Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

X-ray crystallographic studies of HemK from Thermotoga maritima, an N5-glutamine methyltransferase

The enzyme HemK (or PrmC) is one of the first identified methyltransferases that modify glutamine. It methylates the highly conserved GGQ motif in class I release factors (RF1 and RF2) in Escherichia coli. HemK from Thermotoga maritima was over-expressed and crystallized in the presence of S-adenosylmethionine at 296 K using ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystal is orthorhombic, belonging to the space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters of a = 104.24, b = 118.73, and c = 146.62 A. Two (or three) monomers of recombinant HemK are likely to be present in the crystallographic asymmetric unit, giving a V(M) of 3.62 A3 Da(-1) (or 2.41 A3 Da(-1)), with a solvent content of 62.7% (or 44.0%).     

Dynamics of macrophage cell populations during murine pulmonary tuberculosis

The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b(-)/CD11c(+/high) were defined as alveolar macrophages, CD11b(high)/CD11c(+/high) as dendritic cells, and CD11b(+/mid)/CD11c(+/mid) as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.     

Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).     

Hydrazinocurcumin,a novel synthetic curcumin derivative,is a potent inhibitor of endothelial cell proliferation

Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel curcumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC(50)=520 nM) without cytotoxicity. In vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent.