A novel hydrophilic support, CoFoam, for enzyme immobilization

Lipases from different sources (porcine pancreas, Mucor miehei and Candida antarctica B) were covalently immobilized on a hydrophilic polyurethane composite (CoFoam). Their hydrolytic activities assayed with tributyrin were 0.55, 2.1 and 447 U g(-1), respectively. The activity of the C. antarctica B composite in the synthesis of methyl oleate in hexane was 8.8 U g(-1) compared to 60.6 U g(-1) for commercial Novozyme 435. The advantage of the CoFoam composite lies in the low pressure drop in a packed-bed reactor at fairly large flow rates. For example, at flow rates of 10-12 l min(-1), the pressure drop over 15 cm is typically 3 kPa.     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

MMDB: Entrez's 3D-structure database

Three-dimensional structures are now known within most protein families and it is likely, when searching a sequence database, that one will identify a homolog of known structure. The goal of Entrez's 3D-structure database is to make structure information and the functional annotation it can provide easily accessible to molecular biologists. To this end, Entrez's search engine provides several powerful features: (i) links between databases, for example between a protein's sequence and structure; (ii) pre-computed sequence and structure neighbors; and (iii) structure and sequence/structure alignment visualization. Here, we focus on a new feature of Entrez's Molecular Modeling Database (MMDB): Graphical summaries of the biological annotation available for each 3D structure, based on the results of automated comparative analysis. MMDB is available at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html.     

Novel drug designing approach for dual inhibitors as anti-inflammatory agents: implication of pyridine template

Compounds incorporating thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest, owing to the therapeutic utility of the template as useful drug molecular scaffolding. We report the synthesis and pharmacological evaluation of thiophenes substituted with 4-methanesulfonyl benzoyl moiety at the fifth position of the ring, as possible anti-inflammatory lead candidates. The aryl sulfonyl methyl thiophene analogs AP29, AP82, and AP37, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, exhibited moderate to good activity at a dose level of 100 mg/kg body weight P.o compared to Ibuprofen. In a five day formalin induced rat paw edema, a chronic in vivo anti-inflammatory model, candidates AP29, AP82, and AP37 inhibited the disease progression by 53%, 34%, and 65%, respectively on the fifth day, at a dose level of 100 mg/kg body weight P.o compared to Rofecoxib, Ibuprofen, and Dexamethasone at therapeutic doses which gave a protection of 53.8%, 81.5%, and 81.5%, respectively. The replacement of the 4-methanesulfonyl benzoyl moiety in AP82 with the pyridine template, 3,5-dimethyl-4-methoxy-2-pyridyl function, gave rise to AP84, which was less active in the acute model, but gave 54% and 75% protection both during the first day and fifth day, respectively, in the chronic model. A dual mechanism of action is proposed for AP84, a non-steroidal drug which has exhibited remarkable activity when compared to the steroid dexamethasone. These results open up new avenues in designing novel anti-inflammatory drugs as dual inhibitors with the incorporation of a pyridine template as part of the pharmacophore.     

The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals

Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.     

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: a 3.2 ns molecular dynamics simulation study

A dodecylphosphocholine micelle of 86 monomers with 5776 water molecules has been simulated under NPT conditions for 3.2 ns using GROMACS2.0. The micelle was found to be very dynamic. Some of the C-C bonds, independent of their position in the DPC monomer, adopt gauche conformation and the trans <--> gauche transitions are quite frequent. An average of about 11% of the C-C bonds in the micelle are observed to be in the gauche conformation (i.e., |dihedral angle|< 120 degrees). The terminal methyl groups are randomly distributed all over the micelle whereas the nitrogen atom of phosphocholine headgroup atoms is restricted to the interface region. Some of the monomers were found to lie on the surface. The shape of micelle, influenced by the packing considerations, shows deviations from spherical shape. The phosphocholine headgroup is well solvated and there is no water penetration into the micelle core. The overall features of the micelle of 86 DPC monomers conforms to the lattice model of micelle proposed by Dill and Flory [Dill K A, Flory P J (1981) Proc Natl Acad Sci USA 78, 676-680] and is similar to DPC micelles of smaller aggregate sizes except for the positional preference of the C-C bonds for the gauche conformation and the trans<-->gauche transition times [Tieleman D P, van der Spoel D, Berendsen H J C (2000) J Phys Chem B 104, 6380-6388; Wymore T, Gao X F, Wong T C (1999) J Mol Struct (Theochem) 485-486, 195-210]. It appears that packing considerations play a predominant role in determining the shape and dynamics of the micelle.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.