蟾毒灵白蛋白纳米粒对结肠癌干细胞端粒酶活性的影响

目的观察蟾毒灵白蛋白纳米粒对SW480结肠癌干细胞增殖及端粒酶活性的影响。方法流式细胞仪分选SW480结肠癌细胞CD133+/CD44+亚群(SW480结肠癌干细胞),以不同浓度(1、10、50、100 nmol/L)的蟾毒灵及蟾毒灵白蛋白纳米粒进行干预,MTT法观察细胞增殖,采用端粒重复序列扩增-酶联免疫吸附测定法(TRAP-ELISA)检测结肠癌干细胞端粒酶活性的变化。结果与对照组比较,低浓度(1、10 nmol/L)蟾毒灵及蟾毒灵白蛋白纳米粒干预对SW480结肠癌干细胞的增殖及端粒酶活性均无明显影响(P>0.05);与对照组和中、高浓度(50、100 nmol/L)蟾毒灵组比较,相应浓度的蟾毒灵白蛋白纳米粒组SW480结肠癌干细胞的增殖及端粒酶活性明显受到抑制(P<0.05)。结论 50 nmol/L和100 nmol/L的蟾毒灵白蛋白纳米粒能显著抑制结肠癌干细胞的增殖和端粒酶活性,且效果优于蟾毒灵。

肝包虫破裂致过敏性休克急死1例

死者男、18岁。1988年5月20日早8时许,与他人玩耍时腹部被对方肘部顶了一下,当即倒地,并述'肚子疼',面色青紫,口吐白沫,呼之不应。15分钟后呼吸停止,经抢救无效死亡。一、尸体检验:死者发育正常,营养良好,尸斑呈暗紫红色,尸僵存在于全身各关节。

老年性脑包虫病急死一例

死者陈××,男性,61岁,浙江人,汉族。生前长期从事海上航运工作,一年前退休回家。在业时,体力脑力稍有疲劳时易发生癫痫。1983年12月30日与邻居吵架,被推倒在地,不能起立,头冒大汗,送医院抢救时呼吸、心跳停止,前后相隔仅20分钟。

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex （NPC）, while most proteins are transported by energy driven transport mechanisms Active transport of viral proteins is mediated by nuclear localization signals （NLS）, which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral they contain short stretches of lysine or arginine residues. These signals are recognized proteins. Usually by the importin super-family （importin α and β） proteins that mediate the transport across the nuclear envelope through Ran-GTP In contrast, only one class of the leucine-rich nuclear export signal （NES） on viral proteins is known at present. Chromosome region maintenance 1 （CRM1） protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

AIM： To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F （MV-FMG）, encoded by an adenovirus vector Ad.MV-HI F, alone or in combination with local coexpression of cytokines （IL-2, IL-12, IL-18, IL-21 or GM-CSF）, can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS： We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector- treated tumor. We assessed the induction of a tumorspecific cytotoxic T lymphocyte （CTL） response with a lactate dehydrogenase （LDH） release assay.RESULTS： We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion, in addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and lL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION： Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Serum manganese superoxide dismutase and thioredoxin are potential prognostic markers for hepatitis C virus-related hepatocellular carcinoma

AIM:To evaluate the clinical significance of oxidative stress markers in patients with hepatitis C virus(HCV)related hepatocellular carcinoma(HCC).METHODS:Sixty-four consecutive patients who were admitted to Kagoshima University Medical and Dental Hospital were enrolled in this retrospective study.All patients had chronic liver disease(CLD) due to infection with HCV.Thirty patients with HCV-related HCC,34 with HCV-related CLD without HCC(non-HCC),and 20 healthy volunteers(HVs) were enrolled.Possible associations between serum manganese superoxide dismutase(MnSOD) and thioredoxin(TRX) levels and clinical parameters or patient prognosis were analyzed over a mean follow-up period of 31.7 mo.RESULTS:The serum MnSOD levels were significantly higher in patients with HCV-related HCC than in patients without HCC(P = 0.03) or HVs(P < 0.001).Similarly,serum TRX levels were also significantly higher in patients with HCV-related HCC than in patients without HCC(P = 0.04) or HVs(P < 0.01).However,serum levels of MnSOD and TRX were not correlated in patients with HCC.Among patients with HCC,the overall survival rate(OSR) was lower in patients with MnSOD levels ≥ 110 ng/mL than in patients with levels < 110 ng/mL(P = 0.01),and the OSR tended to be lower in patients with TRX levels < 80 ng/mL(P = 0.05).In addition,patient prognosis with HCC was poorest with serum MnSOD levels ≥ 110 ng/mL and serum TRX levels < 80 ng/mL.Furthermore,a multivariate analysis using a Cox proportional hazard model and serum levels of five factors(MnSOD,prothrombin time,serum albumin,serum α-fetoprotein(AFP),and serum des-γ-carboxy prothrombin) revealed that MnSOD levels ≥ 110 ng/mL(risk ratio:4.12,95% confidential interval:1.22-13.88,P = 0.02) and AFP levels ≥ 40 ng/mL(risk ratio:6.75;95% confidential interval:1.70-26.85,P < 0.01) were independent risk factors associated with a poor patient prognosis.CONCLUSION:Serum MnSOD and TRX levels are potential clinical biomarkers that predict patient prognosis in HCV-related HCC.

Isolation of Prawn(Exopalaemon carinicauda) Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene and Its Expression in Responding to Bacterial and Viral Infections

The pattern recognition proteins （PRPs） play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and 13-1, 3-glucan binding protein （LGBP） gene in the ridge tail white prawn （Exopalaemon carinicauda） （EcLGBP） was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crus- taceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at dif- ferent levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus （WSSV）. The expression of EcLGBP in response to E parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

Human μ-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous G_(i/o) proteins

Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.

Virion Proteomics of Large DNA Viruses

Large DNA viruses normally have complex structures with many of protein components derived from both viral and host origins. The development in proteomics, especially mass spectrometry identification techniques provide powerful tools for analyzing large viruses. In this review, we have summarized the recent achievements on proteomic studies of large DNA viruses, such as herpesvirus, poxvirus, nimavirus and baculoviruse. The proteomics of baculovirus occlusion-derived virions (ODV) were emphasized. Different mass spectrometry techniques used on various baculoviruses were introduced, and the identified structurally associated proteins of baculoviruses are summarized.

Gastric choriocarcinoma admixed with an α-fetoprotein-producing adenocarcinoma and separated adenocarcinoma

We report a case of gastric choriocarcinoma admixed with an α-fetoprotein (AFP)-producing adeno-carcinoma. A 70-year-old man was hospitalized for gastric cancer that was detected during screening by esophagogastroduodenoscopy (EGD). Initial laboratory data showed the increased serum level of AFP and EGD revealed a 5-cm ulcerofungating mass in the greater curvature of the gastric antrum. The patient underwent radical subtotal gastrectomywith D2 lymph node dissection and Billroth gastrojejunostomy. Histopathological evaluation confirmed double primary gastric cancer: gastric choriocarcinoma admixed with an AFP-producing adenocarcinoma and separated adenocarcinoma. At 2 wk postoperatively, his human chorionic gonadotropin and AFP levels had reduced and six cycles of adjuvant chemotherapy were initiated. No recurrence or distant metastasis was observed at 4 years postoperatively.

Molecular Characterization of Nonstructural Protein NS38 of Grass Carp Reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.

The Protamine-like DNA-binding Protein P6.9 Epigenetically Up-regulates Autographa californica Multiple Nucleopolyhedrovirus Gene Transcription in the Late Infection Phase

Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription^l~. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus （AcMNPV） encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication1311. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection （hpi）, whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9＇s co-localization with DNA is temporally and spatially synchronized with P6.9＇s impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.

Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and olden coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus （NPV） and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac 16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that BmS/Ac 16 interacts with baculoviral and cellular proteins. BmS/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Acl6 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.

A Multiple Functional Protein:the Herpes Simplex Virus Type 1 Tegument Protein VP22

The herpes simplex virus type 1 （HSV-1） VP22, is one of the most abundant HSV-I tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction ofmicrotubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating VP16, pU factor I （TAF-I） and nonmuscle myosin II A （NMIIA）, and viral proteins including pUS9 and pUL46, glycoprotein E （gE） and gD. Recently, many novel functions perform tegument protein ed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function .

E2 GLYCOPROTEIN OF GENOTYPE Ⅲ CHINESE ISOLATES OF HEPATITIS C VIRUS EXPRESSED IN MAMMALIAN CELL AS ANTIGEN FOR ANTI-E2 ANTIBODY DETECTION

Expression vector inserted with E2/NS1 gene derived from genotype Ⅲ Chinese isolates of HCV was transfected into mammalian cells to express E2 glycoprotein. Expressed protein was used as antigen for an- ti-E2 antibody detection in 19 cases of hepatitis C patients by Western blot. It was first to express E2 gly- coprotein of genotype Ⅲ Chinese hepatitis C virus isolates. For anti-E2 detection, 14 cases of patients were positive of antibodies against E2(73. 7 % ). These results indicated that E2 glycoprotein expressed in mam-malian cells had good immunogenicity and cross reactivity to serum infected with genotype Ⅱ Chinese hep-atitis C virus isolates.