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.     

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).     

Lipophilic antioxidants in human sebum and aging

Skin surface lipids (SSL), a very complex mixture of sebum mixed to small amounts of epidermal lipids, mantle the human epidermis, thus representing the outermost protection of the body against exogenous oxidative insults. The present work is a systematic and quantitative analysis of upper-chest SSL and their content in antioxidants in 100 healthy volunteers, divided into five age groups using TLC, HPLC, and GC-MS methods. Further, the effect of exposing SSL in vitro to increasing doses of UV irradiation was examined. Straight monounsaturated and diunsaturated as well as branched monounsaturated fatty acids of triglycerides and pooled fractions were found to be higher at maturity than in childhood and in advancing age. Diunsaturated fatty acids were below 3% of the total and constituted exclusively of C18:2 Δ5,8 , C20:2 Δ7,10 , C18:2 Δ9,12 . Squalene, vitamin E (vit. E) and Coenzyme Q 10 (CoQ 10 ) were found to increase from childhood to maturity to decrease again significantly in old age. Vitamin E and CoQ 10 were the only known lipophilic antioxidants present in SSL. In spite of their low levels they were found to synergically inhibit the UV induced depletion of squalene, cholesterol and of unsaturated fatty acids of SSL. In fact, exposure of SSL to increasing amounts of UV irradiation led preferentially to lowering of the levels of vit. E and CoQ 10 . Four minimal erythema dose (MED) (5.6 J/cm 2 ) were able to deplete 84% vit. E and 70% ubiquinone, and only 13% squalene. Diunsaturated and monounsaturated fatty acids as well as cholesterol were unaffected even following 10 MED UV exposures, which produced a 26% loss of squalene. The same UV dose when applied in the absence of vit. E and CoQ 10 produced a 90% decrease of squalene.     

Electrophoretic evidence for homoeologous chromosome pairing in the apogamous fern species<Emphasis Type="Italic"> Dryopteris nipponensis</Emphasis> (Dryopteridaceae)

Segregation of genotypes through homoeologous chromosome pairing in the apogamous species Dryopteris nipponensis was tested by electrophoretic analysis. Of 284 progeny examined (250 gametophytes and 34 sporophytes), from the parental sporophyte with the Pgi-2 genotype abc, five showed different genotypes from that of the parent (three aac, one bbc and one bcc). This is the first evidence for genetic segregation in the progeny of apogamous fern species. Electronic Publication     

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.     

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.     

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.     

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

1. Download : Download high-res image (211KB)
2. Download : Download full-size image

     

Tyr682 in the Aβ‐precursor protein intracellular domain regulates synaptic connectivity,cholinergic function,and cognitive performance

Processing of Aβ‐precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the ⁶⁸²YENPTY⁶⁸⁷ motif. To dissect the functions of this sequence in vivo, we created an APP knock‐in allele mutating Y⁶⁸² to Gly (APP^YG/YG mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP‐like protein 2 KO background. This evidence prompted us to characterize further the APP^YG/YG mice. Here, we show that APP^YG/YG mice develop aging‐dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP^YG/YG mice. The profound deficits caused by the Y⁶⁸² mutation underscore the biological importance of APP and indicate that APP^YG/YG are a valuable mouse model to study APP functions in physiological and pathological processes.