Simultaneous bioconversion of cellulose and hemicellulose to ethanol

Lignocellulosic materials containing cellulose, hemicellulose, and lignin as their main constituents are the most abundant renewable organic resource present on Earth. The conversion of both cellulose and hemicellulose for production of fuel ethanol is being studied intensively with a view to develop a technically and economically viable bioprocess. The fermentation of glucose, the main constituent of cellulose hydrolyzate, to ethanol can be carried out efficiently. On the other hand, although bioconversion of xylose, the main pentose sugar obtained on hydrolysis of hemicellulose, to ethanol presents a biochemical challenge, especially if it is present along with glucose, it needs to be fermented to make the biomass-to-ethanol process economical. A lot of attention therefore has been focussed on the utilization of both glucose and xylose to ethanol. Accordingly, while describing the advancements that have taken place to get xylose converted efficiently to ethanol by xylose-fermenting organisms, the review deals mainly with the strategies that have been put forward for bioconversion of both the sugars to achieve high ethanol concentration, yield, and productivity. The approaches, which include the use of (1) xylose-fermenting yeasts alone, (2) xylose isomerase enzyme as well as yeast, (3) immobilized enzymes and cells, and (4) sequential fermentation and co-culture process are described with respect to their underlying concepts and major limitations. Genetic improvements in the cultures have been made either to enlarge the range of substrate utilization or to channel metabolic intermediates specifically toward ethanol. These contributions represent real significant advancements in the field and have also been adequately dealt with from the point of view of their impact on utilization of both cellulose and hemicellulose sugars to ethanol.     

Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352

A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin.     

Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli

Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E. coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.     

Genetic analysis of the corticosterone response to ethanol in BXD recombinant inbred mice.

The genetic control over the corticosterone response to ethanol (EtOH) and its possible relationship to other EtOH-related traits was examined using BXD recombinant inbred (RI) strains derived from an F2 cross of C57BL/6J (B6) and DBA/2J (D2) progenitor strains. Quantitative trait locus (QTL) analysis of corticosterone levels 1 hr following EtOH suggested the influence of a single major gene on this trait. Two loci were predicted to account for 47% of the genetic variance in plasma corticosterone levels 6 hr following EtOH, whereas 3 loci were predicted to account for 78% of the genetic variance in corticosterone levels 7 hrs following EtOH. Markers associated with corticosterone levels 7 hrs following EtOH and corrected corticosterone levels 6 hrs post-EtOH overlapped with ones found to influence acute and chronic EtOH withdrawal severity, suggesting some degree of common genetic determination between these traits. Overall these results indicate that gene action significantly influences stress responsiveness and suggest possible chromosomal locations of these genes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Epidemiologic and actual importance of entero-hemorrhagic E. coli in Northern Bavaria (1996)

OBJECTIVES: To compare peripheral type 1 (T1) and type 2 (T2) T cell activities in rheumatoid arthritis (RA) patients with that found for osteoarthritic (OA) patients and healthy controls and to correlate peripheral T1/T2 cell activity in RA with parameters of the disease. METHODS: Peripheral blood mononuclear cells were isolated from patients with RA (n = 66), OA (n = 19), and healthy controls (n = 15). Primary T cell activity in these mononuclear cells was enhanced by means of anti-CD3/anti-CD28, which mimicks stimulation of T cells by activation of the T cell receptor and a major co-stimulatory signal. Interferon gamma (IFN gamma) production and interleukin 4 (IL4) production in the three groups were quantified as measures of T1 and T2 cell activity, respectively, and compared. Serum tumour necrosis factor alpha (TNF alpha), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and joint destruction assessed radiographically of RA patients were determined as parameters of disease activity and correlated with T1/T2 cell activity. RESULTS: Peripheral T cells from RA patients produced significantly less IFN gamma and more IL4 than T cells from both age and sex matched OA patients and healthy controls. Moreover, in RA patients both a decrease in IFN gamma and an increase in IL4 production correlated with an increase in serum TNF alpha, ESR, CRP, and joint destruction. CONCLUSIONS: These results suggest a role for differential T cell activity in RA. In view of the intra-articular T1 cell predominance the results might be explained by selective T1 cell migration into the joint or peripheral suppression of T1 cell activity.     

Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.     

A recombinant autoantigen U 1 RNP 70,000: expression in E. coli. and its serodiagnostic application

BACKGROUND AND OBJECTIVES: Violence directed at general practitioners (GPs) is on the increase in urban areas of Ireland. However, little information has been available on the incidence, effects, or precipitants of this violence. This study documents the frequency of the problem in Ireland's capital city, Dublin. METHODS: A survey was sent to all 634 GPs in the Eastern Health Board region of Ireland. The survey asked each GP to report an incident of violence or aggression using standardized definitions and reporting forms. The survey also requested that participants provide information on their practice and its staff and setting. RESULTS: We collected information from 622 of 634 doctors (98%). The overall incidence of violence or aggression in the sample was 21%. Of the 131 incidents reported, only 7% resulted in injury to the doctor; the remainder all involved verbal abuse and/or threats. The most frequent assailant characteristics identified were alcohol or opiate abuse. CONCLUSIONS: Violence and aggression against GPs are common. Several interventions are discussed.     

Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis

A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized. Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism. Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene. The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate. The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions. Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site. The divalent cations Mg2+, Zn2+ and Mn2+ were found to be inhibitors of enzymatic activity, whereas Ca2+ had no effect. Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity. A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.     

Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity]

Numerous data strongly suggest the involvement of cytokines and the matrix metalloproteinase collagenase (MMP-1) in the pathogenesis of periodontitis. Recently, we have demonstrated that, upon culturing under the influence of IL-1 alpha + EGF, a large amount of inactive procollagenase (MMP-1) is stored in the extracellular matrix of periosteal tissue. We now show that this endogenous reservoir of proenzyme can be operative after activation with plasmin and is able to induce a rapid and almost complete breakdown of the collagenous extracellular matrix. The level of collagen degradation following activation showed a strong correlation with the amount of proenzyme that was incorporated in the tissue. The highest level of degradation (70% of the total amount of collagenous proteins) was found with the IL-1 alpha + EGF-treated explants, followed by those treated with IL-1 alpha alone (35%). Explants cultured with EGF or in the absence of cytokines, containing only small amounts of procollagenase, showed little collagen breakdown following plasmin activation (7%). Inhibition of metalloproteinases by EDTA, or blockage of plasmin by PMSF, prevented the degradation in all explants irrespective of the amount of proenzyme present in the tissue. Our findings demonstrate that endogenous proenzyme stored in a native connective tissue matrix can be activated at a later time interval which results in a massive breakdown of the tissue. This study shows a possible pathway of collagenase-induced breakdown without recent de novo synthesis of the enzyme. Such a sequence may be operative in chronic inflammatory diseases, such as periodontitis, where production of procollagenase under the influence of cytokines spans a longer time period, whereas breakdown is often characterized by a cyclic behaviour.     

Numerical techniques and mathematical modelling for CI857-controlled gene expression and cell growth in recombinant E. coli

Recombinant gene expression, monitored by beta-galactosidase activity, is studied in a pL, pR-CI857 plasmid expression system in temperature-induced E. coli batch cultures. The experimental procedure has been mathematically modelled, and the corresponding parameters are estimated from specific statistical and numerical methods, basically by using a global least-squares procedure under some constraints induced by the model. The numerical techniques proposed in this work act by accumulation of data coming from several runs of the modelled experiment, so that more accuracy is obtained in the parameter estimation. In particular, for the production process, an extra-model parameter depending on an indicator vector is introduced for each run of the experiment in order to globalize the data. The analysis of the data obtained leads to an integrated model for both cell growth and gene expression, which describes an asymmetric dynamics between culture growth and recombinant protein yield, and can serve to predict the maximal value of accumulated gene expression and the time required for it to be achieved at any age of the preinducing cell growth.     

A study for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 by the bacterial agglutination technique

We evaluated the usefulness of bacterial agglutination antibodies for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 infections. We examined 50 serum samples from 50 control children (whiout diarrhea 31, with diarrhea 19), 24 samples from 8 diarrhea cases due to O157:H7, 37 samples from 14 cases of hemolytic uremic syndrome (HUS) for antibodies to heat-killed E. coli E32511 (O157:H.-) strain using the bacterial agglutination technique. Of the control sera all but one (x80) showed 20 > or = in the antibody. All the diarrhea patients due to O157:H7 showed a significant rise (x160-x5120) of the titers in the sera at 5-7 days on illness, after that the titers fell rapidly. Significant antibody rise (x160-x5120) was detected in twelve out of 14 HUS patients at the early stage of the illness which fell in the convalescent phase. The assay appeared to be a useful serodiagnostic technique because of its easiness and simplicity as well as because of its high sensitivity and specificity.     

Metabolic consequences of phosphotransferase (PTS) mutation in a phenylalanine-producing recombinant Escherichia coli

E. coli strain PPA305, which has a wild-type PTS system, and PPA316, which utilizes a proton-galactose symport system for glucose uptake, were used as host strains to harbor a phenylalanine overproduction plasmid pSY130-14 and to study the effects of using different glucose uptake systems on phenylalanine production. The non-PTS strain (PPA316/pSY130-14) produced much less phenylalanine, ranging from 0 to 67% of that produced by the PTS strain (PPA305/pSY130-14) depending on cultivation conditions used. The non-PTS strain PPA316/pSY130-14 had an intracellular PEP concentration only one-sixth that of the PTS strain, PPA305/pSY130-14. Additionally, PPA316/pSY130-14 had a substantially lower energy state in terms of the size of the pool of high-energy phosphate compounds and the magnitude of the pH difference across the cytoplasmic membrane. The non-PTS strain consumed oxygen at a higher rate, attained lower biomass concentration, and produced no acetate and phenylalanine during fermentation, suggesting more carbon was oxidized to CO2, most likely through the TCA cycle. Analysis of intracellular fluxes through the central carbon pathways was performed for each strain utilizing exponential phase data on extracellular components and assuming quasi-steady state for intermediate metabolites. The non-PTS strain had a higher flux through pyruvate kinase (PYK) and TCA cycle which, in agreement with the observed higher oxygen uptake rate, suggests that more carbon was oxidized to CO2 through the TCA cycle. Further analysis using rate expression data for PYK and NMR data for the intracellular metabolites identified the regulatory properties of PYK as the probable cause for lower intracellular PEP levels in PPA316/pSY130-14.     

The chemical synthesis of E. coli tRNA(Lys) anticodon loop fragment and its analogues

E. coli tRNA(Lys) anticodon loop fragment (Umnm5s2UUUt6A) 1 and its analogues 2-6 were synthesized by the classical phosphotriester approach in solution. The preparation of suitably protected derivatives of N6-threonylcarbamoyladenosine 18 is also described.     

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.     

Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli

The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.     

The structure and function of yeast xylose (aldose) reductases

In three dimensional (3-D) human movement analysis using close-range photogrammetry, surface marker clusters deform and rigidly move relative to the underlying bone. This introduces an important artefact (skin movement artefact) which propagates to bone position and orientation and joint kinematics estimates. This occurs to the extent that those joint attitude components that undergo small variations result in totally unreliable values. This paper presents an experimental and analytical procedure, to be included in a subject-specific movement analysis protocol, which allows for the assessment of skin movement artefacts and, based on this knowledge, for their compensation. The effectiveness of this procedure was verified with reference to knee-joint kinematics and to the artefacts caused by the hip movements on markers located on the thigh surface. Quantitative validation was achieved through experimental paradigms whereby prior reliable information on the target joint kinematics was available. When position and orientation of bones were determined during the execution of a motor task, using a least-squares optimal estimator, but the rigid artefactual marker cluster movement was not dealt with, then knee joint translations and rotations were affected by root mean square errors (r.m.s.) up to 14 mm and 6 degrees, respectively. When the rigid artefactual movement was also compensated for, then r.m.s errors were reduced to less than 4 mm and 3 degrees, respectively. In addition, errors originally strongly correlated with hip rotations, after compensation, lost this correlation.     

Kinetics and mechanism of the conversion of covellite (CuS) to digenite (Cu1.8S)

Rates of conversion of covellite (CuS) to digenite (Cu_1.*S) in N₂ and various partial pressures of O₂ have been studied in the temperature range 260° to 400°C. The reaction is first order with respect to O₂ concentration in N₂?O₂ mixtures at 1 atm. The experimental activation energy for the conversion of CuS to Cu_1.8S is 23±3 kcal. In a nitrogen atmosphere, the conversion proceeds at a slower rate but with the same activation energy and leads to the formation of elemental sulfur instead of SO₂. An externally added small amount of iron powder (0.6 pct) increased the rate of conversion, but had no effect on the apparent activation energy. The rate-controlling reaction in the conversion mechanism is postulated to be Cu_1.8S→1.8Cu⁺+1.8e ^?+S at the Cu_1.8S/gas interface. Reduction of CuS by copper(I) ions and electrons occurs by a rapid reaction, 0.8 Cu⁺+0.8e ^?+CuS→Cu_1.8S at the CuS/Cu_1.8S interface, which steadily contracts with time.     

Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce epithelial cell actin rearrangements resulting in pedestal formation beneath adherent bacteria. This requires the secretion of specific virulence proteins needed for signal transduction and intimate adherence. EPEC interaction induces tyrosine phosphorylation of a protein in the host membrane, Hp90, which is the receptor for the EPEC outer membrane protein, intimin. Hp90-intimin interaction is essential for intimate attachment and pedestal formation. Here, we demonstrate that Hp90 is actually a bacterial protein (Tir). Thus, this bacterial pathogen inserts its own receptor into mammalian cell surfaces, to which it then adheres to trigger additional host signaling events and actin nucleation. It is also tyrosine-phosphorylated upon transfer into the host cell.