Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.     

Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2(-/-)) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2(-/-)map1b(-/-) mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an "inside-out" pattern. Detailed observation of primary cultures of hippocampal neurons from map2(-/-)map1b(-/-) mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2(-/-)map1b(-/-) mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

New insights into the role of mitochondrial dysfunction and protein aggregation in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative movement disorder that affects increasing number of elderly in the world population. The disease is caused by a selective degeneration of dopaminergic neurons in the substantia nigra pars compacta with the molecular mechanism underlying this neurodegeneration still not fully understood. However, various studies have shown that mitochondrial dysfunction and abnormal protein aggregation are two of the major contributors for PD. In fact this notion has been supported by recent studies on genes that are linked to familial PD (FPD). For instance, FPD linked gene products such as PINK1 and parkin have been shown to play critical roles in the quality control of mitochondria, whereas α-synuclein has been found to be the major protein aggregates accumulated in PD patients. These findings suggest that further understanding of how dysfunction of these pathways in PD will help develop new approaches for the treatment of this neurodegenerative disorder.     

Catechin and hydroxybenzhydrols as models for the environmental photochemistry of tannins and lignins.

The photochemistry of several model plant-derived compounds has been studied in aqueous solution. In particular, the reactions of catechin as a model tannin and methoxy-substituted hydroxybenzhydrols as model lignin functionalities were investigated. Tannins and lignins constitute a significant portion of the humic substances in aquatic systems, which are themselves the main component of dissolved organic matter thought to be responsible for the absorption and attenuation of light in these environments. Catechin (1) was found to undergo a reversible photoisomerization reaction to give epicatechin (2). Such a reaction is an explicit example of a photon absorbing process that enables catechin (1) and its derivatives to act as natural sunscreens by attenuating light energy through non-destructive reactions. The methoxy-substituted hydroxybenzhydrols were found to undergo photosolvolysis reactions via efficient generation of quinone methide intermediates. The intermediate quinone methides were observed to be longer lived, and thus more stable, than previously studied hydroxybenzhydrol derivatives. The meta-hydroxybenzhydrol isomer (5) was found to undergo additional chemistry leading to the production of a ring-closed fluorene from the quinone methide intermediate.     

Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.