Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction

Previous studies have convincingly argued that reactive oxygen species (ROS ) contribute to the development of several major types of sensorineural hearing loss, such as noise‐induced hearing loss (NIHL ), drug‐induced hearing loss, and age‐related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG ) mouse line expressing the human NADPH oxidase 4 (NOX 4, NOX 4‐ TG mice), which is a constitutively active ROS ‐producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX 4 ‐TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high‐frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHC s). The vulnerability to loss of hearing function and OHC s was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat‐shock protein 47 (HSP 47) in models using HEK 293 cells, including H₂O₂ treatment and cells with stable and transient expression of NOX 4. Furthermore, the up‐regulated levels of Hsp47 were observed in both the cochlea and heart of NOX 4 ‐TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL . Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo , and counteracting ROS ‐related hearing loss.

     

Molecular phylogeny and taxonomic implications of Asarum (Aristolochiaceae) based on ITS and matK sequences

The genus Asarum (Aristolochiaceae) encompasses approximately 120 species from five sections. Taxonomic controversies concerning the genus Asarum and/or its intrageneric classification remain unresolved. In particular, sect. Heterotropa accounts for a large percentage of the genus (80 of 120 species) and is well diverged in the Sino–Japanese Forest subkingdom. Reconstruction of Heterotropa phylogeny and estimation of its divergence times would provide significant insight into the process of species diversity in the Sino–Japanese floristic region. This study encompassed 106 operational taxonomic units (OTUs), and phylogenetic analyses were conducted based on internal transcribed spacer (ITS) and matK sequences. Although the matK sequences provided informative results solely for section Geotaenium, phylogenetic trees based on ITS regions yielded a clear result for several sections. Three sections, Asarum, Geotaenium and Asiasarum, were supported as robust monophyletic groups, whereas Heterotropa had low support. Sect. Hexastylis was revealed to be polyphyletic, suggesting taxonomic reconstruction would be needed. Sect. Heterotropa comprises two clades, which correspond to species distribution ranges: mainland China and the island arc from Taiwan to mainland Japan via the Ryukyu Islands. It is notable that the common ancestry of the latter clade in the eastern Asian islands was highly supported, suggesting that the present species diversity of Heterotropa was initially caused by allopatric range fragmentation in East Asia.     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

Actomyosin bundles serve as a tension sensor and a platform for ERK activation

Tensile forces generated by stress fibers drive signal transduction events at focal adhesions. Here, we report that stress fibers per se act as a platform for tension-induced activation of biochemical signals. The MAP kinase, ERK is activated on stress fibers in a myosin II-dependent manner. In myosin II-inhibited cells, uniaxial stretching of cell adhesion substrates restores ERK activation on stress fibers. By quantifying myosin II- or mechanical stretch-mediated tensile forces in individual stress fibers, we show that ERK activation on stress fibers correlates positively with tensile forces acting on the fibers, indicating stress fibers as a tension sensor in ERK activation. Myosin II-dependent ERK activation is also observed on actomyosin bundles connecting E-cadherin clusters, thus suggesting that actomyosin bundles, in general, work as a platform for tension-dependent ERK activation.