A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay（ELISA）, immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells（HPDC）.     

Prion protein and cancers

The normal cellular prion protein, PrPc is a highly con served and widely expressed cell surface glycoprotein in all mammals. The expression of PrP is pivotal in the pathogenesis of prion diseases; however, the normal physiological functions of PrPc remain incompletely understood. Based on the studies in cell models, a plethora of functions have been attributed to PrPc. In this paper, we reviewed the potential roles that PrPc plays in cell physiology and focused on its contribution to tumorigenesis.     

Study on interaction between microtubule associated protein tau and prion protein

Microtubule-associated protein tau is considered to play roles in many neurodegenera-tive diseases including some transmissible spongiform encephalopathies.To address the possible molecular linkage of prion protein(PrP) and tau,a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immuno-precipitation.We found tau protein interacted with various style prion proteins such as native prion protein(PrPC) or protease-resistant isoform(PrPSc) .Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene.The domain of interaction with tau was located at N-terminal of PrP(residues 23 to 91) .The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.     

Inhibition of phagocytosis reduced the classical activation of BV2 microglia induced by amyloidogenic fragments of beta-amyloid and prion proteins

The inflammatory responses in Alzheimer＇s disease and prion diseases are dominated by microglia activation. Three different phenotypes of microglial activation, namely classical activation, alternative activation, and acquired deactivation, have been described. In this study, we investigated the effect of amyloido- genic fragments of amyloid 13 and prion proteins （Aβ1_42 and PrP106-126） on various forms of microglial activation. We first examined the effect of Aβ1_42 and PrP106-126 stimulation on the mRNA expression levels of several markers of microglial activation, as well as the effect of cytochalasin D, a phagocytosis inhibitor, on microgllal activation in Aβ1_42- and PrP106-126- stimulated BV2 microglla. Results showed that Aβ1-42 and PrPlo6_126 induced the classical activation of BV2 microglia, decreased the expression level of alternative expression markers, and had no effect on the expression of acquired de- activation markers. Cytochalasin D treatment significantly reduced Aβ1_42- and PrP106-26-induced up-regulation of proinflammatory factors, but did not change the expression profile of the markers of alternative activation or acquired de- activation in BV2 cells which were exposed to Aβ1-42 and PrPlo6_126. Our results suggested that microglia interact with amyloidogenic peptides in the extraceilular milieu-stimulated microglial classical activation and reduce its alternative activa- tion, and that the uptake of amyloidogenic peptides from the extracellular milieu amplifies the classical microglial activation.     

Regulation of proteasomes in prion disease

The hallmark of prion disease is the accumulation of mis folded protein PrPsc, which is toxic to neuronal cells. The proteasome system is responsible for the rapid, precise, and timely degradation of proteins and plays an important role in cellular protein quality control. Increasing evidence indi cates impaired activity of proteasomes in prion diseases. Accumulated PrPsc can directly or indirectly affect prote asome activity. Misfolded protein may influence the assem bly and activity of 19S regulatory particle, or post translational modification of 20S proteasome, which may adversely affect the protein degradation activity of protea somes. In this review, we summarized the recent findings concerning the possible regulation of proteasomes in prion and other neurodegenerative diseases. The proteasome system may enhance its degradation activity by changing its structure, and this activity can also be increased by related chaperones when neuronal cells are subject to stress. When the proteasome system is inhibited, degradation of protein aggregates via autophagy may increase as a compensatory system. It is possible that a balance exists between the prote asome and autophagy in vivo; when one is impaired, the ac tivity of the other may increase to maintain homeostasis. However, more studies are needed to elucidate the relation ship between the proteasome system and autophagy.     

Binding between Prion Protein and Aβ Oligomers Contributes to the Pathogenesis of Alzheimer's Disease

A plethora of evidence suggests that protein misfolding and aggregation are underlying mechanisms of various neurodegenerative diseases, such as prion diseases and Alzheimer's disease(AD). Like prion diseases, AD has been considered as an infectious disease in the past decades as it shows strain specificity and transmission potential. Although it remains elusive how protein aggregation leads to AD, it is becoming clear that cellular prion protein(PrP~C ) plays an important role in AD pathogenesis. Here, we briefly reviewed AD pathogenesis and focused on recent progresses how PrP~C contributed to AD development. In addition, we proposed a potential mechanism to explain why infectious agents, such as viruses, conduce AD pathogenesis. Microbe infections cause Aβ deposition and upregulation of PrP~C , which lead to high affinity binding between Aβ oligomers and PrP~C . The interaction between PrP~C and Aβ oligomers in turn activates the Fyn signaling cascade, resulting in neuron death in the central nervous system(CNS). Thus, silencing PrP~C expression may turn out be an effective treatment for PrP~C dependent AD.     

PrP mutants with different numbers of octarepeat sequences are more susceptible to the oxidative stress

One of the physiological functions of cellular prion protein(PrP C )is believed to work as a cellular resistance to oxidative stress,in which the octarepeats region within PrP plays an important role.However,the detailed mechanism is less clear.In this study,the expressing plasmids of wild-type PrP (PrP-PG5)and various PrP mutants containing 0(PrP-PG0),9(PrP-PG9)and 12(PrP-PG12)octarepeats were generated and PrP proteins were expressed both in E.coli and in mammalian cells.Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E.coli after treatment of H2O2.MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP.Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats.Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H2O2.Furthermore,cells expressing wild-type PrP showed stronger resistant activity to the challenge of H2O2 at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation.Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.     

Doppel-induced cytotoxicity in human neuronal SH-SY5Y cells is antagonized by the prion protein

Doppel （Dpl） is a prion （PrP）-like protein due to the structural and biochemical similarities; however, the natural functions of Dpl and PrP remain unclear. In this study, a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes. Furl-length and various truncated human Dpl and PrP proteins were expressed and purified from Escherichia coil Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity, and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids （aa） 81-122]. Interestingly, DpMnduced cytotoxicity was antagonized by the presence of full- length wild-type PrP. Analysis on fragments of PrP mutants showed that the N-terminal fragment （aa 23- 90） of PrP was responsible for the protective activity. A truncated PrP （PrPA32-121） with similar secondary structure as Dpl induced DpMike cytotoxicity on SH- SY5Y cells. Furthermore, binding of copper ion could enhance the antagonizing effect of PrP on Dpi-induced cytotoxicity. Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism. These results suggested that the function of Dpl is antagonistic to PrP rather than synergistic.     

The octarepeat region of hamster PrP （PrP51-91） enhances the formation of microtubule and antagonize Cu^2＋-induced microtubule-disrupting activity

Prion protein （PrP） is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP （PrP51-91） was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the molecular interaction between PrP51-91 and tubulin was observed. Our data also demonstrated that PrP51-91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtubule dynamics. Moreover, PrP51-91 was confirmed to be able to antagonize Cu^2＋-induced microtubule-disrupting activity in vivo, partially protecting against Cu^2＋- intoxication to culture cells and stabilize cellular microtubule structure. The association of the octarepeat region of PrP with tubufin may further provide insight into the biological function of PrP in the neurons.     

Recombinant neural protein PrP can bind with both recombinant and native apolipoprotein E in vitro

The most essential and crucial step during the pathogenesis of transmissible spongiformencephalopathy is the conformational change of cellular prion protein (PrP~C) to pathologic isoform (PrP~(Sc)).Alot of data revealed that caveolae-like domains (CLDs) in the cell surface were the probable place where theconversion of PrP proteins happened.Apolipoprotein E (ApoE) is an apolipoprotein which is considered toplay an important role in the development of Alzheimer's disease and other neurodegenerative diseases byforming protein complex through binding to the receptor located in the clathrin-coated pits of the cell surface.In this study,a 914-bp cDNA sequence encoding human ApoE3 was amplified from neuroblastoma cell lineSH-SY5Y.Three human ApoE isomers were expressed and purified from Escherichia coli.ApoE-specificantiserum was prepared by immunizing rabbits with the purified ApoE3.GST/His pull-down assay,immunoprecipitation and ELISA revealed that three full-length ApoE isomers interact with the recombinantfull-length PrP protein in vitro.The regions corresponding to protein binding were mapped in the N-terminalsegment of ApoE (amino acid 1-194) and the N-terminal of PrP (amino acid 23-90).Moreover,the recombinantPrP showed the ability to form a complex with the native ApoE from liver tissues.Our data provided directevidence of molecular interaction between ApoE and PrP.It also supplied scientific clues for assessing thesignificance of CLDs on the surface of cellular membrane in the process of conformational conversion fromPrP~C to PrP~(Sc) and probing into the pathogenesis of transmissible spongiform encephalopathy.     

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus （GCRV） is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses （MRV） compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.     

Cotton Leaf Curl Virus： Ionic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus （CLCuV） in two cotton （Gossypium hirsutum L.） cuItlvars （I.e. CIM-240 and S-12） was Investigated. The incidence of disease attack was found to be 100% In the S-12 cuItlvar and 16% in the CIM-240 cuItivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antlsera-treated cell sap. The CLCuV Impaired the accumulation of different nutrients in both cuItivars. A marked decrease in the accumulation of Ca^2＋ and K^＋ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na^＋, N, and P. It was observed that the curling of leaf margins in CLCuV-Infected plants was associated with the leaf Ca^2＋ content; leaf curling was severe in plants with a significant reduction In Ca^2＋ content. Moreover, leaf K＆＋ content was found to be associated with resistance/susceptibility to CLCuV infection.     

NKT cells in HIV-1 infection

Natural killer T （NKT） cells are a unique T cell population that have important immunoregulatory functions and have been shown to be involved in host immunity against a range of microorganisms. It also emerges that they might play a role in HIV-1 infection, and therefore be selectively depleted during the early stages of infection. Recent studies are reviewed regarding the dynamics of NKT depletion during HIV-1 infection and their recovery under highly active antiretroviral treatment （HAART）. Possible mechanisms for these changes are proposed based on the recent developments in HIV pathogenesis. Further discussions are focused on HIV＇s disruption of NKT activation by downregulating CDld expression on antigen presentation cells （APC）. HIV-1 protein Nefis found to play the major role by interrupting the intracellular trafficking of nascent and recycling CDld molecules.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin

The growth factor midkine （MK） is a cytokine that inhibits HIV infection in cell cultures in an autocrine and paracrine manner by blocking the attachment of HIV particles to permissive cells. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-7 and activation of T lymphocytes by PHA or through the engagement of the CD28 antigen. The binding of MK to cells occurs specifically at a high and a low affinity binding site. This low affinity-binding site is the cell-surface expressed nucleolin, which is implicated in the mechanism of the initial attachment of HIV particles to cells. Accordingly, the nucleolin-binding HB-19 pseudopeptide has no effect on the MK binding to the high affinity binding site, whereas it prevents the binding of MK to the low affinity binding site, thus suggesting the low affinity receptor of MK is the cell-surface-expressed nucleolin. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicates that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, as the domain that binds MK. The specific binding of MK to the surface nucleolin is independent of heparan sulfate and chondroitin sulfate proteoglycans. After binding to cells, MK enters cells by an active process in which nucleolin and lipid rafts appear to be implicated. The potent and the distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli, point out that MK is a cytokine that could be involved in HIV pathogenesis.     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

Vaccination against very virulent infectious bursal disease virus using recombinant T4 bacteriophage displaying viral protein VP2

In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus （vvIBDV）, we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid （SOC） protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies （mAbs） against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free （SPF） chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethaldose （LD50） per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, whereas the unvaccinated group and the group vaccinated with the wild-type T4 phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively, the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.     

Medical Virology in Malaysia

Virology is a branch of biological science dealing with the study of viruses, and medical virology focuses on the study and control of diseases due to viruses that is of medical importance. The development of medical virology in Malaysia has its beginning in the Institute for Medical Research （IMR）, following the establishment of the Division of Medical Zoology and Virus Research in the institute on 23 March 1953. The second institution in the country to establish diagnostic and research work in medical virology was Department of Medical Microbiology, Faculty of Medicine, University Malaya. This was followed by University Kebangsaan Malaysia, University Sains Malaysia and University of Sarawak Malaysia. The National Public Health Laboratory （NPHL） is the latest institution to establish a laboratory in 2003 for virus isolation and services to support country surveillance and outbreak investigation of infectious diseases due to viruses. In the field of medical virology, Malaysia contributed substantially in the areas of virus diagnostic services, development and research ranging from survey and documentation on the existence and prevalence of viruses causing diseases in Malaysia, clinical presentation and epidemiological features of virus diseases, evaluation of new diagnostic tests to pathogenesis of viral diseases. Malaysia contributed to the discoveries of at least 12 new viruses in the world. ASEAN plus Three （China, Japan, Republic of Korea） Emerging Infectious Programme was established to overcome the challenges and impact of emerging and re-emerging infectious diseases in this region. Malaysia as the co-ordinator of the laboratory component of the programme, contributed to strengthen the regional laboratory capability, capacity, laboratory-based surveillance and networking. The future of medical virology in Malaysia in terms of integration of diagnostic, reference and research to support the country＇s need will be enhanced and strengthened with the on-going development of the National Centre fo