Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1alpha with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.     

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.     

Characterization of a Novel Cr6+ Reducing Pseudomonas sp. with Plant Growth–Promoting Potential

The isolate RNP4 obtained from a long-term tannery waste contaminated soil was characterized and presumptively identified as Pseudomonas sp. The strain RNP4 tolerated concentrations up to 450 mg Cr⁶⁺/L on a Luria-Bartani (LB) agar medium and reduced a substantial amount of Cr⁶⁺ to Cr³⁺ in the LB liquid medium. The ability of performing multifarious activities in tandem suggested the uniqueness of isolate RNP4. The strain produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. The strain also exhibited the production of siderophore and solubilization of phosphorus in mineral salt medium and SRS1 medium, respectively. Concurrent production of IAA and siderophore and the solubilization of phosphorus revealed its plant growth promotion potential. Furthermore, the strain was able to promote the growth of black gram, Indian mustard, and pearl millet in the presence of Cr⁶⁺. Thus, the innate capability of this novel isolate for parallel bioremediation and plant growth promotion has significance in the management of environmental and agricultural problems.     

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231–Pro232 and Thr212–Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3β phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Aβ-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-β APP accumulation than C83-βAPP in APPsw-tansfectant and thereby promoted Aβ-42 production in transfectants. (ii) the inhibition of tau and GSK3β phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3β for protecting against Alzheimer’s disease.     

Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori

To investigate the pathogenicity of Pseudomonas aeruginosa in insects, a gacA mutant of P. aeruginosa PA01 was constructed by site-directed mutagenesis. The mutant was designated as C1. C1 was less virulent to Bombyx mori than the parent strain. To complement the gacA gene, P. aeruginosa C1 was transformed with the broad host range plasmid pJB3Km1 carrying a 3.9-kbp gacA fragment. The expression of the gacA mRNA in C1 (pgacA) was detected. In addition, the complemented mutant restored the level and timing of pyocyanin production, indicating that functional GacA is produced in the complemented strain. However, no significant difference was observed between C1 and C1 (pgacA) with respect to the killing of B. mori larvae.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)

The complete nucleotide sequences of the mitochondrial genome (mitogenome) of the oriental mole cricket, Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), were determined. The 15,521-bp-long G. orientalis mitogenome contains typical gene complement, base composition, and codon usage found in metazoan mitogenomes. The G. orientalis mitogenome contains the third lowest A+T content (70.5%) among the complete insects mt genome sequences. The initiation codon for the G. orientalis COI gene appears to be ATG, instead of the tetranucleotides, which have been postulated to act as initiation codon for Locusta migratoria and some lepidopteran COI genes. The initiation codon for ND2 appears to be GTG, which is rare, but has been designated as an initiator of Tricholepidion gertschi ND2. All anticodons of G. orientalis tRNAs were identical to Drosophila yakuba and L. migratoria. The tRNA(Ser)(AGN) could not form a stable stem loop structure in the DHU arm as shown in many other insect tRNA(Ser)(AGN). Phylogenetic analysis of nucleotide sequence information from all mt genes supported a monophyletic Diptera, a monophyletic Lepidoptera, a monophyletic Coleoptera, a monophyletic Mecopterida (Diptera+Lepidoptera), and a monophyletic Endopterygota (Diptera+Lepidoptera+Coleoptera), suggesting that the complete insect mitogenome sequence has a resolving power to the diversification events within Endopterygota. However, the relationships of ancient insect orders were unstable, indicating the limited use of mitogenome information at deeper phylogenetic depth.     

Serological investigation of Theileria sergenti using latex agglutination test in South Korea

Theileria sergenti causes persistent theileriosis in cattle, characterized by fever and chronic anemia. Theileriosis causes losses in feed efficiency and growth retardation through cycling infections in endemic areas. Among several major proteins of T. sergenti merozoites, the surface protein p33 is reported to be the most immunogenic. In this study, we investigated the use of p33 as a diagnostic antigen in a latex agglutination test to monitor antibodies against T. sergenti. When compared with TaqMan polymerase chain reaction, the sensitivity and specificity of the latex agglutination test were 86.5 and 92.5%, respectively. An epidemiological survey using the latex agglutination test was conducted with 1,046 sera collected from 4 slaughterhouses and 2 individual pasture farms throughout South Korea; 27.3% of samples were seropositive, depending on the areas in which the cattle were raised. This study indicated that the latex agglutination test could be used as a convenient tool for epidemiological monitoring of T. sergenti infections in the field.     

Virion proteins of Kaposi's sarcoma-associated herpesvirus

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.