Mab_3168c,a Putative Acetyltransferase,Enhances Adherence,Intracellular Survival and Antimicrobial Resistance of Mycobacterium abscessus

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.     

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.     

Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation

A novel method for selection of transgenic plants utilizing the sweet pepper ( Capsicum annuum L.) ferredoxin-like protein ( pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (beta-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.     

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 microl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.     

Screening and production of erythritol by newly isolated osmophilic yeast-like fungi

Twenty-eight erythritol-producing strains were isolated from pollen, honey and high sugar food samples collected in Taiwan. Amongst these, six strains (166-2, 262-1, 278-3, 440, 441 and 442) were high erythritol-producers with a yield higher than 30% for 30% glucose. The erythritol productivity of these strains ranged from 90.9 to 116.4 g l⁻¹. ¹H- and ¹³C-NMR analyses confirmed that the fermentation product was erythritol. The results of morphological and physiological studies indicate that strains 166-2, 262-1, 278-3, 440, and 442 may be members of the genus Moniliella. More studies are required to determine the taxonomic position of strain 441. The use of a medium containing 30% glucose and 1% yeast extract gave the highest erythritol productivity. On batch fermentation in a 5-l fermentor using strain 166-2, a maximal erythritol productivity of 111.0 g l⁻¹ was obtained after cultivation for 144 h.     

Quantification of surfactant phospholipids in the dog lung

We quantified total phospholipid (PL), total and disaturated phosphatidylcholine (PC and DSPC), phosphatidylglycerol (PG), and total protein in alveolar washings and lung tissue in 22 dog lungs. Quantitative recovery of alveolar material and assessment of its possible contamination by blood lipids were important determinants of methodology. To remove blood, the vessels of half the lungs were perfused with a fluorocarbon emulsion before lavage. The volume of blood removed by perfusion and the quantity and fatty acid patterns of its whole blood and plasma PL and PC were determined. Washings of unperfused lungs contained means of 21% more PL and 24% more PC than those of perfused lungs. Although this excess could be accounted for by the PL and PC in pulmonary blood, the hemoglobin and total protein content of washings and their PC fatty acid patterns indicated that blood lipids were not a major source of the excess lipid in washings of unperfused lungs. Using more recent morphometric estimates rather than the indirect ones previously used by others, the quantity of alveolar DSPC (1 mg/g lung) is calculated to be 1.8 times the amount necessary to form a packed monolayer on the internal surface of the lung at functional residual capacity.     

3,5-Diaryl-1H-pyrazole as a molecular scaffold for the synthesis of apoptosis-inducing agents

The scaffold of 3,5-diaryl-1H-pyrazole was selected as a molecular template to synthesize novel growth-inhibitory agents in the present study. Our findings suggested that analogs bearing electron-withdrawing groups on one ring while electron-donating groups on another reveal significant activities. In particular, 26 bearing a 1,1′-biphenyl moiety displayed the most potent activity against OVCA, SW620, H460 and AGS cells with GI₅₀ values of 0.67, 0.89, 0.73 and 0.79 μM, respectively. The mechanistic study revealed that 26-mediated apoptosis-inducing effect on OVCA cells was, in part, attributed to the inhibition of protein kinase B/Akt activity, accompanied by the mitochondrial apoptotic pathway through the activation of caspase-9, caspase-3, as well as the cleavage of protein poly(ADP-ribose) polymerase (PARP) and DNA fragmentation. Further structure–activity relationship study employed by Comparative Molecular Field Analysis (CoMFA) was carried out with q² and R² values of 0.671 and 0.846, respectively.