Synergy of Siam weed (Chromolaena odorata) and poultry manure for energy generation: Effects of pretreatment methods, modeling and process optimization.

The co-digestion of Chromolaena odorata with poultry manure was evaluated in this study. Two samples of the weed: (A: which was pre-treated with mechanical, chemical and thermal methods) and (B: which was pretreated using mechanical and chemical methods only) were separately digested with poultry manure. Biogas generation started from the 2nd to 4th and 4th to 7th day for samples 'A' and 'B' respectively. The most desired actual biogas yield from samples 'A' and 'B' were 3884.20 and 2544.70 (10-4m3/kg VS) respectively and the gas composition was 68±2% Methane and 20±2% Carbon dioxide for sample A while it was 62±3% Methane and 22±2% Carbon dioxide for sample B. In all, there was a 38.06% increase in gas generation in 'A' over 'B'. The coefficient of determination (R2) for the Response Surface Methodology (RSM) model (0.9009) was high suggesting high accuracy in the modeling and prediction. The worldwide usage of C. odorata is encouraged.

Comparative biogas generation from fruit peels of fluted pumpkin (Telfairia occidentalis) and its optimization.

This study evaluated the potentials of fluted pumpkin fruit peels for biogas generation using three different pre-treatment methods (A, B, C) and the optimization of its process parameters. The physic-chemical characteristics of the substrates revealed it to be rich in nutrients and mineral elements needed by microorganisms. Gas chromatography analysis revealed the gas composition to be within the range of 58.5±2.5% Methane and 27±3% Carbon dioxide for all the three digestions. The study revealed that combination of three pre-treatment methods enhanced enormous biogas yield from the digested substrates as against the use of two methods and no pre-treatment experiment. Optimization of the generated biogas data revealed that RSM predicted higher gas yield than ANN, the latter gives higher accuracy and efficiency than the former. It is advocated that fluted pumpkin fruit peels be used for energy generation especially in the locations of its abundance.

Mesophilic anaerobic co-digestion of poultry dropping and Carica papaya peels: Modelling and process parameter optimization study.

The study evaluated anaerobic co-digestion of poultry dropping and pawpaw peels and the optimization of important process parameters. The physic-chemical analyses of the substrates were done using standard methods after application of mechanical, thermal and chemical pre-treatments methods. Gas chromatography analysis revealed the gas composition to be within the range of 66-68% methane and 18-23% carbon dioxide. The study equally revealed that combination of the different pre-treatment methods enhanced enormous biogas yield from the digestion. Optimization of the generated biogas data were carried out using the Response Surface Methodology and the Artificial Neural Networks. The coefficient of determination (R(2)) for RSM (0.9181) was lower compare to that of ANN (0.9828). This shows that ANN model gives higher accuracy than RSM model for the current. Further usage of Carica papaya peels for biogas generation is advocated.

Characterization of antiallodynic actions of ALE-0540, a novel nerve growth factor receptor antagonist, in the rat.

There is growing evidence that nerve growth factor (NGF) may function as a mediator of persistent pain states. We have identified a novel nonpeptidic molecule, ALE-0540, that inhibits the binding of NGF to tyrosine kinase (Trk) A or both p75 and TrkA (IC50 5.88 +/- 1. 87 microM, 3.72 +/- 1.3 microM, respectively), as well as signal transduction and biological responses mediated by TrkA receptors. ALE-0540 was tested in models of neuropathic pain and thermally-induced inflammatory pain, using two routes of administration, a systemic i.p. and a spinal intrathecal (i.th.) route. Morphine was also tested for comparison in the antiallodynia model using mechanical stimuli. We show that either i.p. or i.th. administration of ALE-0540 in rats produced antiallodynia in the L5/L6 ligation model of neuropathic pain. The calculated A50 values (and 95% confidence intervals) for ALE-0540 administered i.p. and i. th. were 38 (17.5-83) mg/kg and 34.6 (17.3-69.4) microgram, respectively. ALE-0540 given i.th., at doses of 30 and 60 microgram, also blocked tactile allodynia in the thermal sensitization model. Although morphine displayed greater potency [A50 value of 7.1 (5.6-8. 8) mg/kg] than ALE-0540 in anti-allodynic effect when given i.p. to L5/L6-ligated rats, it was not active when administered i.th. These data suggest that a blockade of NGF bioactivity using a NGF receptor antagonist is capable of blocking neuropathic and inflammatory pain and further support the hypothesis that NGF is involved in signaling pathways associated with these pain states. ALE-0540 represents a nonpeptidic small molecule which can be used to examine mechanisms leading to the development of agents for the treatment of pain.

Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.

A plasmid-encoded anion-translocating ATPase.

An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon.

The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.

Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon.

The regulatory region of the plasmid-encoded arsenical resistance (ars) operon was cloned as a 727-bp EcoRI-HindIII fragment. When cloned into a promoter probe vector this fragment conferred arsenite inducible tetracycline resistance in Escherichia coli, indicating that the fragment carried a regulatory gene, the arsR gene. A single region corresponding to -35 and -10 promoter recognition sites was identified. The transcriptional start site of the mRNA was determined by primer extension. The sequence has an open reading frame for a potential 13,179 Da polypeptide, termed the ArsR protein. The fragment was cloned into a temperature regulated expression vector. A protein with an apparent molecular mass of about 12 kDa was induced by either temperature or arsenite. This protein was purified and used to produce antibodies specific for the ArsR protein.

Molecular analysis of an ATP-dependent anion pump.

The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells. The catalytic component of the pump, the 63 kDa ArsA protein, hydrolyses ATP in the presence of its anionic substrate antimonite (SbO2-). The ATP analogue 5'-p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site(s) of the ArsA protein. From sequence analysis there are two potential nucleotide binding sites. Mutations were introduced into the N-terminal site. Purified mutant proteins were catalytically inactive and incapable of binding nucleotides. Conformational changes produced upon binding of substrates to the ArsA protein were investigated by measuring the effects of substrates on trypsin inactivation. The hydrophobic 45.5 kDa ArsB protein forms the membrane anchor for the ArsA protein. The presence of the ArsA protein on purified inner membrane can be detected immunologically. In the absence of the arsB gene no ArsA is found on the membrane. Synthesis of the ArsB protein is limiting for formation of the pump. Analysis of mRNA structure suggests a potential translational block to synthesis of the ArsB protein. Northern analysis of the ars message demonstrates rapid degradation of the mRNA in the arsB region.

Expression in Escherichia coli of the Cellulomonas fimi Structural Gene for Endoglucanase B.

Endoglucanase B (EB) of Cellulomonas fimi has an M(r) of 110,000 when it is produced in Escherichia coli. The level of expression of the cenB gene (encoding EB) was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. EB was purified to homogeneity from the periplasmic fraction of E. coli in one step by affinity chromatography on microcrystalline cellulose (Avicel). Alignment of the NH(2)-terminal amino acid sequence with the partial nucleotide sequence of a fragment of C. fimi DNA showed that EB is preceded by a putative signal polypeptide of 33 amino acids. The signal peptide functions and is processed correctly in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of beta-galactosidase. The intact EB polypeptide is not required for enzymatic activity. Active polypeptides with M(r)s of 95,000 and 82,000 also appear in E. coli, and a deletion mutant of cenB encodes an active polypeptide with an M(r) of 72,000.

A bifunctional exoglucanase-endoglucanase fusion protein.

A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.