Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells.

The pharmacology and clinical application of traditional Chinese medicine has been extensively documented. We have used an in vitro model system, PC12 cells, to demonstrate the presence of neuroactive compounds in Ganoderma lucidum (lingzhi). Ganoderma extract induced the neuronal differentiation of PC12 cells and prevented nerve growth factor-dependent PC12 neurons from apoptosis. Moreover, these effects of ganoderma might be mediated via the ras/extracellular signal-regulated kinase (Erk) and cAMP-response element binding protein (CREB) signaling pathways, as demonstrated by the phosphorylation of Erk1, Erk2 and CREB. Thus, our data not only present the first evidence of the presence of neuroactive compounds that mediate the neuronal differentiation and neuroprotection of the PC12 cells, but also reveal the potential signaling molecules involved in its action.

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein.

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

Proteomic analysis of proteins in PC12 cells before and after treatment with nerve growth factor: increased levels of a 43-kDa chromogranin B-derived fragment during neuronal differentiation.

Proteomic analysis is an important approach to characterizing the proteome and studying protein function in the post-genomic era. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be missed by conventional biochemical techniques. The aim of this study was to perform a preliminary proteomic analysis of PC12 cells in order to investigate the effect of nerve growth factor (NGF) on protein expression in PC12 cells during neurite outgrowth. PC12 cell proteins were separated by two-dimensional electrophoresis (2DE) and visualized by silver staining, then certain proteins were identified by N-terminal amino acid microsequencing and a homology search of a protein sequence database. Over 400 proteins were detected, 10% of which showed a significant (greater than 30%) increase or decrease in expression during NGF-induced neuronal differentiation. Seven proteins in the 2DE map were identified; the levels of five of these were unaffected by NGF treatment, whereas the levels of the other two, beta-tubulin and a novel 43-kDa chromogranin B-derived fragment, were significantly increased by more than 30 and 200%, respectively. Our results suggest that chromogranin B processing is enhanced in PC12 cells during NGF-induced neuronal differentiation. In addition, since this increase in the levels of the chromogranin B-derived fragment was specifically blocked by PD98059, we suggest that the increased processing can be ascribed to activation of the MAP kinase pathway, and that the 43-kDa chromogranin B-derived fragment can serve as a new marker of neuronal differentiation for proteomic studies.

Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin.

Kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. After centrifugation at 16,000 x g for 10 min about 70% of the original RNA-dependent RNA polymerase (RDRP) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with NP40 detergent. Membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solutions, either for 3 h (top-loaded on 4 ml 20-60% sucrose) or for 19 h (centre-loaded in 37 ml 0-60% sucrose). Similar separations of bands of light membranes were obtained in all gradients. Multi-layered heavy membrane bands obtained with w/w sucrose gradients were resolved into two well-separated bands (F4 and F5) using w/v sucrose gradients. Thin-section electron microscopy of embedded membrane fractions, gel analysis of intracellular RNA, and RDRP assays showed that the w/w centre loading method and the w/v top-loading (short spin) method produced similar recoveries and distributions of smooth and rough membranes, intact virus particles and RDRP activity. The distribution of intracellular viral RNA and proteins was coincident with the RDRP, all being located in the F4 and F5 bands which contained the characteristic membrane structures induced during flavivirus infection. Significant advantages of the preferred method (w/v sucrose, top loading and short spin) were its rapidity, good preservation of membranes and RDRP, and the concentrations of RDRP achieved in the small volume fractions collected from a total of 4.5 ml.

Detection and identification of FIJI disease virus in infected sugarcane by immunodiffusion, immuno-osmophoretic and enzyme-linked immunosorbent assays.

Radial double-immunodiffusion, immuno-osmophoretic and enzyme-linked immunosorbent assay (ELISA) methods have been compared for the detection of Fiji disease virus (FDV) in infected sugarcane tissue extracts using an antiserum containing antibodies specific to FDV proteins and ds-RNA. ELISA was the most sensitive of these tests and detected only FDV-specific proteins byt not ds-RNA. Immuno-osmophoretic tests were less sensitive than ELISA but detected both the protein and ds-RNA antigens as distinct precipitin lines. Immunodiffusion tests were much less sensitive for the detection of FDV antigens than either ELISA or immuno-osmophoretic tests. FDV antigens were detected in leaves of virus-infected sugarcane, but only in tissues of the galls which develop in response to infection. Even ELISA failed to detect any antigens in normal tissues adjacent to galls. It is concluded that for the identification to FDV in infected surgarcane, it is necessary to observe galls which can then be tested for the presence of FDV antigens. Immuno-osmophoresis appears to be a satisfactory method for such tests.

Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA.

In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500 g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or "heavy membrane" fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20S to 40S. Enrichment of NS3 and of the small nonstructural proteins NS2A and NS2B/NS4A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the "heavy membrane" fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.

Novel virus-like particles containing circular single-stranded DNAs associated with subterranean clover stunt disease.

Novel virus-like particles, 17-19 nm in diameter, have been isolated from subterranean clover and pea plants infected with the pathogen of subterranean clover stunt disease (SCSD). The structure and genetic organization of these particles suggest that the pathogen of SCSD is representative of a new group of plant DNA viruses. SCS virus-like particles (SCSV) are isometric and band as a single component with buoyant densities of 1.24 g/ml in Cs2SO4 and 1.34 g/ml in CsCl. The A260 nm/A280 nm is about 1.35, which is consistent with an estimated nucleic acid content of 17%. Molecular calculations suggest that the particles have a T = 1 capsid structure containing 60 polypeptide subunits each with Mr of 19,000. Nucleic acid analysis including restriction enzyme digestions of double-stranded cDNAs suggests that SCSV have a divided genome composed of multiple species of circular, single-stranded DNA molecules each of approximately 850-880 nucleotides and that each is encapsidated in a separate particle. Linear and aggregated forms of these DNAs are also detected by gel electrophoresis. Evidence suggests that these virus-like particles are the pathogen of SCSD.

Characterization of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro.

RNA-dependent RNA polymerase (RDRP) activity was characterized in a cytoplasmic extract of Kunjin virus-infected Vero cells at 24 hr. The activity was influenced, possibly indirectly, by the length of prior treatment of infected cells with actinomycin D; however, 6 micrograms/ml actinomycin D and 10(-5) M alpha-amanitin in the RDRP assay had no effect. The replication complex was membrane-bound and Mg2+ was essential for RDRP activity. Incorporation was more dependent on exogenous UTP and GTP than ATP or CTP. The specific activity was low, and rate of incorporation of GMP decreased as the period of assay was increased; however, incorporation of label lasted for at least 60 min. RNA products were fractionated by LiCl precipitation, and kinetic studies showed that the sequence of accumulation of label was the same as that observed in vivo, viz., RI----RF----44 S RNA; limited reinitiation was also observed. This sequence of labeling also indicated that the in vitro RDRP activity was due to an enzyme capable of elongation, release, and reinitiation of Kunjin RNA synthesis and not merely end labeling or elongating preexisting RNA molecules. No labeled bands in urea-polyacrylamide gels were observed using extracts from mock-infected cells and hence the three RNA products of assays were readily identified in a single gel. The replication complex was still active after treatment with nonionic detergent, but no labeled 44 S RNA was detected in gels, even in the presence of RNasin in the assay which inhibited some nuclease activity. Antibodies to flavivirus-specific nonstructural proteins were preincubated with infected cell extracts in the presence and absence of detergent but no inhibition of RDRP activity was observed. However, anti-dsRNA plus detergent blocked activity by as much as 78% and label was found only in RF.

Chemical and serological comparison of the coat proteins of velvet tobacco mottle and Solanum nodiflorum mottle viruses.

Velvet tobacco mottle virus (VTMoV) and Solanum nodiforum mottle virus (SNMV) are serologically closely related as shown by immunodiffusion tests. Dissociated VTMoV coat protein separates into three electrophoretic components in polyacrylamide gels corresponding to polypeptides with Mr's of about 37,000, 33,000 and 31,500, whereas SNMV protein migrates as a single component of Mr about 31,000. Evidence is presented indicating that the VTMoV particle is built from a single species of protein subunit of Mr about 37,000. However, the protein is unstable and degrades during virus purification into a stable polypeptide of Mr about 31,500. It is proposed that the partially degraded VTMoV particle consisting of the stable polypeptide is antigenically similar to the SNMV particle and that these two particles with stable protein subunits are the principal antigens involved in eliciting antibody production in animals. The antigenic and other properties of VTMoV and SNMV are discussed in relation to their taxonomic identity.

The chemical subunit of tobacco ringspot virus coat protein.

The coat protein of tobacco ringspot virus (TRSV) dissociated in the presence of sodium dodecyl sulphate (SDS), urea, and 2-mercaptoethanol (MCE) separated into six components on electrophoresis in polyacrylamide gels. It was established that none of the components was a protein of host plant origin and that they were all chemically and antigenically similar. The estimated molecular weights of the components ranged from about 14,000 to more than 110,000, approximating a polymeric series of 1, 2, 3, 4, and 8; over 85% of the protein was recovered in a band with a molecular weight of about 57,000. These data, together with the amino acid composition of the protein and the results of tryptic peptide mapping experiments, indicate that the TRSV capsid is constructed from a single polypeptide species containing 119 amino acid residues with a molecular weight of 12,988. These results are discussed in relation to capsomeric models of the TRSV protein shell.

Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication.

Only three forms of Kunjin virus-specified RNA were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 S genomic-sized single-stranded RNA, 20 S double-stranded "replicative form" (RF), and 20-28 S partially ribonuclease-resistant (about 70%) "replicative intermediate" (RI). The RF and RI were resolved by electrophoresis in aqueous-agarose gel only following LiCl fractionation. The RI did not enter urea-polyacrylamide gels. After denaturation of untreated or RNase-treated RI and RF, only 44 S RNA was present in electropherograms. RNA polymerase activity at 8 hr postinfection was detected by in vitro assays of cytoplasmic extracts and reached a maximum at 24 hr, the only major labeled product being RF; a trace amount of free 44 S RNA was also produced. These results, and the kinetics of incorporation of [3H]uridine into RI, RF, and 44 S RNA in pulse and pulse-chase experiments, formed the basis of a model in which flavivirus RF functions as a recycling template for semiconservative and (mainly) asymmetric replication, on which only one nascent strand is synthesized per cycle.

Sequence of subterranean clover stunt virus DNA: affinities with the geminiviruses.

The nucleotide sequences of seven circular, single-stranded DNA components of one isolate of subterranean clover stunt virus (SCSV) have been determined. Each component, of about 1 kb, appears to encode a single open reading frame in the same sense as the encapsidated DNA. Notably, the proteins encoded by two SCSV components are related. Each has a consensus nucleotide binding motif and shares about 40% amino acid identity with the other and with the putative replication proteins of banana bunchy top virus and coconut foliar decay virus. The noncoding regions of the five other SCSV components share a highly conserved noncoding sequence of about 160 nucleotides. All seven components were found to contain a sequence capable of forming a stable stem-loop structure in the noncoding region which contains a conserved 9-nucleotide sequence in the loop, very similar to that of the geminiviruses. In addition, the putative replication proteins of SCSV are similar to those of the geminiviruses. We suggest that the SCSV-like viruses and the geminiviruses share a common ancestor and that this ancestor was more like SCSV in particle structure and genome organisation.

The in vitro synthesis of velvet tobacco mottle virus-specific double-stranded RNA by a soluble fraction in extracts from infected Nicotiana clevelandii leaves.

RNA-dependent RNA polymerase activity was detected in a membrane-free cytoplasmic fraction from the leaves of Nicotiana clevelandii 4 days after inoculation with velvet tobacco mottle virus (VTMoV). The amount of enzyme activity was still increasing 8 days after inoculation when virus-induced local lesions were becoming necrotic. RNA synthesis in extracts from leaves infected for 7 days proceeded linearly for about 10 min and required all four ribonucleoside triphosphates and magnesium ions. Enzyme activity was significantly increased by the addition of EGTA to the assay mixture. The polymerase activity was insensitive to actinomycin D, DNase, rifampicin and a-amanitin showing that it was RNA-dependent. The virus-specific RNA-dependent RNA polymerase-template complex in crude extracts from VTMoV-infected leaves and in partially purified preparations was shown to synthesize ds-RNAs of M, about 3.5 X 106 and 0.72 X 106, specific to VTMoV RNAs 1 and 3, respectively. The in vitro synthesis appears to have involved only the elongation of positive strands of RNAs 1 and 3 on their negative strand templates.

Requirement of a genome-associated protein of tobacco ringspot virus for infectivity but not for in vitro translation.

Tobacco ringspot virus (TRSV) RNA incubated with Pronase to render it incapable of initiating infection retained its messenger activity in a wheat germ in vitro translation system. Furthermore, polypeptides synthesized by the Pronase-treated and untreated RNAs were indistinguishable.

Detection, isolation, and characterization of high molecular weight double-stranded RNAs in plants infected with velvet tobacco mottle virus.

Two species of double-stranded (ds) RNA were detected in Nicotiana clevelandii infected with velvet tobacco mottle virus (VTMoV) which were not present in healthy plants. The ds-RNAs were first detected by polyacrylamide gel electrophoresis 2 to 4 and about 8 days after inoculation in the inoculated and systemically infected leaves respectively. Thereafter the concentrations of ds-RNA increased significantly. The VTMoV-specific ds-RNA was purified from plants 10 to 14 days after inoculation. The ds-RNA was electrophoresed on agarose gels, transferred to nitrocellulose paper and blot hybridized with 32P-labelled probes specific to nucleotide sequences of either the positive or negative sense strands of the various VTMoV RNA components: the linear single-stranded (ss) RNA 1 of Mr about 1.5 x 10(6) or RNAs 2 and 3, the circular and linear forms respectively, of viroid-like ss-RNA with Mr of about 1.2 x 10(5). One of the ds-RNA species, with Mr of about 2.8 x 10(6), was shown to contain base sequences specific to RNA 1 and the other, with Mr of about 3.6 x 10(6), specific to RNAs 2 and 3. The possible significance of the VTMoV-specific ds-RNAs to replication of the virus is discussed.

Expression of retinoid receptors during the retinoic acid-induced neuronal differentiation of human embryonal carcinoma cells.

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.

Cloning and expression of a novel nuclear matrix-associated protein that is regulated during the retinoic acid-induced neuronal differentiation.

Retinoic acid (RA), a derivative of vitamin A, is essential for the normal patterning and neurogenesis during development. RA treatment induces growth arrest and terminal differentiation of a human embryonal carcinoma cell line (NT2) into postmitotic central nervous system neurons. Using RNA fingerprinting by arbitrarily primed polymerase chain reaction, we identified a novel serine/threonine-rich protein, RA-regulated nuclear matrix-associated protein (Ramp), that was down-regulated during the RA-induced differentiation of NT2 cells. Prominent mRNA expression of ramp could be detected in adult placenta and testis as well as in all human fetal tissues examined. The genomic clone of ramp has been mapped to the telomere of chromosome arm 1q, corresponding to band 1q32.1-32.2. Associated with the nuclear matrix of NT2 cells, Ramp translocates from the interphase nucleus to the metaphase cytoplasm during mitosis. During the late stage of cytokinesis, Ramp concentrates at the midzone of the dividing daughter cells. The transcript expression of ramp is closely correlated with the cell proliferation rate of NT2 cells. Moreover, overexpression of Ramp induces a transient increase in the proliferation rate of NT2 cells. Taken together, our data suggest that Ramp plays a role in the proliferation of the human embryonal carcinoma cells.