Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp-ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae).     

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.     

Robust and systematic drug screening method using chemical arrays and the protein library: identification of novel inhibitors of carbonic anhydrase II

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.     

Modal gating of human CaV2.1 (P/Q-type) calcium channels: I. The slow and the fast gating modes and their modulation by beta subunits

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.     

Prevalence and genetic properties of Salmonella enterica serovar typhimurium definitive phage type 104 isolated from Rattus norvegicus and Rattus rattus house rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria

Bcl-2 protein play important roles in the regulation of apoptosis. We previously reported that the phosphorylation of Bcl-2 was augmented by treatment with protein phosphatase 2A (PP2A) inhibitor; however, the kinase responsible for Bcl-2 phosphorylation had not yet been identified. In this study, we identified extracellular-signal-regulated kinase (ERK) as the responsible kinase for the phosphorylation of Bcl-2. We also found that the transmembrane region (TM) deleted form of Bcl-2 (Bcl-2DeltaTM), which was unable to localize on the mitochondria was constitutively phosphorylated, whereas wild-type Bcl-2 that localized on the mitochondria, was present in its hypophosphorylated form. The phosphorylation of Bcl-2DeltaTM was retarded by treatment with MAP kinase ERK kinase (MEK) inhibitor and PP2A did not bind to Bcl-2DeltaTM. These observations suggest that Bcl-2DeltaTM is constitutively phosphorylated by ERK, but is not dephosphorylated by PP2A in human tumor cell lines. The phosphorylation of Bcl-2 resulted in a reduction in anti-apoptotic function, implying that dephosphorylation promoted the anti-apoptotic activity of Bcl-2 protein in human tumor cell lines. Thus, the present findings suggest that ERK and PP2A are physiological regulators of Bcl-2 phosphorylation, and these enzymes exert an influence on the anti-apoptotic function of Bcl-2.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.