Protein kinase CK2 is inhibited by human nucleolar phosphoprotein p140 in an inositol hexakisphosphate-dependent manner

Protein kinase CK2 is a ubiquitous protein kinase that can phosphorylate various proteins involved in central cellular processes, such as signal transduction, cell division, and proliferation. We have shown that the human nucleolar phosphoprotein p140 (hNopp140) is able to regulate the catalytic activity of CK2. Unphosphorylated hNopp140 and phospho-hNopp140 bind to the regulatory and catalytic subunits of CK2, respectively, and the interaction between hNopp140 and CK2 was prevented by inositol hexakisphosphate (InsP(6)). Phosphorylation of alpha-casein, genimin, or human phosphatidylcholine transfer protein-like protein by CK2 was inhibited by hNopp140, and InsP(6) recovered the suppressed activity of CK2 by hNopp140. These observations indicated that hNopp140 serves as a negative regulator of CK2 and that InsP(6) stimulates the activity of CK2 by blocking the interaction between hNopp140 and CK2.     

Detection of high-molecular-weight glutenin subunit genes for 1Dx2 and 1Dx5 using loop-mediated isothermal amplification assay

High-molecular-weight glutenin subunits (HMW-GS) in wheat grain are the major determinants of dough elasticity and viscosity and thus of bread-making quality. PCR-based molecular markers designed based on DNA polymorphisms were used to analyze HMW-GS genes in wheat. The loop-mediated isothermal amplification (LAMP) assay is a simple and rapid method for specific detection of genomic DNA target sequences. In the present study, we designed a set of LAMP markers by targeting the unique sequences of 1Dx2 and 1Dx5 genes. The primers could effectively distinguish the 1Dx2 and 1Dx5 genes from other genes at the Glu-1 locus. The results were confirmed by agarose gel electrophoresis. For visualization, ethidium bromide was used, and fluorescence only appeared in the positive samples. Under optimal conditions, the detection could be finished in 1 h. Thirty-eight wheat cultivars with known HMW-GS were used to validate LAMP markers for 1Dx2 and 1Dx5 genes. Only DNA samples with target genes could be amplified, and the results could be read easily using this method. The tests using LAMP were easy to perform, rapid, and sensitive. Thus, the current study results have the potential to be a powerful tool for the detection of HMW-GS genes in wheat.     

CDK11p46 and RPS8 associate with each other and suppress translation in a synergistic manner

CDK11p46, a 46 kDa isoform of the PITSLRE kinase family, is a key mediator of cell apoptosis, while the precise mechanism remains to be elucidated. By using His pull-down and mass spectrometry analysis, we identified the ribosomal protein S8 (RPS8), a member of the small subunit ribosome, as an interacting partner of CDK11p46. Further analysis confirmed the association of CDK11p46 and RPS8 in vitro and in vivo, and revealed that RPS8 was not a substrate of CDK11p46. Moreover, RPS8 and CDK11p46 synergize to inhibit the translation process both in cap- and internal ribosomal entry site (IRES)-dependent way, and sensitize cells to Fas ligand-induced apoptosis. Taken together, our results provide evidence for the novel role of CDK11p46 in the regulation of translation and cell apoptosis.     

GluR1 phosphorylation and persistent expression of levodopa-induced motor response alterations in the Hemi-Parkinsonian rat

The phosphorylation of glutamate receptor 1 (GluR1) has been increasingly implicated in the formation and maintenance of plastic responses. To investigate molecular mechanisms that underlie the persisting alterations in motor response occurring with levodopa treatment of parkinsonian patients, we evaluated the time course of these changes in relation to the phosphorylation of GluR1 in 6-hydroxydopamine (6-OHDA) lesioned animals. Three weeks of twice-daily levodopa administration to rats shortened the duration of the rotational responses and increased the peak turning responses, which lasted at least 7 days after withdrawal of chronic levodopa treatment. The shortened response duration and increased peak turning, resembling human wearing-off fluctuations and dyskinesia, were associated with a marked increase in Ser-845 phosphorylated GluR1 (pGluR1S845) immunoreactivity in lesioned striatum in response to levodopa treatment. The time course of changes in GluR1 phosphorylation correlated with the time course of changes in motor behavior after withdrawal of chronic levodopa therapy. Our immunostaining data showed that these changes were confined to parvalbumin-positive neurons where GluR1 are exclusively expressed. Both the altered motor response and the degree of pGluR1S845 were attenuated by the intrastriatal administration of protein kinase A (PKA) inhibitor Rp-cAMPS or GluR1 antisense oligonucleotides. The results suggest that Ser-845 GluR1 phosphorylation within parvalbumin-positive neurons contributes to the persistence of the motor response alterations produced by chronic intermittent dopaminergic stimulation.     

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Development of ELISA for Detection of Mercury Based on Specific Monoclonal Antibodies Against Mercury-Chelate

Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31?×?10⁹ L/mol and its cross-reactivity (CR) with other metals was <2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg²⁺ measurements. The detection range was 0.087–790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.