Comparisons between cellulase production by Aspergillus fumigatus in agitated vessels and in an air-lift fermentor

Aspergillus fumigatus was cultured in disc-turbine-agitated vessels and in an air-lift fermentor. In the agitated vessels the yield of cellulase was reduced when the agitation rate was increased, although extracellular protein levels rose. The enzyme complex itself was shown to be exceptionally stable under conditions similar to those in the agitated vessels, so probably shear damage to the mycelium had occurred, liberating intracellular contents. These appeared to contain an inhibitor that could be removed by fabricated inorganic protein absorbents, such as kieselguhr and alumina. However, the inhibitor was not likely to be protease, since only relatively low levels could be detected and its identity has not been established. The use of an air-lift fermentor avoided the shear effects due to use of the disc turbine agitator in the conventional fermentors, and yields of enzyme were then found to increase by about 20%, maximum yields being obtained at maximum K(L)a values.     

Novel retinoid-binding proteins from filarial parasites.

The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions.     

Corneal Dystrophy Mutations Drive Pathogenesis by Targeting TGFBIp Stability and Solubility in a Latent Amyloid-forming Domain

Numerous mutations in the corneal protein TGFBIp lead to opaque extracellular deposits and corneal dystrophies (CDs). Here we elucidate the molecular origins underlying TGFBIp's mutation-induced increase in aggregation propensity through comprehensive biophysical and bioinformatic analyses of mutations associated with every major subtype of TGFBIp-linked CDs including lattice corneal dystrophy (LCD) and three subtypes of granular corneal dystrophy (GCD 1–3). LCD mutations at buried positions in the C-terminal Fas1–4 domain lead to decreased stability. GCD variants show biophysical profiles distinct from those of LCD mutations. GCD 1 and 3 mutations reduce solubility rather than stability. Half of the 50 positions within Fas1–4 most sensitive to mutation are associated with at least one known disease-causing mutation, including 10 of the top 11 positions. Thus, TGFBIp aggregation is driven by mutations that despite their physico-chemical diversity target either the stability or solubility of Fas1–4 in predictable ways, suggesting straightforward general therapeutic strategies.     

Dearomatization of diesel oil using <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Objectives

To improve the quality of diesel fuel via removal of aromatic compounds using Pseudomonas sp.

Results

In the present study Pseudomonas sp. was able to remove 94% of fluorene, 59% of phenanthrene, 49% of anthracene, 52% of fluoranthene, 45% of pyrene and 75% carbazole present in diesel oil. Additionally, it also does not affect the aliphatic content of fuel thus maintaining the carbon backbone of the fuel.

Conclusions

Pseudomonas sp. is a potential biocatalyst that can be used in the refining industry.

     

Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.     

Biochemical diversity evaluation in chickpea accessions employing mini-core collection

The seeds of chickpea provide an exceptional source of dietary proteins and is one of the important legumes in both developed and developing countries over the world. The available germplasm of cultivated chickpea is deficient in desired biochemical signatures. To identify new sources of variations for breeding, reduced subsets of germplasm such as mini-core collection can be explored as an effective resource. In the present investigation, mini-core collections consisting of 215 accessions of chickpea were extensively evaluated for tapping biochemical diversity. Analysis included ten biochemical parameters comprising total protein, total free amino acids, phytic acid, tannin, total phenolics, total flavonoids, lectin, DPPH radical scavenging activity, in vitro digestibility of protein and starch. The spectrum of diversity was documented for total protein (4.60–33.90%), total free amino acids (0.092–9.33 mg/g), phytic acid (0.009–4.06 mg/g), tannin (0.232–189.63 mg/g), total phenolics (0.15–0.81 mg/g), total flavonoids (0.04–1.57 mg/g), lectin (0.07–330.32 HU/mg), DPPH radical scavenging activity (26.74–49.11%), in vitro protein digestibility (59.45–76.22%) and in vitro starch digestibility (45.63–298.39 mg of maltose/g). The principal component analysis revealed association of chickpea higher protein content to the lower level of total phenolics and flavonoid contents. The dendrogram obtained by unweighted pair group method using arithmetic average cluster analysis grouped the chickpea accessions into two major clusters. This is the first comprehensive report on biochemical diversity analysed in the mini-core chickpea accessions. The ultimate purpose of conducting such studies was to deliver information on nutritional characteristics for effective breeding programmes. Depending on the objectives of the breeding aforesaid accessions could be employed as a parent.     

Outlier detection in BLAST hits

Background

An important task in a metagenomic analysis is the assignment of taxonomic labels to sequences in a sample. Most widely used methods for taxonomy assignment compare a sequence in the sample to a database of known sequences. Many approaches use the best BLAST hit(s) to assign the taxonomic label. However, it is known that the best BLAST hit may not always correspond to the best taxonomic match. An alternative approach involves phylogenetic methods, which take into account alignments and a model of evolution in order to more accurately define the taxonomic origin of sequences. Similarity-search based methods typically run faster than phylogenetic methods and work well when the organisms in the sample are well represented in the database. In contrast, phylogenetic methods have the capability to identify new organisms in a sample but are computationally quite expensive.

Results

We propose a two-step approach for metagenomic taxon identification; i.e., use a rapid method that accurately classifies sequences using a reference database (this is a filtering step) and then use a more complex phylogenetic method for the sequences that were unclassified in the previous step. In this work, we explore whether and when using top BLAST hit(s) yields a correct taxonomic label. We develop a method to detect outliers among BLAST hits in order to separate the phylogenetically most closely related matches from matches to sequences from more distantly related organisms. We used modified BILD (Bayesian Integral Log-Odds) scores, a multiple-alignment scoring function, to define the outliers within a subset of top BLAST hits and assign taxonomic labels. We compared the accuracy of our method to the RDP classifier and show that our method yields fewer misclassifications while properly classifying organisms that are not present in the database. Finally, we evaluated the use of our method as a pre-processing step before more expensive phylogenetic analyses (in our case TIPP) in the context of real 16S rRNA datasets.

Conclusion

Our experiments make a good case for using a two-step approach for accurate taxonomic assignment. We show that our method can be used as a filtering step before using phylogenetic methods and provides a way to interpret BLAST results using more information than provided by E-values and bit-scores alone.

     

Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles

Objectives

Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications.

Results

Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg^?1 silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg^?1 silica nanoparticles.

Conclusions

Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.

     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.