Isolation and Characterization of Dibenzofuran-Degrading <Emphasis Type="Italic">Serratia marcescens</Emphasis> from Alkalophilic Bacterial Consortium of the Chemostat

Alkalophilic bacterial consortium developed by continuous enrichment in the chemostat in presence of 4-chlorosalicylic acid as sole source of carbon and energy contained six bacterial strains, Micrococcus luteus (csa101), Deinococcus radiothilus (csa102), csa103 (Burkholderia gladioli), Alloiococcus otilis (csa104), Micrococcus diversus (csa105), Micrococcus luteus (csa106), identified by the Biolog test method. The strains were tested for utilization of organic compounds in which one of the strains (csa101) had higher potency to utilize dibenzofuran (DF) as sole carbon and energy source identified as Serratia marcescens on the basis of 16S rDNA. The degradation of DF by bacterial strain proceeded through an oxidative route as indicated by 2,2′3-trihydroxybiphenyl, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, salicylic acid, and catechol, which was identified by gas chromatography–mass spectrometry.     

New furofurano lignans from Justicia simplex

A new 3,7-dioxabicyclo[3,3,O]octane lignan, named justisolin, and a new lignan O-glucoside, named simplexoside, were isolated from the whole plant of Justicia simplex D. Don. (Acanthaceae), collected at fruiting. The structure of the free lignan was established as 2e-(3,4-methylenedioxy-6-hydroxy)-phenyl-6e- Piperonyl-3,7-dioxabicyclo[3,3,0]octane (1) and that of the glucoside as 2e-(3-methoxy-4-O-β-d- glucopyranosyl)-phenyl-6e-piperonyl-3,7-dioxabicyclo[3,3,0]octane (2) on the basis of chemical transformation and spectral evidence. The biological functions of these and related lignans are appraised.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.     

Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.     

NRH:quinone oxidoreductase2 (NQO2)

The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.