Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

High level production of supra molecular weight poly (3-hydroxybutyrate) by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis>

The supra molecular weight poly ([R]-3-hydroxybutyrate) (PHB), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineeredEscherichia coli strain and its fermentation for high level production of supra molecular weight PHB. RecombinantE. coli strains, harboring plasmids of different copy numbers containing theAlcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinantE. coli XL 1-Blue, harboring a medium-copy-number pJC2 containing theA. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE. coli     

GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Aryl Acylamidase Activity on Acetylcholinesterase Is High During Early Chicken Brain Development

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to exhibit aryl acylamidase activities (here called AAA(AChe) and AAA(BChe), respectively), which have been suggested to be involved in developmental and pathological processes. We here have investigated the developmental profiles of both AAA(AChe) and AAA(BChe) activities along with their AChE and BChE activities from embryonic days E3 to hatching (E21) in Triton-extracted homogenates from chicken embryonic brains. AAA(AChe) follows continuously an increase that is typical for AChE expression itself, whereas AAA(BChe) was relatively high before E10 to then become negligible toward hatching. Sucrose gradient centrifugation of both homogenized and immunopurified samples from E6-E18 brains showed that all globular forms (G1, G2, G4) of AChE present AAA(AChe) activity. Interestingly, the ratio of AAA(AChe) to AChE is highest at E6, and here again higher on G1/G2- over the G4-form. Noticeably, the sensitivity of AAA(AChe) toward the specific AChE inhibitor BW284c51 at all stages is higher than that of AChE itself. These data of high ratios of AAA associated at young stages with cholinesterases strongly indicate a role of AAA in early brain development.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

Utility of Amplified Fragment Length Polymorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex

Phylogenetic analyses of the Alexandrium tamarense species complex using ribosomal RNA sequences show a differentiation of ribotypes/clades into geographic areas and not into the three morphotypes/species A. tamarense, A. fundyense and A. catenella. Different parts of the rRNA operon have proven informative in revealing the existence and the relationships of these geographic clades, whereas even internal transcribed spacer (ITS) regions lack the resolution required to gain a deeper insight into the population structure of the species complex. Here, the utility of the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) as a possible tool for such purposes was tested. A mixed sampling strategy was used in order to assess the amount of variation of AFLP banding patterns at the level of populations and geographic clades. We also describe optimized methods to achieve a good reproducibility. Our results suggest that AFLPs can provide useful information at the population level using clonal samples from a certain bloom, whereas the amount of variation that we found is too high to allow for meaningful comparisons of a few strains collected from different localities at different time points even though they belong to one geographic clade.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Sensitivity of molecular dynamics simulations to the choice of the X-ray structure used to model an enzymatic reaction

A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X-ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl-CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton-abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis.     

Average protein density is a molecular-weight-dependent function

The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.