A Comparative Study of Caffeine Degradation by Four Different Fungi

The objective of the present study is to investigate the caffeine-degrading abilities of different fungi and to apply this knowledge to environmental remediation and industrial decaffeination process. Chrysosporium keratinophilum, Gliocladium roseum, Fusarium solani, and Aspergillus restrictus were isolated from the coffee pulp obtained from a coffee estate. Pure cultures of fungi were isolated on standard conventional potato dextrose broth (PDB) medium and authenticated. Pure cultures were subjected to a caffeine tolerance study at different concentrations of caffeine (1–8 g/L) in potato dextrose agar (PDA) and minimal media. On PDA, Fusarium solani could tolerate caffeine concentration up to 8 g/L, whereas Chrysosporium keratinophilum, Gliocladium roseum, and Aspergillus restrictus could tolerate up to 6 g/L. On minimal agar medium containing different concentrations of caffeine (1–8 g/L), Fusarium solani tolerated up to 8 g/L and the other fungi up to 2 g/L. A time-bound caffeine degradation study was undertaken at 1 g/L concentration of caffeine and glucose in nitrogen-containing and nitrogen-free liquid minimal media by subjecting the four fungi to shake flask culture at 120 rpm and 30°C. Degradation of caffeine up to 7 days at 24-h intervals was analyzed by high-performance liquid chromatography (HPLC). Gliocladium roseum followed by Aspergillus restrictus showed maximum degradation of caffeine at 0.47 and 0.3 mg/ml, respectively, by 96 h in nitrogen-containing minimal medium, whereas Fusarium solani showed maximum degradation of caffeine by 48 h (0.35 mg/ml) and Chrysosporium keratinophilum by 72 h (0.29 g/ml). In nitrogen-free minimal medium, Chrysosporium keratinophilum showed maximum degradation of caffeine at 72 h (0.45 mg/ml), followed by Gliocladium roseum, Fusarium solani (0.3 mg/ml), and Aspergillus restrictus (0.25 mg/ml) at 96 h. Overall, Chrysosporium keratinophilum showed a comparatively higher rate of caffeine degradation in minimal medium with or without a nitrogen source as compared with the other three fungi, indicating that nitrogen affects caffeine metabolism.     

Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66–68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin–methotrexate (CDDP–MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66–68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66–68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.     

Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ～85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.     

Biometric ratio in estimating widths of maxillary anterior teeth derived after correlating anthropometric measurements with dental measurements

doi: 10.1111/j.1741‐2358.2012.00648.x Biometric ratio in estimating widths of maxillary anterior teeth derived after correlating anthropometric measurements with dental measurements Objective: To correlate dental measurements i.e. combined mesiodistal width of six maxillary anterior teeth with facial measurements i.e. inner canthal distance, interpupillary distance and intercommissural width and acquire a biometric ratio to serve as a preliminary guide in selection of the maxillary anterior teeth. Background: In the absence of pre‐extraction records, the resultant denture can lead to patient dissatisfaction towards the aesthetic appeal of their dentures. The maxillary anterior teeth play a pivotal role in denture aesthetics. Various techniques and biometric ratios have been described in literature for selection of the maxillary anteriors. This study derives a biometric ratio for the same, obtained after correlating anthropometric measurements with dental measurements. Materials and methods: Two standardized digital photographs of the face were generated; one, when the facial muscles were relaxed and the other, when the subject was smiling; thereby, revealing the maxillary anterior teeth upto the canine tip. Inner canthal distance, interpupillary distance, intercommissural distance, distance between the tips of the maxillary canines and distance between the distal surfaces of the canines were measured. On the cast, the distance between tips of maxillary canines and distance between distal surfaces of maxillary canines were noted. The data was analysed using Spearman’s rank correlation coefficient. Results: A high correlation was found between the intercommissural measurement with distance between the tips of the canines on the photograph and between the tips of the canines on the cast with the interpupillary distance, giving a biometric ratio of 1:1.35 and 1:1.41 respectively. The least correlation was between the inner canthal distance and the tips of the canines measured on the photograph. Conclusions: Extra oral anthropometric measurements of the interpupillary distances and the intercommissural distances with the help of standardised photographs can help us determine the combined widths of the anterior teeth accurately, thus aiding their selection in the absence of pre‐extraction records.     

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.     

Extraction and characterization of an exopolysaccharide from a marine bacterium

International Microbiology - The marine bacterial exopolysaccharides (EPS) have transfigured the biotech sector with their myriad applications and prospects. This work was carried out to...     

Genetic diversity of Indian garlic core germplasm using agro-biochemical traits and SRAP markers

The characterization of garlic germplasm improves its utility, despite the fact that garlic hasn't been used much in the past. Garlic has an untapped genetic pool of immense economic and medicinal value in India. Hence, using heuristic core collection approach, a core set of 46 accessions were selected from 625 Indian garlic accessions based on 13 quantitative and five qualitative traits. The statistical measures (CV per cent, CR per cent, VR per cent) were used to sort the core set using Shannon-Wiener diversity index and the Nei diversity index. In addition, the variation within the core set was tested for 18 agro-morphological and six biochemical characteristics (allicin, phenol content, pyruvic acid, protein, allyl methyl thiosulfinate (AMTHS), and methyl allyl thiosulfinate (MATHS)). Further study of the core set's molecular diversity was performed using sequence related amplified polymorphism (SRAP) markers, which revealed a wide range of diversity among the core set's accessions, with an average polymorphism efficiency (PE) of 80.59 percent, polymorphism information content (PIC) of 0.29, effective multiplex ratio (EMR) of 3.51, and marker index (MI) of 0.99. The findings of this study will be useful in identifying high-yielding, elite garlic germplasm lines with the trait of interest. Since this core set is indicative of total germplasm, these selected breeding lines will be used for genetic improvement of garlic in the future.     

Sphingosine,a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼3000 small molecules, including one comprising ∼600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.