High Molecular Weight Intermediates Research Articles

Lumped chemical kinetic modelling of raw and torrefied biomass under pressurized pyrolysis

Accurate prediction of the yield and composition of pyrolysis products is an important requirement for the design and operation of pyrolysis reactors and gasifiers. In this paper, a new semi-global kinetic reaction scheme is proposed to predict the composition of pyrolytic volatiles (classified as main chemical family groups), non-condensable gases and char derived from both raw and torrefied biomass for a wide range of operating conditions. The model is based on an adjustable mechanistic reaction scheme, which includes a combination of three different sub-mechanisms for the primary pyrolysis of three reference biopolymers (cellulose, hemicelluloses and lignin) and the secondary pyrolysis of their respective intermediates. The extent of primary/secondary reactions is varied according to the main process features (linear heating rate, temperature, pyrolysis time, volatile residence time and pressure). The secondary reactions in the scheme involve liquid-phase reactions of high molecular weight intermediates (producing non-condensable gases, water vapour and secondary char) as well as homogeneous and heterogeneous gas-phase conversion of primary volatiles. The model predictions were validated using experimental data obtained from fast pyrolysis in different micropyrolyzers (at 500–600 °C and heating rates of 27 and 110 °C/s, respectively) and slow pyrolysis in a laboratory-scale high-pressure fixed bed reactor (at 400–600 °C, 7 °C/min and 1, 15 and 30 bar). In general, the comparison of model outputs and experimental data were satisfactory, and the model predicted accurate trends in product distribution for changes of the lignocellulosic composition (the pre-removal of hemicelluloses in the case of torrefied biomass), heating rate and pressure. The model correctly predicted a significant increase in char yield (14.6 wt%) when torrefied instead of raw biomass was pyrolyzed due to the significance of char-forming reactions during pyrolysis of torrefied biomass. Moreover, the model’s reliability was proven through its accurate prediction of various condensate groups in bio-oil produced in a micropyrolyzer (maximum deviation < 4 wt%). Corresponding to the experimental data, the model predictions showed that the effect of pressure was most significant in the range of 1–15 bar (bio-oil yield decreased by 5.4 wt%), whereas no significant effect in bio-oil yield was evident for a pressure increase in the range of 15–30 bar. The CO2 and CO yields were slightly under-predicted by around 3 wt%, attributed to the catalytic effects of inherent inorganics on secondary cracking reactions which were not considered in the reaction scheme. Future work should focus on the validation of the model at temperatures below 400 °C.

Thermal degradation of hexachlorobenzene in the presence of calcium oxide at 340–400 °C

Hexachlorobenzene (HCB) in the milligram range was co-heated with calcium oxide (CaO) powder in sealed glass ampoules at 340–400°C. The heated samples were characterized and analyzed by Raman spectroscopy, elemental analysis, gas chromatography/mass spectrometry, ion chromatography, and thermal/optical carbon analysis. The degradation products of HCB were studied at different temperatures and heated times. The amorphous carbon was firstly quantitatively evaluated and was thought to be important fate of the C element of HCB. The yield of amorphous carbon in products increased with heating time, for samples treated for 8h at 340, 380°C and 400°C, the value were 17.5%, 34.8% and 50.2%, respectively. After identification of the dechlorination products, the HCB degradation on CaO at 340–400°C was supposed to through dechlorination/polymerization pathway, which is induced by electron transfer, generate chloride ions and form high-molecular weight intermediates with significant levels of both hydrogen and chlorine, and finally form amorphous carbon. Higher temperature was beneficial for the dechlorination/polymerization efficiency. The results are helpful for clarifying the reaction mechanism for thermal degradation of chlorinated aromatics in alkaline matrices.

Measurement of secondary products during oxidation reactions of terpenes and ozone based on the PTR-MS analysis: effects of coexistent carbonyl compounds.

Continuous measurements using proton transfer reaction mass spectrometry (PTR-MS) can be used to describe the production processes of secondary products during ozone induced oxidation of terpenes. Terpenes are emitted from woody building materials, and ozone is generated from ozone air purifiers and copy machines in indoor environments. Carbonyl compounds (CCs) are emitted by human activities such as smoking and drinking alcohol. Moreover, CCs are generated during ozone oxidation of terpenes. Therefore, coexistent CCs should affect the ozone oxidation. This study has focused on the measurement of secondary products during the ozone oxidation of terpenes based on the use of PTR-MS analysis and effects of coexistent CCs on oxidized products. Experiments were performed in a fluoroplastic bag containing α-pinene or limonene as terpenes, ozone and acetaldehyde or formaldehyde as coexistent CCs adjusted to predetermined concentrations. Continuous measurements by PTR-MS were conducted after mixing of terpenes, ozone and CCs, and time changes of volatile organic compounds (VOCs) concentrations were monitored. Results showed that, high-molecular weight intermediates disappeared gradually with elapsed time, though the production of high-molecular weight intermediates was observed at the beginning. This phenomenon suggested that the ozone oxidation of terpenes generated ultrafine particles. Coexistent CCs affected the ozone oxidation of α-pinene more than limonene.

Conversion of non-fibrillar β-sheet oligomers into amyloid fibrils in Alzheimer’s disease amyloid peptide aggregation

Aβ(1–40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer’s disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular β-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Aβ(1–40) fibril formation process. A unique sample containing 90 μM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar β-structures. The number of oligomers and the amount of non-fibrillar β-structures grows throughout the lag phase and during the elongation phase these non-fibrillar β-structures are transformed into fibrillar (amyloid) β-structures, formed by association of high molecular weight intermediates.

Recombination in alphaherpesviruses

Within the Herpesviridae family, Alphaherpesvirinae is an extensive subfamily which contains numerous mammalian and avian viruses. Given the low rate of herpesvirus nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in alphaherpesviruses is intimately linked to DNA replication. Both viral and cellular proteins participate in this recombination-dependent replication. The presence of inverted repeats in the alphaherpesvirus genomes allows segment inversion as a consequence of specific recombination between repeated sequences during DNA replication. High molecular weight intermediates of replication, called concatemers, are the site of early recombination events. The analysis of concatemers from cells coinfected by two distinguishable alphaherpesviruses provides an efficient tool to study recombination without the bias introduced by invisible or non-viable recombinants, and by dominance of a virus over recombinants. Intraspecific recombination frequently occurs between strains of the same alphaherpesvirus species. Interspecific recombination depends on enough sequence similarity to enable recombination between distinct alphaherpesvirus species. The most important prerequisite for successful recombination is coinfection of the individual host by different virus strains or species. Consequently the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology, virulence and latency. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.

Biosynthesis of proopiomelanocortin-derived peptides in prohormone convertase 2 and 7B2 null mice.

Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary alpha-MSH; interestingly, 7B2 nulls are still able to generate beta-endorphin from beta-lipotropin, whereas PC2 nulls contain little if any beta-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of alpha-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.

Concatemeric Intermediates of Equine Herpesvirus Type 1 DNA Replication Contain Frequent Inversions of Adjacent Long Segments of the Viral Genome

In common with other α-herpesviruses, the genome of equine herpesvirus type-1 (EHV-1) comprises covalently linked long and short unique sequences of DNA, each flanked by inverted repeats. Equimolar amounts of two genomic isomers, generated by free inversion of the short segment, relative to the long segment, are packaged into EHV-1 virions. In contrast with herpes simplex virus (HSV), inversion of genomic long segments has not been described. In the current work, the structures of high molecular weight intermediates of EHV-1 DNA replication were studied by field inversion gel electrophoresis. It is shown that adjacent long segments of the viral genome are frequently inverted in concatemeric intermediates of EHV-1 DNA replication. Further, like HSV concatemers, high molecular weight intermediates of EHV-1 replication are flanked exclusively by the long segment of the viral genome. Hence, despite the fact that only two, rather than four, isomers of EHV-1 DNA are packaged into virions, the intermediates of EHV-1 DNA replication closely resemble those of herpes simplex virus type 1 in structure. These data have implications relating to the mechanisms involved in packaging of α-herpesvirus DNA.

Analysis of chorion hardening of eggs of rainbow trout, Oncorhynchus mykiss.

We estimated changes of chorion hardness of rainbow trout (Oncorhynchus mykiss) egg by the use of three parameters, namely increase of resistance of an egg to rupture by extraneously applied pressure, decrease of solubility of chorion proteins in 8 mol/L urea and a change in the content of γ-glutamyl-ε-lysine crosslink. Unfertilized egg chorions became hardened after egg activation. During chorion hardening, 49, 56 and 65 kDa protein components of the chorion gradually disappeared, high molecular weight intermediates (113,160-170 and higher than 250 kDa) were newly formed and, finally, all components became undetectable by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The content of γ-glutamyl-ε-lysine (γ-Glu-ε-Lys) crosslink in the chorion increased after hardening. Chorion hardening was inhibited by the incorporation of monodansyl-cadaverine, a competitive inhibitor for transglutaminase (TGase), into the chorions. TGase activity was detected in unfertilized eggs and localized in the chorion fraction rather than in the ooplasmic fraction. The findings suggest that chorion hardening depends upon polymerization of the chorion components by TGase-dependent γ-Glu-ε-Lys crosslink formation.

Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis.

ClpP, the proteolytic component of the ATP-dependent ClpAP protease, is composed of 12 identical subunits and has intrinsic degradative activity against short peptides. Degradation of proteins and some peptides by ClpP requires the regulatory component ClpA and ATP. Peptide and protein substrates have been used to distinguish the roles of nucleotide binding and nucleotide hydrolysis in the activation of ClpAP protease. ATP binding alone promoted interaction between ClpA and ClpP, affected the substrate response curves for very short peptides, and activated degradation of larger peptides that were not degraded by ClpP alone. ATP hydrolysis did not increase in proportion to the increase in peptide bond hydrolysis of short peptides. However, ATP hydrolysis was strictly required for degradation of proteins such as alpha-casein; there was no indication of even limited cleavage of protein substrates when nonhydrolyzable analogs of ATP were used. Most large peptides and proteins were degraded in multiple sites without release of high molecular weight intermediates. Partial inactivation of ClpP with diisopropyl fluorophosphate produced ClpP with one to three active subunits/dodecamer. When only a few active sites were available in the active complex of ClpAP, degradation of large peptides and proteins released significant amounts of high molecular weight intermediates. Thus, processive degradation of protein substrates is a function of the multiple array of proteolytic active sites within the ClpP dodecamer.

Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms: Cleavage at a single arginine residue

Processing of rat prodynorphin (proDyn) by the mouse prohormone convertase PCI was investigated. Recombinant vaccinia virus vectors were used to coexpress proDyn and PC1 in rat PC12 pheochromocytoma and mouse AtT-20 corticotroph cells. In vitro experiments were also conducted by co-incubating purified proDyn and PC1. The results demonstrate that PC1 cleaves proDyn at pairs of basic residues to yield 10 and 16 kDa high molecular weight (HMW) intermediates. Additionally, PC1 cleaves proDyn at a single arginine residue to yield an 8 kDa product and the C-peptide. This demonstrates that PC1 cleaves proDyn at single and pairs of basic residues.

Denaturation by urea and renaturation of 20β-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography

The denaturation by urea and renaturation of 20β-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH high molecular weight aggregates, native tetramers and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.

An investigation of the carbonation of cement paste using trimethylsilylation

Ground samples of hydrated cement paste were exposed to atmospheric carbon dioxide and the progress of reaction was monitored by trimethylsilylation of the silicate phases and differential thermal gravimetry (DTG) for calcium hydroxide. The data indicate that carbonation for calcium silicate hydrate (CSH) proceeds via high molecular weight intermediates to give an amorphous hydrous silica material. The carbonation of CSH and calcium hydroxide occurs simultaneously, but a residue of calcium hydroxide carbonates more slowly. This is consistent with a rate-determining stage controlled by the rate of diffusion of carbon dioxide through calcium hydroxide crystals coated with calcium carbonate, and hence also by crystal size.

Self-cleavage of rna in the replication of viroids and virusoids

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Intracellular transport of high molecular weight intermediates of acid α-glucosidase in human fibroblasts

Two transient, high molecular weight precursors of human acid α-glucosidase were detected by immune precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The high molecular weight precursors were rapidly converted into lower molecular weight forms corresponding to previously identified intermediates of acid α-glucosidase. An accumulation of these precursors was observed in fibroblasts treated with monesin and nigericin, suggesting that these precursors are intermediates of acid α-glucosidase undergoing transport through the Golgi complex.

Activation of prothrombin Barcelona evidence for active high molecular weight intermediates

Abstract Prothrombin Barcelona is a varient of human prothrombin. Its activation by factor Xa is characterized by two abnormal features. The first one is the absence of one of the two Xa-catalyzed cleavages, and the second one the generation of a thrombin-like activity responsible for the thrombin-catalyzed cleavages on the prothrombin molecule and for the activity toward small substrates but without clotting activity. The molecular species exhibiting the thrombin-like activity have been characterized Diisopropyl phosphofluoridate incorporation experiments show that upon activation, two high molecular weight intermediates bearing an active site are formed: one has the molecular weight of prothrombin, the other of prethrombin 1. In the presence of hirudin, only the first intermediate is formed due to a single cleavage by factor Xa which is therefore responsible for the unmasking of the active site.