Mutations in influenza virus M1 CCHH, the putative zinc finger motif, cause attenuation in mice and protect mice against lethal influenza virus infection

Mutations in CCHH, the putative zinc finger motif, apparently do not play an important role in virus replication in MDCK cells in culture (E. K.-W. Hui, K. Ralston, A. K. Judd, and D. P. Nayak, J. Gen. Virol. 84:3105-3113, 2003). In this report, however, we demonstrate that the CCHH motif plays a critical role in virulence in mice and that some CCHH mutants are highly attenuated in BALB/c mice. Some of the mutant viruses replicated the least in mice lungs, induced little or no lung lesions, and caused highly reduced morbidity and mortality. Furthermore, growth patterns of mutant viruses in different cell lines (MDCK, MLE12, 3LL, A549, and 293T) varied. Mutant viruses that were attenuated in mice also grew poorly in mouse and human cells in culture. However, wild-type (WT) and all mutant viruses replicated to the same titer in MDCK (canine) cells or embryonated chicken eggs. Attenuation in mice correlated with reduced growth in mouse cells in culture, suggesting that potential attenuation in a given host can be predicted from the growth characteristics of the virus in cultured cells (preferably lung cells) from the same species. In challenge experiments, mice immunized by infection with attenuated mutant viruses were fully protected from lethal challenge with WT virus. In summary, the replication and attenuating properties of these mutants suggest that the CCHH motif provides a critical determinant for virulence in mouse and that mutations in the CCHH motif yield potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines.     

YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42

Earlier studies have shown that the C-terminal half of helix 6 (H6) of the influenza A virus matrix protein (M1) containing the YRKL sequence is involved in virus budding (E. K.-W. Hui, S. Barman, T. Y. Yang, and D. P. Nayak, J. Virol. 77:7078-7092, 2003). In this report, we show that the YRKL sequence is the L domain motif of influenza virus. Like other L domains, YRKL can be inserted at different locations on the mutant M1 protein and can restore virus budding in a position-independent manner. Although YRKL is a part of the nuclear localization signal (NLS), the function of YRKL was independent of the NLS activity and the NLS function of M1 was not required for influenza virus replication. Some mutations in YRKL and the adjacent region caused a reduction in the virus titer by blocking virus release, and some affected virus morphology, producing elongated particles. Coimmunoprecipitation and Western blotting analyses showed that VPS28, a component of the ESCRT-I complex, and Cdc42, a member of the Rho family GTP-binding proteins, interacted with the M1 protein via the YRKL motif. In addition, depletion of VPS28 and Cdc42 by small interfering RNA resulted in reduction of influenza virus production. Moreover, overexpression of dominant-negative Cdc42 inhibited influenza virus replication, whereas a constitutively active Cdc42 mutant enhanced virus production in infected cells. These results indicated that VPS28, a component of ESCRT-I, and Cdc42, a small G protein, are associated with the M1 protein and involved in the influenza virus life cycle.     

Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50?mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50?°C and 50?mM, respectively. The enzyme was quite stable for more than 6?h at 30?°C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50?mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120?min, respectively, using 5?mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383?mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.     

Acyclovir-Polyethylene Glycol 6000 Binary Dispersions: Mechanistic Insights

The dissolution and subsequent oral bioavailability of acyclovir (ACY) is limited by its poor aqueous solubility. An attempt has been made in this work to provide mechanistic insights into the solubility enhancement and dissolution of ACY by using the water-soluble carrier polyethylene glycol 6000 (PEG6000). Solid dispersions with varying ratios of the drug (ACY) and carrier (PEG6000) were prepared and evaluated by phase solubility, in vitro release studies, kinetic analysis, in situ perfusion, and in vitro permeation studies. Solid state characterization was done by powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) analysis, and surface morphology was assessed by polarizing microscopic image analysis, scanning electron microscopy, atomic force microscopy, and nuclear magnetic resonance analysis. Thermodynamic parameters indicated the solubilization effect of the carrier. The aqueous solubility and dissolution of ACY was found to be higher in all samples. The findings of XRD, DSC, FTIR and NMR analysis confirmed the formation of solid solution, crystallinity reduction, and the absence of interaction between the drug and carrier. SEM and AFM analysis reports ratified the particle size reduction and change in the surface morphology in samples. The permeation coefficient and amount of ACY diffused were higher in samples in comparison to pure ACY. Stability was found to be higher in dispersions. The results suggest that the study findings provided clear mechanical insights into the solubility and dissolution enhancement of ACY in PEG6000, and such findings could lay the platform for resolving the poor aqueous solubility issues in formulation development.     

A catabolic pathway for the degradation of chrysene by Pseudoxanthomonas sp. PNK-04

The chrysene-degrading bacterium Pseudoxanthomonas sp. PNK-04 was isolated from a coal sample. Three novel metabolites, hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid and salicylic acid, were identified by TLC, HPLC and MS. Key enzyme activities, namely 1-hydroxy-2-naphthoate hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase, were noted in the cell-free extract. These results suggest that chrysene is catabolized via hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid, salicylic acid and catechol. The terminal aromatic metabolite, catechol, is then catabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed catabolic pathway for chrysene degradation by strain PNK-04 is chrysene → hydroxyphenanthroic acid → 1-hydroxy-2-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol →cis,cis-muconic acid.     

Identification and Characterization of Toxigenic Fusaria Associated with Sorghum Grain Mold Complex in India

Fusarium species are dominant within the sorghum grain mold complex. Some species of Fusarium involved in grain mold complex produce mycotoxins, such as fumonisins. An attempt was made to identify Fusarium spp. associated with grain mold complex in major sorghum-growing areas in India through AFLP-based grouping of the isolates and to further confirm the species by sequencing part of α-Elongation factor gene and comparing the sequences with that available in the NCBI database. The dendrogram generated from the AFLP data clustered the isolates into 5 groups. Five species of Fusarium—F. proliferatum, F. thapsinum, F. equiseti, F. andiyazi and F. sacchari were identified based on sequence similarity of α-Elongation factor gene of the test isolates with those in the NCBI database. Fusarium thapsinum was identified as predominant species in Fusarium—grain mold complex in India and F. proliferatum as highly toxigenic for fumonisins production. Analysis of molecular variance (AMOVA) revealed 54% of the variation in the AFLP patterns of 63 isolates was due to the differences between Fusarium species, and 46% was due to differences between the strains within a species.     

Characterization of Recombinant Terrelysin,a Hemolysin of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly β-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the β-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.     

Low dose Estrogen Prevents Neuronal Degeneration and Microglial Reactivity in an Acute Model of Spinal Cord Injury: Effect of Dosing,Route of Administration,and Therapy Delay

Spinal cord injury (SCI), depending on the severity of injury, leads to neurological dysfunction and paralysis. Methylprednisolone, the only currently available therapy renders limited protection in SCI. Therefore, other therapeutic agents must be tested to maximize neuroprotection and functional recovery. Previous data from our laboratory indicate that estrogen (17β-estradiol) at a high dose may attenuate multiple damaging pathways involved in SCI and improve locomotor outcome. Since use of high dose estrogen may have detrimental side effects and therefore may never be used in the clinic, the current study investigated the efficacy of this steroid hormone at very low doses in SCI. In particular, we tested the impact of dosing (1–10 μg/kg), mode of delivery (intravenous vs. osmotic pump), and delay in estrogen application (15 min–4 h post-SCI) on microgliosis and neuronal death in acute SCI in rats. Treatment with 17β-estradiol (1–10 μg/kg) significantly reduced microglial activation and also attenuated apoptosis of neurons compared to untreated SCI animals. The attenuation of cell death and inflammation by estrogen was observed regardless of mode and time of delivery following injury. These findings suggest estrogen as a potential agent for the treatment of individuals with SCI.