Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

Inositol 1,4,5-trisphosphate and guanine nucleotides activate calcium release from endoplasmic reticulum via distinct mechanisms

A sensitive and specific guanine nucleotide regulatory process has recently been shown to rapidly mediate a substantial release of Ca2+ from endoplasmic reticulum within the N1E-115 neuronal cell line (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The relationship between this mechanism and Ca2+ efflux mediated by the intracellular regulator inositol 1,4,5-trisphosphate (IP3) has been investigated. Using saponin-permeabilized N1E-115 cells, studies reveal a number of distinctions between the activation of Ca2+ release mediated by GTP and IP3. Thus, the GTP-mediated Ca2+ release process is specifically activated by polyethylene glycol which increases both GTP sensitivity and the extent of GTP-activated Ca2+ release; in contrast, IP3-dependent Ca2+ release is unaffected by polyethylene glycol. The non-hydrolyzable GTP analogue guanosine 5'-O-(3-thio)triphosphate, which completely inhibits GTP-mediated Ca2+ release, does not alter release mediated by IP3. Decreasing the release temperature from 37 to 4 degrees C decreases IP3-activated Ca2+ release by only 20%, whereas the action of GTP on Ca2+ release is abolished at 4 degrees C. Activation of Ca2+ release by IP3 is completely inhibited by increasing free Ca2+ from 0.1 to 10 microM, whereas the fraction of GTP-dependent Ca2+ release (approximately 50% of ionophore-releasable Ca2+) remains unaltered with increasing free Ca2+. These distinctions between IP3- and GTP-mediated Ca2+ release indicate that the two effectors function via distinct mechanisms to activate Ca2+ release; however, they do not preclude the possibility that coupling between the two mechanisms can occur or that a common Ca2+-translocating pathway activated by both effectors exists.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Systematics of white-toothed shrews (Crocidura) (Mammalia: Insectivora: Soricidae) of Taiwan: Karyological and morphological Studies

We define the species boundaries of white-toothed shrews (genus Crocidura ) in Taiwan using karyological and morphological characteristics. Ninety-nine animals were obtained from all over Taiwan at capture rates usually less than 10%. Three species are recognized by distinct cytotypes: Crocidura attenuata tanakae 2n = 40, FN = 56; Crocidura suaveolens hosletti 2n = 40, FN = 50; Crocidura kurodai 2n = 40, FN = 54. A suite of six morphological characters diagnose the three species: shape of skull, position of incisive foramina, shape of fourth upper premolar, shape of pinna, tail vibrissae, and foot pads. A species key and notes on the life history of each species are provided. Finally, we discuss chromosomal evolution and biolgeography of Crocidura in East and South East Asia.     

中华大刀螳形态学、生物学特性及饲养观察

中华大刀螳Tenodera sinensis Saussure既是传统的中药材,又是重要的天敌昆虫,因而具有重要的经济价值.为了充分开发和利用中华大刀螳资源,在人工饲养条件下,对其形态学特征和生物学特性进行了研究.结果表明:中华大刀螳为渐变态昆虫,一生经历卵、若虫和成虫3个虫态.室内1年完成1个世代,且无越冬现象.若虫共8龄,以1龄若虫发育历期最短,平均为11.73 d,8龄若虫发育历期最长,平均为24.75 d;3龄若虫存活率最低,为81.08％,5龄若虫存活率最高,为100％;雌、雄成虫阶段的平均历期为99.36 d和72.86 d.此外,本文介绍了中华大刀螳各虫态的形态特征和生物学特性.     

The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution,Molecular Marker Identification,and Phylogenetic Implications in Asterids

The Madagascar periwinkle ( Catharanthus roseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C . roseus , which could be applied to other C . roseus -related studies. The C . roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffea arabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepias syriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C . roseus -specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C . roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported.     

暗腹雪鸡与海兰褐鸡血液生理生化指标的比较

为了解暗腹雪鸡（Tetraogallus himalayensis）正常血液生理生化特性,本文对暗腹雪鸡21项血液生理生化指标进行了测定,并与海兰褐鸡（Gallus gallus domesticus var. Hyline）进行了比较。测定结果,暗腹雪鸡的红细胞纵径及横纵径乘积、平均红细胞容积及平均红细胞血红蛋白含量极显著大于海兰褐鸡（P<0.01）;暗腹雪鸡天冬氨酸氨基转移酶和胆固醇含量均极显著高于海兰褐鸡（P<0.01）。其他各项血液生理生化指标两禽种间之间差异不显著。实验结果表明,暗腹雪鸡血液生理生化指标与家养蛋鸡之间有一定差异,暗腹雪鸡对高原低氧环境有较强的适应性可能与其红细胞体积较大、平均红细胞容积及平均红细胞血红蛋白含量较高有一定关系。     

Comparative Analysis of the Peanut Witches'-Broom Phytoplasma Genome Reveals Horizontal Transfer of Potential Mobile Units and Effectors

Phytoplasmas are a group of bacteria that are associated with hundreds of plant diseases. Due to their economical importance and the difficulties involved in the experimental study of these obligate pathogens, genome sequencing and comparative analysis have been utilized as powerful tools to understand phytoplasma biology. To date four complete phytoplasma genome sequences have been published. However, these four strains represent limited phylogenetic diversity. In this study, we report the shotgun sequencing and evolutionary analysis of a peanut witches''-broom (PnWB) phytoplasma genome. The availability of this genome provides the first representative of the 16SrII group and substantially improves the taxon sampling to investigate genome evolution. The draft genome assembly contains 13 chromosomal contigs with a total size of 562,473 bp, covering ∼90% of the chromosome. Additionally, a complete plasmid sequence is included. Comparisons among the five available phytoplasma genomes reveal the differentiations in gene content and metabolic capacity. Notably, phylogenetic inferences of the potential mobile units (PMUs) in these genomes indicate that horizontal transfer may have occurred between divergent phytoplasma lineages. Because many effectors are associated with PMUs, the horizontal transfer of these transposon-like elements can contribute to the adaptation and diversification of these pathogens. In summary, the findings from this study highlight the importance of improving taxon sampling when investigating genome evolution. Moreover, the currently available sequences are inadequate to fully characterize the pan-genome of phytoplasmas. Future genome sequencing efforts to expand phylogenetic diversity are essential in improving our understanding of phytoplasma evolution.     

Clinical Significance in Oral Cavity Squamous Cell Carcinoma of Pathogenic Somatic Mitochondrial Mutations

Somatic mutations affecting the mitochondrial DNA (mtDNA) have been frequently observed in human cancers and proposed as important oncological biomarkers. However, the clinical significance of mtDNA mutations in cancer remains unclear. This study was therefore performed to explore the possible clinical use in assessing oral squamous cell carcinoma (OSCC) of pathogenic mtDNA mutations. The entire mitochondrial genome of 300 OSCC with their matched control DNAs was screened by direct sequencing and criteria were set to define a pathogenic somatic mutation. The patients'' TP53 R72P genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. The relationships between pathogenic somatic mutations, clinicopathogical features, TP53 R72P genotype and clinical prognosis were analyzed. Overall, 645 somatic mtDNA mutations were identified and 91 of these mutations were defined as pathogenic. About one quarter (74/300) of the OSCC tumor samples contained pathogenic mutations. Individuals with the TP53 R allele had a higher frequency of pathogenic somatic mutation than those with the PP genotype. Kaplan-Meier analysis indicated that TP53 R allele patients with pathogenic somatic mutations demonstrated a significant association with a poorer disease-free survival than other individuals (HR = 1.71; 95% CI, 1.15–2.57; p = 0.009) and this phenomenon still existed after adjusting for mtDNA haplogroup, tumor stage with treatment regimens, differentiation and age at diagnosis (HR = 1.59; 95% CI, 1.06–2.40; p = 0.03). Subgroup analyses showed that this phenomenon was limited to patients who received adjuvant radiotherapy/chemo-radiotherapy after surgery. The results strongly indicated that pathogenic mtDNA mutations are a potential prognostic marker for OSCCs. Furthermore, functional mitochondria may play an active role in cancer development and the patient''s response to radiotherapy/chemo-radiotherapy.