Effect of precursor type on the reduction of concentrated nitrate using zero-valent copper and sodium borohydride.

In this study, we demonstrated that the choice of precursor has a strong effect on the reduction of nitrate (NO3-) using zero-valent copper (Cu0) synthesized by sodium borohydride (NaBH4). Different precursors: CuSO4, CuO, Cu2O, Cu powder, and Cu mesh were used to reduce NO3- at 677 mg-N/L under the reducing conditions of NaBH4. Compared with the prehydrolyzed samples, those prepared without prehydrolysis exhibited lower reduction rates, longer times and higher concentrations of nitrite (NO2-) intermediate. It was found that one-time addition of NaBH4 resulted in higher reduction rate and less NO2- intermediate than two-step addition. Results showed that Cu0 from CuSO4 possessed the smallest particle size (890.9 nm), highest surface area (26.0 m2/g), and highest reaction rate (0.166 min-1). Values of pseudo-first-order constant (kobs) were in the order: CuSO4 > CuO > Cu2O > Cu powder >Cu mesh. However, values of surface area-normalized reaction rate (kSA) were approximately equal. It was proposed that NO3- was reduced to NO2- on Cu0, and then converted to NH4+ and N2, respectively; H2 generated from both NaBH4 hydration and Cu (II) reduction contributed to NO3- reduction as well.

Reduction of concentrated nitrate by using in situ synthesized zero-valent copper.

Although zero-valent iron represents a promising approach for reduction of nitrate (NO(3)(-)) in water, its application in concentrated nitrate is limited by surface passivation. In this study, an alternative approach using in situ synthesized zero-valent copper (Cu(0)) produced by borohydride (NaBH(4)) was investigated. Complete reduction was observed within 55 min by reacting 677 mg-N/L of NO(3)(-) with CuO (0.312 g/L) and NaBH(4) (4.16 g/L) at 60 °C. The pseudo-first-order rate constant was 0.059 min(-1), and it increased threefold when the CuO dose was increased to 1.24 g/L. Increasing the NaBH(4) dose produced less nitrite (NO(2)(-)) throughout the experiments, indicating that it is the primary agent for reducing NO(2)(-). The initial pH exerted a significant effect on the reaction rate, and NO(3)(-) was rapidly reduced when the initial pH was less than 4. Based on the research findings, possible reaction pathways for NO(3)(-) reduction by Cu(0) are proposed in this work.

Molecular characterization of photosensitizer-mediated photodynamic therapy by gene expression profiling.

Photodynamic therapy (PDT) is a novel cancer treatment based on the tumor-specific accumulation of a photosensitizer followed by irradiation with visible light, which induces selective tumor cell death via production of reactive oxygen species. To elucidate the underlying mechanisms, microarray analysis was used to analyze the changes in gene expression patterns during PDT induced by various photosensitizers. Cancer cells were subjected to four different photosensitizer-mediated PDT and the resulting gene expression profiles were compared. We identified many differentially expressed genes reported previously as well as new genes for which the functionfunctions in PDT are still unclear. Our current results not only advance the general understanding of PDT but also suggest that distinct molecular mechanisms are involved in different photosensitizer-mediated PDT. Elucidating the signaling mechanisms in PDT will provide information to modulate the antitumor effectiveness of PDT using various photosensitizers.

Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR) is often associated with impairment of thermonociceptive functions. This study investigated skin innervation and its clinical significance in genetically defined FAP due to a hot-spot Ala97Ser TTR mutation (Ala97Ser). METHODS: Skin biopsies were performed on the distal leg of patients with Ala97Ser, and intraepidermal nerve fiber (IENF) densities were quantified. RESULTS: There were 19 unrelated patients with Ala97Ser manifesting a late-onset (59.47 +/- 5.70 years) generalized neuropathy with disabling motor, sensory, and autonomic symptoms. Against a background of a slowly progressive course, 7 patients (36.8%) exhibited additional rapid declines in neurologic deficits, which were associated with elevation of the protein content in the CSF (p < 0.001). The IENF density was markedly reduced in Ala97Ser patients compared to age- and gender-matched controls (0.99 +/- 1.11 vs 8.31 +/- 2.87 fibers/mm, p < 0.001). Skin denervation was present in all patients and was lower in patients with a higher disability grade (0.17 +/- 0.26 vs 1.37 +/- 1.16 fibers/mm, p = 0.003). Albuminocytologic dissociation in the CSF was observed in 14 patients (73.7%), and the IENF density was negatively correlated with the CSF protein concentration (p = 0.015). CONCLUSIONS: Skin denervation was common in Ala97Ser, and degeneration of cutaneous nerve terminals was correlated with the severity of clinical phenotypes and the level of CSF protein.

A novel chromosome region maintenance 1-independent nuclear export signal of the large form of hepatitis delta antigen that is required for the viral assembly.

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus, as it requires hepatitis B virus for virion production and transmission. We have previously demonstrated that sequences within the C-terminal 19-amino acid domain flanking the isoprenylation motif of the large hepatitis delta antigen (HDAg-L) are important for virion assembly. In this study, site-directed mutagenesis and immunofluorescence staining demonstrated that in the absence of hepatitis B virus surface antigen (HBsAg), the wild-type HDAg-L was localized in the nuclei of transfected COS7 cells. Nevertheless, in the presence of HBsAg, the HDAg-L became both nuclei- and cytoplasm-distributed in about half of the cells. An HDAg-L mutant with a substitution of Pro-205 to alanine could neither form HDV-like particles nor shift the subcellular localization in the presence of HBsAg. In addition, nuclear trafficking of HDAg-L in heterokaryons indicated that HDAg-L is a nucleocytoplasmic shuttling protein. A proline-rich HDAg peptide spanning amino acid residues 198 to 210, designated NES(HDAg-L), can function as a nuclear export signal (NES) in Xenopus oocytes. Pro-205 is critical for the NES function. Furthermore, assembly of HDV is insensitive to leptomycin B, indicating that the NES(HDAg-L) directs nuclear export of HDAg-L to the cytoplasm via a chromosome region maintenance 1-independent pathway.

Roles of the AX(4)GKS and arginine-rich motifs of hepatitis C virus RNA helicase in ATP- and viral RNA-binding activity.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX(4)GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX(4)GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the gamma-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patients.

The purpose of this study was to investigate the clinical usefulness of the color Doppler vascularity index (CDVI) in patients with colon cancer before surgery. Forty-four patients with sonographically visible tumor mass of colon cancer were investigated. The CDVI of each tumor was determined using transabdominal color Doppler ultrasound. The CDVI was defined as the ratio of the number of the colored pixels within a tumor section to the number of total pixels in that specific tumor section and was calculated by using Encomate software (Electronic Business Machine Co. Ltd., Taipei, Taiwan). The correlation between the CDVI and clinicopathological factors, mode of recurrence, and patient survival was studied. For comparison, microvessel density (the mean number of microvessels in three areas of highest vascular density at x200 magnification) of the tumors of these 44 patients was also evaluated by using immunohistochemical staining of surgical specimens with anti-CD34. The microvessel density was not correlated with Dukes' classification, clinicopathological factors, and survival. The CDVI was significantly higher in the patients with lymph node metastases and vascular invasion than in those without such metastases and invasion (P = 0.006 and P = 0.0098, respectively). Moreover, in patients with a high CDVI (> 15%) and positive vascular invasion, survival was significantly poorer than in those with low CDVI (< or = 15%) and negative invasion (P = 0.0037 and 0.0039, respectively). Multivariate analysis indicated that liver metastasis, vascular invasion, and CDVI are independent prognostic factors in the patients with colon cancer. According to the mode of recurrence in 36 patients who underwent curative resection, the frequency of the distant organ recurrence was significantly higher in the high CDVI group (40%) than in the low CDVI group (0%). The CDVI is a good preoperative indicator of recurrence and patient survival in colon cancer. Thus, the CDVI may be helpful in stratifying patients for adjuvant therapy.

Specific interaction between the hepatitis delta virus RNA and glyceraldehyde 3-phosphate dehydrogenase: an enhancement on ribozyme catalysis.

Replication of hepatitis delta virus (HDV) RNA occurs in the nuclei of infected cells. The replication is mediated by cellular factors containing an RNA polymerase II-like enzyme activity through a double rolling-circle mechanism and is regulated by delta antigens. In this study, UV cross-linking experiments were carried out to examine interactions between HDV RNA and proteins present in HeLa nuclear extract. Cellular proteins with molecular mass of 23 (p23), 36 (p36), 38 (p38), and 58 (p58) kDa bound to full-length HDV RNA of both genomic and antigenomic strands. Deletion analysis on the antigenomic strand mapped the interacting domain within a 79-nucleotide fragment but not at the ends of the rod-shaped viral RNA structure. The specificity of the RNA-protein interactions was demonstrated by competition experiments and the specific HDV RNA-binding proteins were purified through column chromatography. Electrophoresis mobility shift assay with the purified fractions demonstrated that the interaction between p36 and HDV RNA was relatively stable even in the presence of 0.5 M NaCl. Biochemical analysis including protein microsequencing identified the p36 as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RNase footprinting indicated that the UC-rich domain between nucleotides 379 and 414 of the HDV antigenomic RNA was involved in the GAPDH binding. Functional studies further demonstrated an enhancing effect of GAPDH on the ribozyme activity of HDV antigenomic RNA. In addition, in the presence of HDV RNA cellular GAPDH relocalized from the cytoplasm to the nucleus where HDV replication occurs. These results suggest that GAPDH is involved in the replication of HDV.

A 12-amino acid stretch in the hypervariable region of the spike protein S1 subunit is critical for cell fusion activity of mouse hepatitis virus.

The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evidently important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of the naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned, and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino acid stretch in the S1 hypervariable region from amino acid residue 446 to 457 when compared with the fusion-positive strain MHV-JHM. In addition, there are three amino acid substitutions in the S2 subunit, Tyr-1144 to Asp, Glu-1165 to Asp, and Arg-1209 to Lys. The cloned MHV-2 S protein exhibited the fusion-negative property in DBT cells as the intrinsic viral protein. Furthermore, similar to the fusion-positive MHV-JHM strain, proteolytic cleavage activity was detected both in DBT cells infected with the fusion-negative MHV-2 and in the transfected cells that expressed the cloned MHV-2 S protein. Domain swapping experiments demonstrated that the 12-amino acid stretch missing in the MHV-2 S1 subunit, but not the proteolytic cleavage site, was critical for the cell-fusion activity of MHV.

Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3' viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5' noncoding region and very poorly by the 3' conserved 98-nucleotide tail. A 3' coding region with conserved stem-loop structures rather than the 3' noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3' coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.

The nucleolin binding activity of hepatitis delta antigen is associated with nucleolus targeting.

Hepatitis delta antigens (HDAgs) are important for the replication and assembly of hepatitis delta virus (HDV). To understand the association between HDAgs and cellular proteins and the mechanism of viral multiplication, we have studied the interaction between HDAgs and nucleolin, a major nucleolar phosphoprotein. The interaction between HDAgs and nucleolin was first demonstrated by immunofluorescence staining studies. HDAgs and endogenous nucleolin were colocalized in the nucleoli of cultured cells transfected with plasmids encoding the small and large HDAg. Coimmunoprecipitation results indicated that the NH2-terminal domain of HDAg was essential for its binding to nucleolin. In vitro ligand binding assays revealed two nucleolin binding sites, NBS1 and NBS2. Each spanned amino acid residues 35-50 and 51-65, respectively, with a conserved core sequence K(K/R)XK. HDV replication was modulated by exogenous human nucleolin. In addition, a small HDAg mutant S-d65/75, which possesses both NBS1 and NBS2, was capable of transactivating HDV replication, whereas the small HDAg mutant S-d50/75, which retained NBS1 but not NBS2, was unable to support the replication of HDV. Thus, the nucleolin binding activity of HDAg is critical for its nucleolar targeting and is involved in the modulation of HDV replication.

Prognostic factors in patients with bulky stage IB or IIA cervical carcinoma undergoing neoadjuvant chemotherapy and radical hysterectomy.

All patients with bulky (> or =4 cm) Stage Ib or IIa cervical carcinoma treated at Chang Gung Memorial Hospital between August 1988 and December 1991 using a strategy of neoadjuvant chemotherapy with cisplatin, vincristine, and bleomycin and radical hysterectomy were reviewed. Fifty-nine evaluable patients received 1 to 3 courses of chemotherapy, and 51 underwent subsequent hysterectomy. The remaining 8 patients, not completing planned surgery, were treated with definitive radiotherapy. The overall clinical response rate was 81.4% (48/59) with 18.6% complete response. Clinical response to chemotherapy was not different by stage, histologic type, tumor size, level of squamous cell carcinoma antigen, or DNA ploidy. However, tumors with DNA indices (DI) greater than 1.3 were associated with higher clinical response rates than tumors with DI < or = 1.3 (P = 0.043). Histologically proven pelvic node metastases was noted in 18.5% (10/54) who had laparotomy. Concomitant pregnancy and more than one node metastases had significant adverse influence on recurrence and death. The 5-year survival rate of those patients who received hysterectomy was 80.3%, while only 1 of the 8 patients without hysterectomy survived. Of the 7 patients received hysterectomy despite clinical poor response, only 2 had node metastases and 3 died, whereas all the 4 patients deterred hysterectomy for poor response died. This study demonstrates the value of DNA flow cytometry in predicting chemosensitivity. However, with a DI cutoff at 1.3, only 29.2% patients could be selected. Further studies are necessary to find additional indicators that predict histological response to select better candidates for this approach and to determine optimal adjunctive treatment in case that poor prognostic features are found.

Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant.

The fact that both aluminum hydroxide adjuvant and proteins have a pH dependent surface charge means that electrostatic forces play a role in the adsorption of proteins by aluminum hydroxide adjuvant during the preparation of vaccines. The objective of this study was to examine the contribution of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. Since the surface charge characteristics of aluminum hydroxide adjuvant can be modified by the adsorption of phosphate anion, a series of aluminum hydroxide adjuvants were prepared by treatment with various concentrations of phosphate anion. The isoelectric points (iep) of these adjuvants ranged from 11.0 to 4.6 and the electrophoretic mobilities at pH 7.4 ranged from 2.0 to -3.3 microns cm/V s. The line broadening of the (020) band of the X-ray diffraction pattern indicated that treatment with phosphate anion did not change the primary crystallite dimension. Adsorption at pH 7.4 of positively charged lysozyme (iep = 11.1) was directly related to the negative surface charge of the adjuvant. No adsorption occurred when the surface charge was positive. In contrast, negatively charged ovalbumin (iep = 4.6) was adsorbed by all of the adjuvants at pH 7.4, although the adsorptive capacity was the greatest when the surface charge was positive. The results indicate that adsorptive forces in addition to the electrostatic attractive force play an important role in the adsorption of some proteins by aluminum hydroxide adjuvant. It is believed the structurally flexible proteins, like ovalbumin, exhibit more complex adsorption behavior than structurally rigid proteins, like lysozyme, for which adsorptive behavior can be explained by electrostatic forces.

Rifabutin as salvage therapy for cases of chronic multidrug-resistant pulmonary tuberculosis in Taiwan.

This study was aimed at assessing the efficacy and tolerability of rifabutin for the re-treatment of cases of chronic, multidrug-resistant pulmonary tuberculosis. The study design was self-controlled, single center. Rifabutin was administered as part of an individual-tailored multidrug regimen. In-patients suffering from pulmonary tuberculosis, infected with Mycobacterium tuberculosis bacilli resistant to isoniazid, rifampicin and other drugs with progressive disease unresponsive to prior courses with standard anti-tuberculosis medications were treated. Overall, 43 patients were enrolled and treated with rifabutin at 300 or 450mg/day according to body weight in conjunction with available anti-tuberculous drugs for a mean time of 353 days (range 42-678). Of these, 36 met all eligibility criteria (i.e. positive baseline culture of sputum with bacilli resistant to rifampicin at least) and were retained for the analysis of efficacy. Seventeen patients (47%) achieved a sustained conversion to a negative culture of sputum in a mean time of 47.7 days with a range of 14-120 days. Treatment prevented deterioration in most patients and resulted in clinical and radiological cure or marked improvement in more than half of cases. No correlation was found between treatment outcome and use of medication concomitant to rifabutin or susceptibility of bacilli to the drugs used. Four deaths occurred due to disease progression, in no case being related to study drugs. Ten patients reported a total of 18 adverse events that led to treatment discontinuation in 5 cases. Rifabutin should be considered for inclusion in regimens for cases of pulmonary multidrug-resistant tuberculosis which fail to respond to previous therapy.

Breast cancer vascularity: color Doppler sonography and histopathology study.

In this prospective study, the authors examined 50 patients with breast tumors (malignant, n = 32; benign, n = 18) to investigate the correlation between color Doppler flow mapping and histopathological findings and to evaluate the clinical significance of color Doppler mapping. Among the 32 patients with breast cancer, color Doppler signals were detected in 24 patients (75%). The maximum flow velocities varied from 5 to 34 cm/sec, with 16 (67%) of them above 15 cm/sec. Among the 18 patients with benign tumors, color Doppler signals could be detected in 7 (39%). The maximum flow velocity varied from 3 to 30 cm/sec but was over 15 cm/sec in only two patients (28%). Histological studies revealed that color Doppler signals detected by Doppler sonography correlated with disordered neovascularization penetrating the lesion from its periphery, consisting of thin-walled blood vessels and large arteriovenous shunts. Although large tumors tend to have high Doppler flow, there is no significant correlation between the maximum flow velocity and tumor size. There is also no significant correlation between the detection of high flow color Doppler signals and the age, receptor status, tumor size, lymph node metastases, or clinical stage of patients with breast cancer. However, there is a positive association (p < 0.05) between nodal metastases and higher tumor flow velocity in T1 (< or = 2 cm) breast tumors, but not in larger tumors. It is concluded that color Doppler is useful in the assessment of tumor vascularity but is of limited value in the differentiation of benign from malignant lesions. However, the presence of color Doppler signals in T1 breast cancer suggesting early dissemination of the cancer might be of important clinical significance in detecting those small, apparently early, but aggressive tumors with poor prognosis.

Automated analytical systems for drug development studies. Part IV. A microdialysis system to study the partitioning of lomefloxacin across an erythrocyte membrane in vitro.

An automated system utilizing microdialysis sampling, intermittent dosing, and liquid chromatographic analysis was assembled in order to study the partitioning of lomefloxacin, a fluoroquinolone antimicrobial, into human erythrocytes in vitro. The apparent erythrocyte:buffer partition coefficient was found to be approximately 2.0 with this system and by a manual method. The value was concentration-dependent; lower partition coefficients were observed at lomefloxacin concentrations less than 1 microgram ml-1. At all concentrations, values obtained by microdialysis were statistically indistinguishable from those obtained by a conventional manual method. The results indicate that erythrocyte partition coefficients can be measured successfully with the microdialysis system. Furthermore, microdialysis sampling eliminates the tedious methodology associated with traditional erythrocyte partitioning studies, including sample clean-up. Due to automated intermittent dosing and on-line LC analysis, the system operates unattended.

Functional domains of delta antigens and viral RNA required for RNA packaging of hepatitis delta virus.

The functions of delta antigens (HDAgs) in the morphogenesis of hepatitis delta virus (HDV) have been studied previously. The C terminus of large HDAg has been shown to complex with the small surface antigen (HBsAg) of helper hepatitis B virus, whereas the assembly of small HDAg requires interaction with the N terminus of large HDAg (M.-F. Chang, C.-J. Chen, and S. C. Chang, J. Virol. 68:646-653, 1994). To further examine the molecular mechanisms by which HDAgs are involved in the assembly of HDV RNA, we have cotransfected Huh-7 cells with plasmids representing a longer than unit-length HDV and the small HBsAg cDNAs. We found that HDAg mRNA could be generated from an endogenous promoter within the HDV cDNA that was translated into large HDAg. Large HDAg is capable of complexing with monomeric HDV genomic RNA to form ribonucleoprotein particles (RNPs) and is capable of forming enveloped HDV-like particles in the presence of small HBsAg without undergoing HDV replication. In addition, the middle region from amino acid residues 89 to 145 of large HDAg is required for assembly of the RNPs but is dispensable for assembly of the enveloped particles. RNA assembly is also demonstrated with small HDAg when it is cotransfected with a packaging-defective large HDAg mutant and small HBsAg. Leu-115 within the putative helix-loop-helix structure of the small HDAg is important for the replication of HDV but is not essential for RNA assembly, suggesting that conformational requirements of small HDAg for replication and assembly of viral RNA may be different. Further studies indicate that a 312-nucleotide linear HDV RNA from one end of the HDV and structure is sufficient to form RNP complexes competent for assembly of virus-like particles with large HDAg and small HBsAg.

Cellular proteins specifically bind to the 5'-noncoding region of hepatitis C virus RNA.

Hepatitis C virus (HCV) RNA contains a highly conserved 5'-noncoding region (5'NCR) which may be important in viral multiplication. To study the possible mechanisms of the cellular proteins involved in HCV replication and pathogenesis, a gel mobility shift assay and competition analysis were performed with the HCV 5'NCR. Two specific complexes were formed between the 341-nucleotide RNA of the HCV 5'NCR and proteins of mammalian cells. The specific RNA-protein complexes were maintained in the region of the 5'NCR from nucleotides 131 to 253. Nevertheless, the slower migrating RNA-protein complex failed to form when a polypyrimidine tract sequence (191-UCCUUUCUU-199) in the stem-loop III structure of HCV 5'NCR was changed to 191-UCCUUUggU-199. A uv cross-linking assay further identified two cellular proteins, p87 and p120, that specifically bound to the stem-loop III structure. Mutations at the polypyrimidine tract sequence inhibited the binding of p87, but maintained the ability of the mutant HCV RNA to interact with p120. Translation competition assay demonstrated that the 5'NCR from nt 131 to 253 within the stem-loop III structure is important for the translation of HCV core protein. In addition, p120 and unidentified cellular proteins are likely to be involved in the translation of HCV polyprotein, whereas p87 may play important roles in HCV multiplication other than translation.

Nuclear localization signals in the core protein of hepatitis C virus.

The core protein of the hepatitis C virus is derived from the N-terminal 191 amino acids of the viral polyprotein by proteolytic cleavage. In the current study, subcellular localizations of the HCV core and its beta-galactosidase fusion proteins in transfected cells were examined by indirect immunofluorescence and cytochemical staining. The core protein was located predominantly in the cytoplasm 6 days after a plasmid encoding the full-length core protein had been introduced into mammalian cells. A hydrophobic domain in the C-terminal region of the core protein may block the efficiency of nuclear transport, since a beta-galactosidase fusion protein that contains HCV core protein lacking the C-terminal 66-amino-acid was located within the nuclei of mammalian cells 24 hours posttransfection. Three independent nuclear localization signals were further identified in the N-terminal region of the HCV core protein.