Jatropha curcas hemagglutinin is similar to a 2S albumin allergen from the same source and has unique sugar affinities

We have previously reported the purification and preliminary X-ray characterization of a hemagglutinin from the seeds of Jatropha curcas and, with the detailed sequencing information available now, we find that it is similar to a 2S albumin allergen isolated from the same source. Through a search of Jatropha genome database (http://www.kazusa.or.jp/jatropha/), we map it to the sequence id JcCA0234191 (now referred to as Jcr4S00619.70 in the new version, release 4.5) which has a conserved alpha amylase inhibitor/seed storage protein domain found in the 2S albumin allergens. The putative sequence of the small and large chains of the protein is assigned and the total mass of the two subunits matches with the intact mass 10?kDa determined through MALDI. The protein retains hemagglutination activity between pH 6–9 and up to 60?°C on heat treatment and its hemagglutination activity is inhibited by sialic acid and fetuin. Bioinformatics studies show that the isolated protein sequence clusters in close association with a 2S albumin from Ricinus communis in phylogeny analysis and has a conservation of the characteristic four disulfide linkage pattern. Hemagglutinins and lectins are known to have allergenic effects through their interaction with immunoglobulin E and histamine release and earlier studies have shown that this interaction can be inhibited by lectin-specific sugars. We hope this report bridges the plant allergens and hemagglutinins further for exploring possible mediation of allergenic activity through sialic acid and complex sugar interactions and generates further interest in the area.     

Role of calcium alginate and mannitol in protecting bifidobacterium

Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (a(w)) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low a(w)s. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C).     

Production of alkali tolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205

The fermentation conditions were optimized for hyper production of xylanase from Bacillus pumilus SV-205. The bacterium secretes high levels (7382.7±1200 IU/mL) of cellulase-free xylanase using wheat bran led to 21.63 fold increase in activity. A combination of yeast extract and peptone stimulated highest xylanase production (2448.0 IU/mL) as compared to other combinations. The most important characteristic of the enzyme is its high pH stability (100%) over a broad pH range of 6-11 for 24h. Thermostability studies revealed that enzyme retained 65% activity after an incubation of 2h at 60°C. The level of production is remarkable as compared to earlier reports.     

Robust,small-scale cultivation platform for <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Background  

For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes.     

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis

Fatty acids play critical role in the survival and virulence of Mycobacterium?tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia?coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ～?0.033?μm) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ～?5.4?×?10(4) m(-1) ·s(-1) ) and slower dissociation rates (k(d,avg) ～?5.84?×?10(-4) s(-1) ) confer higher affinity. The affinity for the promoter is maximum at 300?mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner. Structured digital abstract ? Fad35R?binds to?palindromic sequence?shown by surface plasmon resonance ? Fad35R?binds to?tetracycline?and?activated fatty acids?as shown by fluorescence spectroscopy.     

Another piece of the ribosome: solution structure of S16 and its location in the 30S subunit

BACKGROUND: X-ray crystallography has recently yielded much-improved electron-density maps of the bacterial ribosome and its two subunits and many structural details of bacterial ribosome subunits are now being resolved. One approach to complement the structures and elucidate the details of rRNA and protein packing is to determine structures of individual protein components and model these into existing intermediate resolution electron density. RESULTS: We have determined the solution structure of the ribosomal protein S16 from Thermus thermophilus. S16 is a mixed alpha/beta protein with a novel folding scaffold based on a five-stranded antiparallel/parallel beta sheet. Three large loops, which are partially disordered, extend from the sheet and two alpha helices are packed against its concave surface. Calculations of surface electrostatic potentials show a large continuous area of positive electrostatic potential and smaller areas of negative potential. S16 was modeled into a 5.5 A electron-density map of the T. thermophilus 30S ribosomal subunit. CONCLUSIONS: The location and orientation of S16 in a narrow crevice formed by helix 21 and several other unassigned rRNA helices is consistent with electron density corresponding to the shape of S16, hydroxyl radical protection data, and the electrostatic surface potential of S16. Two protein neighbors to S16 are S4 and S20, which facilitate binding of S16 to the 30S subunit. Overall, this work exemplifies the benefits of combining high-resolution nuclear magnetic resonance (NMR) structures of individual components with low-resolution X-ray maps to elucidate structures of large complexes.     

Replacement of the active surface of a thermophile protein by that of a homologous mesophile protein through structure-guided 'protein surface grafting'

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.     

Structural variations in species B adenovirus fibers impact CD46 association

A majority of species B adenoviruses (Ads) use CD46 as their primary receptor; however, the precise mechanisms involved in the binding of different Ad types to CD46 have not been resolved. Although previous studies indicate close similarities between two members of species B2 Ads in their usage of CD46, our current investigations revealed a surprisingly low CD46 binding affinity of the species B1 Ad16 fiber knob (equilibrium dissociation constant of 437 nM). We determined the crystal structure of the Ad16 fiber knob and constructed a model of this protein in complex with CD46. A comparison of this model to that of the CD46-Ad11 complex revealed structural differences in the FG and IJ loops that are part of the CD46 binding site. An analysis of a panel of recombinant fiber knobs with mutations targeting these regions in Ad16 and Ad11 uncovered a major contribution of the FG loop on CD46 binding. Two extra residues in the FG loop of the Ad16 fiber significantly reduce receptor interaction. Although avidity effects permit the use of CD46 on host cells by Ad16, virus binding occurs with lower efficiency than with B2 Ad types. The longer FG loop of the Ad16 fiber knob also is shared by other species B1 Ad fibers and, thus, may contribute to the low CD46 binding efficiencies observed for these Ad types. Our findings provide a better understanding of how different Ad types associate with CD46 and could aid in the selection of specific Ad fibers for more efficient Ad gene delivery vectors.     

Frictional contact mechanics methods for soft materials: application to tracking breast cancers

Mammography is currently the most widely used screening and diagnostic tool for breast cancer. Because X-ray images are 2D projections of a 3D object, it is not trivial to localise features identified in mammogram pairs within the breast volume. Furthermore, mammograms represent highly deformed configurations of the breast under compression, thus the tumour localisation process relies on the clinician's experience. Biomechanical models of the breast undergoing mammographic compressions have been developed to overcome this limitation. In this study, we present the development of a modelling framework that implements Coulomb's frictional law with a finite element analysis using a C(1)-continuous Hermite mesh. We compared two methods of this contact mechanics implementation: the penalty method, and the augmented Lagrangian method, the latter of which is more accurate but computationally more expensive compared to the former. Simulation results were compared with experimental data from a soft silicon gel phantom in order to evaluate the modelling accuracy of each method. Both methods resulted in surface-deformation root-mean-square errors of less than 2mm, whilst the maximum internal marker prediction error was less than 3mm when simulating two mammographic-like compressions. Simulation results were confirmed using the augmented Lagrangian method, which provided similar accuracy. We conclude that contact mechanics on soft elastic materials using the penalty method with an appropriate choice of the penalty parameters provides sufficient accuracy (with contact constraints suitably enforced), and may thus be useful for tracking breast tumours between clinical images.