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.     

Stronger anti-HIV-1 activity of C-peptide derived from HIV-1 89.6 gp41 C-terminal heptad repeated sequence

C34-LAI containing amino acids 118 to 151 of the HIV-1(LAI) gp41 ectodomain exhibits potent anti-HIV-1 activity. However, the N-terminal halves of C34 peptides vary more according to the HIV-1 strain than the C-terminal halves. Therefore, an analysis was conducted on the anti-HIV-1 activities of the C34 peptides derived from various HIV-1 strains. C34-89.6 exhibited the strongest anti-HIV-1 activity among the C34 peptides tested. Interestingly, its N-terminal half was more acidic than those of the other C34 peptides, whereas its C-terminal half was more basic. Since the C-peptides derived from the HIV-1(LAI) strain are used extensively, the anti-HIV-1 activities of these peptides were compared between the HIV-1 strains 89.6 and LAI. When using chimeric peptides, it was found that the C-terminal basic region of C34-89.6 was more critical than its N-terminal basic region. The anti-HIV-1 activity of T20-89.6 and C28-89.6 was also stronger than that of T20-LAI and C28-LAI, respectively. The anti-HIV-1 activity of C28-89.6 was weakened when the C-terminal basic residues were changed to the corresponding residues of C28-LAI. However, no conformational differences were found among the C28 peptides. Accordingly, these results imply that introducing the C-terminal basic residues of the HIV-1 89.6 C-peptide may be useful for developing potent anti-HIV-1 drugs.     

Clinical relevance of glycerophospholipid,sphingomyelin and glutathione metabolism in the pathogenesis of pharyngolaryngeal cancer in smokers: the Korean Cancer Prevention Study-II

Introduction

Although smoking is a major risk factor for pharyngolaryngeal cancer, most smokers do not develop pharyngolaryngeal cancer.

Objectives

In the prospective Korean Cancer Prevention Study-II (KCPS-II), we investigated the application of metabolomics to differentiate smokers with incident pharyngolaryngeal cancer (pharyngolaryngeal cancer group) from smokers who remained free from cancer (controls) during a mean follow-up period of 7 years and aimed to discover valuable early biomarkers of pharyngolaryngeal cancer.

Methods

We used baseline serum samples from 30 smoking men with incident pharyngolaryngeal cancer and 59 age-matched cancer-free smoking men. Metabolic alterations associated with the incidence of pharyngolaryngeal cancer were investigated by performing metabolomics on baseline serum samples using ultra-performance liquid chromatography-linear-trap quadrupole-Orbitrap mass spectrometry.

Results

Compared to the control group, the pharyngolaryngeal cancer group showed significantly higher oxidized LDL levels. Seventeen metabolites were differentially abundant between the two groups. At baseline, compared to controls, smokers with incident pharyngolaryngeal cancer during follow-up showed significantly higher levels of pyroglutamic acid (glutathione metabolism) but lower levels of lysophosphatidylcholines (lysoPCs) C14:0, C15:0, C16:0, C17:0, C18:0, and C20:5; glycerophosphocholine; PC C36:5; lysoPEs C16:0, C20:1, and C22:0 (glycerophospholipid metabolism); SM (d18:0/16:1); and SM (d18:1/18:1) (sphingomyelin metabolism). Furthermore, smokers with incident pharyngolaryngeal cancer showed significantly higher levels of oleamide and lower levels of tryptophan and linoleyl carnitine at baseline than cancer-free smokers.

Conclusion

This prospective study showed the clinical relevance of dysregulated metabolism of glutathione, glycerophospholipids and sphingolipids to the pathogenesis of pharyngolaryngeal cancer among smokers. These data suggest that the dysregulation of these metabolic processes may be a key mechanism underlying pharyngolaryngeal cancer progression and development.

     

To the Final Goal: Can We Predict and Suggest Mutations for Protein to Develop Desired Phenotype?

Directed evolution of proteins is a good approach to develop desired phenotypes from existing proteins. Fully experimental protein evolution usually utilizes randomization of a given protein sequence by error-prone PCR or gene shuffling followed by high-throughput selection or timeconsuming screening method. However, these random methods create mutant library full of deleterious mutations. In addition, they need high-throughput screening or selection method to search for positive clones from an enormous size of mutant library. Construction of a mutant library while retaining the original function is important for efficient protein evolution because it greatly reduces time and effort for the identification of positive mutants. Therefore, researchers have tried to reduce the size of mutant library by minimizing the occurrence of deleterious mutants. Such efforts have led to the creation of a concept of ‘small but smart library’. For this goal, neutral drift theory has been applied. Although smart library greatly reduces the library size, it is still the beyond the capacity of low-throughput assay. In parallel, computational analysis of protein structure and efforts to discriminate mutatable residues from all residues of a given protein have been consistently pursued. Accumulated knowledge of protein evolution through random mutation and selection has improved our understanding of functions of amino acids in protein structure. Protein evolution by rational design is being developed based on such understanding. In this review, we describe how the use of semi-rationally designed library rather than completely random one has impacted the overall procedure of directed evolution. We also describe efforts made to evaluate the effect of single mutation. Such efforts will bring lazy boys to the final goal - computational mutation suggestion system.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.