspirin during induction of abortion using PGF_2α during the second trimester of pregnancy showed no significant change in the induction-abortion interval compared with patients not taking aspirin.     

A comparison of the substrate specificities of endo-β-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae

The substrate specificities of the endo-β-N-acetylglucosaminidases from $\text{Diplococcus pneumoniae}$ and $\text{Streptomyces griseus}$ were compared and found to differ considerably. The enzyme from $\text{D. pneumoniae}$ released Asn-GlcNAc-Fuc-containing glycopeptides from exoglycosidase-treated acidic IgM glycopeptides but was limited in its capacity to hydrolyze ovalbumin glycopeptides larger than Asn(GlcNAc)₂(Man)₅. In contrast, the enzyme from $\text{S. griseus}$ hydrolyzed this and larger neutral oligosaccharides but could not hydrolyze the above fucose-containing IgM glycopeptides. Removal of the fucose residue, however, converted the latter to an active substrate for the $\text{S. griseus}$ enzyme, thus broadening its substrate range to encompass most of those substrates hydrolyzed by the $\text{D. pneumoniae}$ endoglycosidase.     

Synthesis and Biological Evaluation of some Acyclic Nucleoside Cyclic Phosphoramidate Derivatives

Abstract

The acyclic nucleosides 2 were treated with 2-chloro-3-methyl-1-oxa-3-aza-2-phosphacyclopentane (3) in the presence of diisopropylethylamine to give the corresponding phosphoramidite derivatives (4). The phosphoramidite intermediates (4) were oxidized with m-chloroperbenzoic acid to the phosphoramidate derivatives (5). Treatment of 5a,b with ZnBr₂ in CH₃NO₂ gave the corresponding acyclic nucleoside cyclic phosphoramidates (6a,b). Attempts to desilylation of 5c by tetrabutylammonium fluoride (TBAF) resulted in opening of the phosphoramidate ring. The newly synthesized compounds were evaluated for antiviral and antitumor cell activity.     

Antibacterial,antioxidant and hypoglycaemic effects of Thymus capitatus (L.) Hoffmanns. et Link leaves' fractions

Abstract

The aim of this study was to examine the bioactivity of the methanol fraction (MF) and n-hexane fraction (HF) of Thymus capitatus leaves in relation to their constituents analysed by gas chromatography and gas chromatography-mass spectrometry. The effects of T. capitatus on the growth of pathogenic bacteria associated with respiratory diseases (13 gram-positive and 4 gram-negative) were determined using a microdilution method. The MF was particularly effective on Streptococcus pneumoniae and Moraxella catarrhalis. The antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), ferric-reducing antioxidant power and β-carotene bleaching assays. A strong activity using β-carotene bleaching test was observed with the MF (IC₅₀ of 0.7?μg/mL after 30?min of incubation). In the hypoglycaemic test, a selective α-amylase inhibitory activity was detected with the HF begging the most active (IC₅₀ of 422.5?μg/mL). T. capitatus may represent a source of natural bioactive compounds.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O₂ tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O₂ concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

Direct and maternal genetic effects on body weight maturing patterns in mice

Summary Direct and maternal genetic effects were evaluated for maturing patterns of body weight in mice using a crossfostering design. Crossfostering was performed in one group using dams from populations selected for rapid growth rate (M16 and H₆) and their reciprocal F₁. crosses. A second crossfostering group consisted of dams from the respective control populations (ICR and C₂) and their reciprocal F₁. 's. Population differences were partitioned into direct and maternal effects due to genetic origin, correlated selection responses, heterosis and cytoplasmic or sex-linked effects. Degree of maturity was calculated at birth, 12, 21, 31 and 42 days of age by dividing body weight at each age by 63-day weight. Absolute and relative maturing rates were calculated in adjacent age intervals between birth and 63 days. Genetic origin effects (ICR vs. C₂; M16 vs. H₆) were significant for many maturity traits, with average direct being more important than average maternal genetic effects. In general, correlated responses to selection for maturity traits were larger in the M16 population (M16 vs. ICR) than in the H₆ population (H₆ vs. C₂) and correlated responses in average direct effects were larger than average maternal effects. Positive correlated responses in average direct effects were found for relative maturing rates at all ages and for absolute maturing rates from 31 to 63 days. Apparent correlated responses in degree of maturity were negative for M16 and H₆. However, further analysis suggested that the correlated response for degree of maturity in H₆ may be positive at later ages and negative at earlier ages. Direct and maternal heterosis for degree of maturity was positive in the selected and control crosses. Absolute and relative maturing rates showed positive heterosis initially, followed by negative heterosis. Reciprocal differences due to the cytoplasm or sex-linkage were not important for patterns of maturity.Paper No. 5244 the Journal Series of the North Carolina Agricultural Experiment Station, Ealeigh, Animal Research Institute Contribution No. 683 and Agricultural University at Wageningen Contribution No. 654–490–12On leave from the Animal Research Institute, Agriculture Canada at Ottawa, OntarioOn leave from the Department of Animal Husbandry, Agricultural University at Wagenitgen, the Netherlands     

In vitro plant regeneration from leaf explants of C3 and C3-C4-intermediate Flaveria species

Summary A procedure is described for the invitro regeneration of whole plants of Flaveria cronquistii (C₃ species) F. pubescens and F. chloraefolia (both C₃-C₄ intermediate species) using different concentrations of 6-benzylaminopurine and alpha-napnthalenic acid.Abbreviations BAP 6-benzylaminopurine - NAA alpha-naphthalenic acid - MS medium Murashige-Skoog-medium     

Total synthesis of PGC2 methyl ester. Comparison with enzymatically produced material

The synthesis of PGC₂ methyl ester is described. Comparison of the synthetic material with the methyl ester prepared from natural PGC₂ showed the two to be identical, thus confirming the structure assignment. The physical and biological data of PGC₂ methyl ester are presented.Horton and Jones have recently shown that PGA₁ (Ic) and PGA₂ (Ia) are deactivated on incubation with cats blood (1,2). Jones has proposed that this deactivation involves the enzymic conversion of PGA (I) to an 11,12-double bond isomer (PGC, II) which is subsequently isomerized by base to the inactive PGB (III) (3). Structure assignment of the PGC's in the earlier study were based on chromatographic mobilities and uv and mass spectrometric properties. We report here a total synthesis of PGC₂ methyl ester (IIb). Comparison of the biological and physical properties of this material with those of diazomethane-esterified natural material confirms the earlier structure assignment for PGC₂ (IIa).     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.