Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10muM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.     

Identification of a novel Cochlin isoform in the perilymph: insights to Cochlin function and the pathogenesis of DFNA9

The COCH gene mutated in DFNA9, an autosomal dominant hereditary sensorineural hearing loss and vestibular disorder, encodes Cochlin. Previously, we reported three bovine Cochlin isoforms, p63s, p44s, and p40s, which exhibit significant molecular heterogeneity in vivo. Here we have characterized Cochlin isoforms by generating four isoform-specific anti-Cochlin antibodies. The same three Cochlin isoforms, p63s, p44s, and p40s, were detected in human and cow inner ear tissue; however, p44s and p40s were not detected in perilymph. We identified a novel short 16kDa isoform in human perilymph and a 18-23kDa isoform in cow perilymph, named Cochlin-tomoprotein (CTP), corresponding to the N-terminus of full-length Cochlin (p63s) and the LCCL domain. Notably, CTP contains all of the known mutation sites associated with DFNA9. The pathogenesis of DFNA9 is not fully clarified as yet, and this novel perilymph-associated CTP isoform might provide mechanistic clues to how mutations in the COCH gene damage the inner ear function.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

不同耕作方式对内蒙古旱作农田土壤水热状况的影响

为了研究免耕留茬覆盖和传统耕作土壤水热状况的差异,20052008年在内蒙古呼和浩特市清水河县进行定位试验,结果表明:(1)免耕留茬覆盖能有效的调节土壤温度,出现高温时抑制温度上升,低温时抑制温度降低的现象;土壤温度24h变化的振幅随着土壤深度的增加呈现逐渐减小的趋势,表层010cm的变化幅度最大,变化范围为17.634℃;不同耕作方式的土壤温度均从09:00开始升高,至15:00达到最高值,之后缓慢下降,到06:00达到最低值;土壤温度随季节动态变化的趋势,受制于气候。(2)测定期内免耕留高茬覆盖平均土壤含水量较传统耕作增加了7.37%,免耕留低茬覆盖较传统耕作增加了5.93%;燕麦苗期,不同耕作方式的土壤含水量随土层深度增加,呈增大的态势。分蘖期,不同耕作方土壤含水量在020cm呈下降趋势,之后逐渐升高。燕麦花期,不同耕作方土壤含水量在040cm呈下降趋势,之后逐渐上升,至80100cm土层深度达到最大值;土壤含水量主要受季节影响,随降雨量变化而变化。(3)免耕留茬覆盖耕作方式增加土壤有机质和土壤含水量,其热容量增大,因此能调节和保持温度变化平缓。     

Distinct contributions from the hindbrain and mesenchyme to inner ear morphogenesis

A mature inner ear is a complex structure consisting of vestibular and auditory components. Microsurgical ablations, rotations, and translocations were performed in ovo to identify the tissues that control inner ear morphogenesis. We show that mesenchyme/ectoderm adjacent to the developing ear specifically governs the shape of vestibular components - the semicircular canals and ampullae - by conferring anteroposterior axial information to these structures. In contrast, removal of individual hindbrain rhombomeres adjacent to the developing ear preferentially affects the growth and morphogenesis of the auditory subdivision, the cochlear duct, or basilar papilla. Removal of rhombomere 5 affects cochlear duct growth, while rhombomere 6 removal affects cochlear growth and morphogenesis. Rotating rhombomeres 5 and 6 along the anteroposterior axis also impacts cochlear duct morphogenesis but has little effect on the vestibular components. Our studies indicate that discrete tissues, acting at a distance, control the morphogenesis of distinct elements of the inner ear. These results provide a basis for identifying factors that are essential to vestibular and auditory development in vertebrates.     

Cell cycle regulation in the inner ear sensory epithelia: Role of cyclin D1 and cyclin-dependent kinase inhibitors

Sensory hair cells and supporting cells of the mammalian cochlea and vestibular (balance) organs exit the cell cycle during embryogenesis and do not proliferate thereafter. Here, we have studied the mechanisms underlying the maintenance of the postmitotic state and the proliferative capacity of these cells. We provide the first evidence of the role of cyclin D1 in cell cycle regulation in these cells. Cyclin D1 expression disappeared from embryonic hair cells as differentiation started. The expression was transiently upregulated in cochlear hair cells early postnatally, paralleling the spatiotemporal pattern of unscheduled cell cycle re-entry of cochlear hair cells from the p19^Ink4d/p21^Cip1 compound mutant mice. Cyclin D1 misexpression in vitro in neonatal vestibular HCs from these mutant mice triggered S-phase re-entry. Thus, cyclin D1 suppression is important for hair cell's quiescence, together with the maintained expression of cyclin-dependent kinase inhibitors. In contrast to hair cells, cyclin D1 expression was maintained in supporting cells when differentiation started. The expression continued during the neonatal period when supporting cells have been shown to re-enter the cell cycle upon stimulation with exogenous mitogens. Thereafter, the steep decline in supporting cell's proliferative activity paralleled with cyclin D1 downregulation. Thus, cyclin D1 critically contributes to the proliferative plasticity of supporting cells. These data suggest that targeted cyclin D1 induction in supporting cells might be an avenue for proliferative regeneration in the inner ear.     

The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins

BMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1^ΔC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1^ΔC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1^ΔC pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1^ΔC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1^ΔC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation.     

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.