Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.     

The population of Odonata (dragonflies) in small tropical rivers with reference to asynchronous growth patterns

The odonate larval communities in three small rivers in Penang Island were studied. More species of dragonflies were found in the Botanical Garden and Titi Teras rivers (13 and 11 respectively) of relatively similar environmental parameters. Fewer (nine) dragonfly species were collected from the Youth Park River which has a lower dissolved oxygen (DO) and a higher biological oxygen demand (BOD), conductivity and turbidity. A mixture of sand, gravel and pebble substrate of Botanical Garden River with dense growth of submerged Hydrilla, grasses and Cladias (Araceae) provided suitable habitats for the dragonflies. The sandy substrate and relatively fast flowing water of Titi Teras River was highly preferred by gomphids. In the Youth Park River, the small community of dragonfly larvae was dominated by tolerant Pseudagrion rubriceps, P. microcephalum, Orthetrum chrysis and Crocothemis servilia. Based on the larval instar distribution of Ictinogomphus decoratus and O. chrysis, very asynchronous populations of these dragonflies occurred in each river. Young larvae were continuously introduced into the populations resulting in undulating growth rate curves. The growth rates of these two species were higher in the Titi Teras River when compared to those in other rivers. Density-dependent mortality, asynchronous cannibalism and fish predation could play important roles in regulating the larval dragonfly population in these rivers.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Inhibitory effects of glycoprotein-120 (G-120) from Ulmus davidiana Nakai on cell growth and activation of matrix metalloproteinases

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-kappaB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-kappaB.     

Chitosan-modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of concanavalin A

Lectin affinity membranes have been receiving much attention for the separation and detection of various glycoconjugates. In this work, we present a simple and efficient method for the preparation of lectin affinity nanofibrous membranes. Chitosan-modified poly(acrylonitrile-co-acrylic acid) (PANCAA) nanofibrous membranes were first prepared by a coupling reaction between the primary amino groups of chitosan and the carboxyl groups of PANCAA electrospun membranes. Surface characterizations by attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy (FESEM) confirm the chemical and morphological changes of the studied nanofibrous membranes. Fluorescence-labeled concanavalin A (FL-Con A) was then immobilized on these membranes via noncovalent binding. Analyses by fluorescence spectrophotometer (FS) and confocal laser scanning microscopy (CLSM) reveal that the immobilization of Con A onto the modified nanofibrous membranes has been successfully achieved on the basis of the electrostatic interaction and the specific recognition between Con A and chitosan. The results show that the amount of adsorbed FL-Con A increases dramatically with the increasing coupling degree of chitosan (CDC) on the nanofibrous membrane. Moreover, Con A immobilized on the chitosan-modified nanofibrous membranes (CMNMs) can remain relatively stable at pH 5.3. Therefore, it is believed that this work may provide a new kind of material for affinity application.     

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson’s disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first “mtISR suppressor” localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.Subject terms: Stress signalling, Mitochondria     

BRCA1基因在人食管癌中的表达

目的本研究利用组织芯片检测BRCA1基因在人食管癌中的表达与食管癌的生长、分化和转移等临床特征的关系,期望找到BRCA1与食管癌发生、发展的关系。方法收集48例食管癌患者标本,分别取其肿瘤组织、癌前病变组织及正常组织制成组织微阵列,免疫组织化学SP法检测BRCA1蛋白的表达,分析BRCA1在各种组织的表达特点及其与肿瘤的关系。选择其中10例患者的上述组织的新鲜标本,采用Western blot检测BRCA1蛋白的表达。结果免疫组织化学结果显示,BRCA1在肿瘤组织阳性占70.50%,癌前组织阳性占43.10%,正常组织阳性占39.00%,食管癌组织与癌前病变组织、食管癌组织与正常组织相比BRCA1的表达均存在显著差异（P〈0.01）。BRCA1的表达与食管癌的病理分化有统计学上的差异（P〈0.05）。Western blot显示,BRCA1在食管癌肿瘤组织、癌前病变组织及正常组织中表达量依次降低,差异具有统计学意义（P〈0.05）;BRCA1在高、中、低分化程度的食管癌组织中表达量依次降低,差异具有统计学意义（P〈0.05）。结论BRCA1与食管癌发生发展有关,BRCA1的表达与食管癌的分化呈正相关。     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.