Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes

Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.     

海兔素对人乳腺癌SK-BR-3细胞的抑制作用及机制研究

本文阐述了海兔素(Aplysin)对人乳腺癌SK-BR-3细胞增殖和凋亡的影响,并探讨了其可能的作用机制.分别采用CCK-8法和流式细胞术Annexin V-FITC/PI双染法测定不同剂量海兔素对SK-BR-3细胞的增殖抑制和凋亡诱导作用,并以Western blot测定SK-BR-3细胞中EGFR、Akt及ERK表达水平和磷酸化水平.结果发现,20、30、40、45、50、55、60 mg/L海兔素处理SK-BR-3细胞24 h后,细胞的生长增殖明显受到抑制,呈量效依赖性,其IC25和IC50值为分别为29.4和33.7 mg/L; IC25和IC50剂量海兔素可明显诱导细胞凋亡,并下调SK-BR-3细胞EGFR、Akt及ERK的磷酸化蛋白表达水平,但是不影响总蛋白表达水平.表明海兔素对人乳腺癌SK-BR-3细胞具有抑制增殖和诱导凋亡的作用,其作用机制可能与海兔素抑制细胞中EGFR蛋白磷酸化,进而阻断下游效应分子Akt和ERK的活化有关.     

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.     

截短型人乳头瘤病毒58型L1蛋白的表达及其体外生物活性研究

利用PCR技术克隆截短型HPV58 L1基因并重组入杆状病毒表达系统穿梭质粒pFastBac-Htb,通过转座反应,将目的基因片段重组入杆状病毒基因组,分离重组的Bacmid DNA, 并转染Sf-9昆虫细胞,收集被转染的Sf-9细胞,提取细胞蛋白,SDS-PAGE检测可见在大约58Kda处出现一新生蛋白条带,Western blot 证实为HPV58L1蛋白。用ProBondTM纯化系统纯化所表达的蛋白。小鼠红细胞凝集试验证实纯化的蛋白可介导小鼠红细胞凝集,透射电镜观察证实纯化蛋白可自组装成VLP。结果表明昆虫杆状病毒表达系统可高效表达截短型HPV58L1蛋白,纯化后的截短型HPV58L1蛋白在体外可自组装VLP,并具有介导小鼠红细胞凝集的生物学活性。     

Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: a study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.     

Proteomic characterization of the striatum and midbrain treated with 6-hydroxydopamine: Alteration of 58-kDa glucose-regulated protein and C/EBP homologous protein

Abstract

The present study performed proteomic analysis of the midbrain and striatum of 6-hydroxydopamine (6-OHDA)-treated neonatal rats—a model of attention-deficit hyperactivity disorder (ADHD). Proteomic analysis revealed that a 58-kDa glucose-regulated protein (Grp58) was temporarily phosphorylated and its level was elevated by 6-OHDA. Furthermore, 6-OHDA increased the expression level of C/EBP homologous protein (CHOP), a mediator of endoplasmic reticulum (ER) stress response, in the midbrain and striatum. In vitro experiments using PC12 cells revealed that 6-OHDA or hydrogen peroxide could induce the elevation of Grp58 and CHOP. 6-OHDA could induce the elevation of Grp58 and CHOP in the presence of catalase, a hydrogen peroxide-removing enzyme, suggesting that the elevation of Grp58 and CHOP are induced by both hydrogen peroxide and p-quinone generated by 6-OHDA. Collectively, these findings suggest that ER stress involving the alteration of Grp58 and CHOP play a significant role in the induction of insults by 6-OHDA in vivo.     

Inhibition of the JNK/Bim pathway by Hsp70 prevents Bax activation in UV-induced apoptosis

Here we studied the mechanism by which heat shock protein 70 (Hsp70) prevents Bax activation during ultraviolet (UV)-induced apoptosis. UV treatment led to c-Jun N-terminal kinase (JNK) phosphorylation, Bim redistribution and subsequent Bax activation. Bim depletion caused a smaller reduction in apoptosis than that by JNK inhibition, indicating that Bim activation is not entirely responsible for induction of apoptosis and other mechanisms are involved. Hsp70 knockdown resulted in high levels of activated JNK and Bax, while Hsp70 overexpression inhibited these processes. These findings demonstrate that Hsp70 prevented Bax activation via inhibiting the JNK/Bim pathway. Simultaneously, increased binding of Hsp70 to Bax was observed. Collectively, our results for the first time demonstrate that Hsp70 prevents Bax activation both by inhibiting the JNK/Bim pathway and by interacting with Bax in UV-induced apoptosis.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

Oncogenic comparison of human papillomavirus type 58 E7 variants

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage‐independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7‐driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow‐up strategy.     

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.