A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Seizure Outcomes and Predictors of Recurrent Post-Stroke Seizure: A Retrospective Observational Cohort Study

Background

Seizure is a common complication after stroke (termed “post-stroke seizure,” PSS). Although many studies have assessed outcomes and risk factors of PSS, no reliable predictors are currently available to determine PSS recurrence. We compared baseline clinical characteristics and post-stroke treatment regimens between recurrent and non-recurrent PSS patients to identify factors predictive of recurrence.

Methods

Consecutive PSS patients admitted to our stroke center between January 2011 and July 2013 were monitored until February 2014 (median 357 days; IQR, 160–552) and retrospectively evaluated for baseline clinical characteristics and PSS recurrence. Cumulative recurrence rates at 90, 180, and 360 days post-stroke were estimated by Kaplan—Meier analysis. Independent predictors of recurrent PSS were identified by Cox proportional-hazards analysis.

Results

A total of 104 patients (71 men; mean age, 72.1 ± 11.2 years) were analyzed. PSS recurred in 31 patients (30%) during the follow-up. Factors significantly associated with PSS recurrence by log-rank analysis included previous PSS, valproic acid (VPA) monotherapy, polytherapy with antiepileptic drugs (AEDs), frontal cortical lesion, and higher modified Rankin Scale score at discharge (all p < 0.05). Independent predictors of recurrent PSS were age <74 years (HR 2.38, 95% CI 1.02–5.90), VPA monotherapy (HR 3.86, 95% CI 1.30–12.62), and convulsions on admission (HR 3.87, 95% CI 1.35–12.76).

Conclusions

Approximately one-third of PSS patients experienced seizure recurrence within one year. The predictors of recurrent PSS were younger age, presence of convulsions and VPA monotherapy. Our findings should be interpreted cautiously in countries where monotherapy with second-generation AEDs has been approved because this study was conducted while second-generation AEDs had not been officially approved for monotherapy in Japan.     

Phylogeny and phylogeography of the sword-tailed newt,Cynops ensicauda (Amphibia: Caudata), as revealed by nucleotide sequences of mitochondrial DNA

We investigated the phylogenetic relationships and phylogeography among individuals of the endemic newt (Cynops ensicauda) of the Central Ryukyus, Japan based on the mitochondrial cytochrome b gene. The results of phylogenetic analyses showed the presence of remarkable differentiation between assemblages from the Amami and Okinawa Island Groups, supporting the validity of the subspecies C. e. popei described from the latter on morphological grounds. The group of individuals from Okinawa Island Group was further divided into two distinct subgroups unlike the results of previous morphological and allozyme studies. Geographic ranges of these subgroups overlapped in the northern part of Okinawajima Island. The phylogeographic pattern within the Okinawa Island Group suggests an initial division into two geographically isolated population lineages and subsequent secondary sympatry before formation of reproductive isolation. It is also likely that within the Okinawa Island Group emigration occurred from the central and northern parts of Okinawajima Island to its southern part, as well as to several small islets off its western coast. Within the Amami Island Group, recurring restricted gene flow with isolation by distance seems to have occurred around the southwestern region including three islets southwest of Amamioshima Island.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.     

Taxonomic relationships of Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), with a description of a new genus (Amphibia, Bufonidae)

Examination of types and recently collected specimens revealed that Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), both described from Kinabalu, Sabah, Malaysia, are hardly differentiated morphologically. Analyses of a total of 2,427 bp of the 12S rRNA, tRNA(val), and 16S mitochondrial rRNA genes revealed that the two species are very close genetically. Thus A. anotis is regarded as conspecific and is synonymized with P. maculatus. Genetically, this species proved to form a lineage distinct from other bufonids from Southeast Asia, including species of Ansonia and Pedostibes. Because the species has also some unique morphological traits different from known bufonid genera, we propose to establish a new genus for Nectophryne maculata Mocquard, 1890.     

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.