Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).     

Determination of the nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus genome

Summary.  The nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3′ and 5′ non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5′ NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5′ very end, and the extra 9 nucleotides in the US-2 strain. In the 3′ NCR, swine HEV shared 90–91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58–65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV. Received April 20, 2001 Accepted July 10, 2001     

Determination of the 5′- and 3′-Terminal Sequences Completes the Sequences of the Two Double-stranded RNAs of Penicillium stoloniferum Virus S

The two genomic segments of Penicillium Stoloniferum virus S (PsV-S), a member of the Partitiviridae, were recently sequenced and published. We independantly sequenced PsV-S and showed that the original sequence was missing nucleotides at both the 5′ and 3′ termini of both segments. We determined the correct sequence in three independent experiments and found the segments to be 1753 bp (encoding the RNA-dependant RNA polymerase) and 1581 bp (encoding the Capsid Protein). Homology was shown between the 5′ and 3′ ends of PsV-S and other members of the Partitiviridae. The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers AM040148 and AM040149.     

Electrospun tubular scaffolds: On the effectiveness of blending poly(ε-caprolactone) with poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Tissue engineering can effectively contribute to the development of novel vascular prostheses aimed to overcome the well-known drawbacks of small-diameter grafts. To date, poly(ε-caprolactone) (PCL), a bioresorbable synthetic poly(α-hydroxyester), is considered one of the most promising materials for vascular tissue engineering. In this work, the potential advantage of intimate blending soft PCL and hard poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a polymer of microbial origin, has been evaluated. Nonwoven mats and small-diameter tubular scaffolds of PCL, PHBV, and PCL/PHBV were fabricated by means of electrospinning technique. Mechanical properties and suture retention strength were investigated according to the international standard for cardiovascular implants. Biological tests demonstrated that both PCL-based scaffolds supported survival and growth of rat cerebral endothelial cells in a short time. The fiber alignment of the electrospun tubular scaffolds contributed to a more rapid and homogeneous cell colonization of the luminal surface. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Long,nearly identical untranslated sequences at the 3′ terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain)

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3 UTR) immediately 5 of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3 UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned the accession number Z34265.     

A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

The 613-base 5-untranslated leader (5-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.     

Roles of the polypyrimidine tract and 3′ noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation

Summary. Hepatitis C virus (HCV) genome contains a 3noncoding region (3NCR) consisting of a variable region, a polypyrimidine tract (polyU/UC) and the X region. To examine the roles of 3NCR and polyU/UC tract in the internal ribosome entry site (IRES)-mediated translation process, a variety of 3NCRs containing different lengths of polyU/UC tract were obtained from HCV infected patients and cloned respectively to the downstream of the firefly luciferase coding gene linked to HCV 5NCR and 30 nucleotides of core gene (containing IRES element). The results of in vitro translation in rabbit reticulocyte lysate (RRL) and cell transfection assay in mammalian cells showed that the IRES-mediated translation efficiency could be enhanced by the full-length of 3NCR of HCV RNA. However, contradictory results were observed when the role of polyU/UC tract in the IRES-mediated translation was studied. While the IRES-mediated translation efficiency was inhibited by the presence of polyU/UC tract in in vitro translation experiments, transfection of these expression cassettes into hepatic cell line showed that polyU/UC tract enhanced IRES-mediated translation efficiency in vivo. Cellular-fraction complement experiments showed that cellular factors were required for the enhancement by the polyU/UC tract. Further antibody blocking assay and UV cross-linking assay suggested the correlation of IRES-mediated translation with host factors, including the La protein. The data above also indicated that the modulations of the IRES-mediated translation by the HCV 3NCR and the polyU/UC tract were in a length-independent manner.These authors contributed equally to this work.     

A high poly(ethylene glycol) density on graphene nanomaterials reduces the detachment of lipid–poly(ethylene glycol) and macrophage uptake

Amphiphilic lipid–poly(ethylene glycol) (LPEG) is widely used for the noncovalent functionalization of graphene nanomaterials (GNMs) to improve their dispersion in aqueous solutions for biomedical applications. However, not much is known about the detachment of LPEGs from GNMs and macrophage uptake of dispersed GNMs in relation to the alkyl chain coverage, the PEG coverage, and the linker group in LPEGs. In this study we examined these relationships using single walled carbon nanohorns (SWCNHs). The high coverage of PEG rather than that of alkyl chains was dominant in suppressing the detachment of LPEGs from SWCNHs in protein-containing physiological solution. Correspondingly, the quantity of LPEG-covered SWCNHs (LPEG-SWCNHs) taken up by macrophages decreased at a high PEG coverage. Our study also demonstrated an effect of the ionic group in LPEG on SWCNH uptake into macrophages. A phosphate anionic group in the LPEG induced lower alkyl chain coverage and easy detachment of the LPEG, however, the negative surface charge of LPEG-SWCNHs reduced the uptake of SWCNHs by macrophages.     

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus,a unique tobamovirus that contains an internal poly(A) region in its 3′ end

In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3′-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6–68.7, 49.9–70.8, 31.0–70.8 and 39.4–70.1 %, respectively, at the nucleotide level and 39.8–75.0, 43.6–77.8, 19.2–70.4 and 31.2–74.2 %, respectively, at the amino acid level. The 5′- and 3′-UTRs of HLFPV-J showed 24.3–58.6 and 13.0–79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.     

The N-terminal helix α(0) of hepatitis C virus NS3 protein dictates the subcellular localization and stability of NS3/NS4A complex

The N-terminal amphipathic helix α₀ of hepatitis C virus (HCV) NS3 protein is an essential structural determinant for the protein membrane association. Here, we performed functional analysis to probe the role of this helix α₀ in the HCV life cycle. A point mutation M21P in this region that destroyed the helix formation disrupted the membrane association of NS3 protein and completely abolished HCV replication. Mechanistically the mutation did not affect either protease or helicase/NTPase activities of NS3, but significantly reduced the stability of NS3 protein. Furthermore, the membrane association and stability of NS3 protein can be restored by replacing the helix α₀ with an amphipathic helix of the HCV NS5A protein. In summary, our data demonstrated that the amphipathic helix α₀ of NS3 protein determines the proper membrane association of NS3, and this subcellular localization dictates the functional role of NS3 in the HCV life cycle.     

Does the tissue engineering architecture of poly(3-hydroxybutyrate) scaffold affects cell-material interactions?

A critical element in tissue engineering involves the fabrication of a three-dimensional scaffold. The scaffold provides a space for new tissue formation, supports cellular ingrowth, and proliferation and mimics many roles of the extracellular matrix. Poly(3-hydroxybutyrate) (PHB) is the most thoroughly investigated member of the polyhydroxyalkanoates (PHAs) family that has various degrees of biocompatibility and biodegradability for tissue engineering applications. In this study, we fabricated PHB scaffolds by utilizing electrospinning and salt-leaching procedures. The behavior of monkey epithelial kidney cells (Vero) and mouse mesenchymal stem cells (mMSCs) on these scaffolds was compared by the MTS assay and scanning electron microscopy. Additionally, this study investigated the mechanical and physical properties of these scaffolds by measuring tensile strength and modulus, dynamic contact angle and porosity. According to our results, the salt-leached scaffolds showed more wettability and permeability, but inferior mechanical properties when compared with nanofibrous scaffolds. In terms of cell response, salt-leached scaffolds showed enhanced Vero cell proliferation, whereas both scaffolds responded similarly in the case of mMSCs proliferation. In brief, nanofibrous scaffolds can be a better substrate for cell attachment and morphology.     

Disruption of the 3′ phosphoglycerate kinase (pgk) gene in Aspergillus nidulans

We report the isolation of a pgk ^- mutant strain of Aspergillus nidulans by means of a gene disruption strategy, and demonstrate that the pgk gene is located on chromosome VIII. The pgk ^- mutant conidiates poorly, will only grow on media supplemented with both a glycolytic and a gluconeogenic carbon source, and is inhibited by hexoses.     

Characterization of the thermo- and pH-responsive assembly of triblock copolymers based on poly(ethylene glycol) and functionalized poly(ε-caprolactone)

A series of novel triblock copolymers composed of poly(ethylene glycol) (PEG) and poly(ε-caprolactone)-bearing benzyl carboxylate on the α-carbon of ε-caprolatone were synthesized through ring opening polymerization of α-benzyl carboxylate-ε-caprolactone by dihydroxylated PEG. The debenzylation of the synthesized copolymer, i.e., poly(α-benzyl carboxylate-ε-caprolactone)-b-PEG-b-poly(α-benzyl-carboxylate-ε-caprolactone) (PBCL-b-PEG-b-PBCL), in the presence of hydrogen gas using different levels of catalyst, was carried out to achieve copolymers with various degrees of free α-carboxyl to α-benzyl-ε-carboxylate groups on the hydrophobic block. Incomplete reduction of PBCL led to the formation of poly(α-carboxyl-co-benzyl caboxylate-ε-caprolactone) PCBCL in the lateral blocks at 27%, 50% and 75% carboxyl group substitution. The molecular weight and polydispersity of the resultant copolymers were estimated by ¹H NMR and MALDI-TOF. Synthesized triblock copolymers formed stable micelles at low concentrations (critical micellar concentrations (CMC) of 0.34–12.5 μg ml⁻¹). Polymers containing carboxyl groups in their structure showed a pH-dependent increase in CMC. As the pH was raised from 4.0 to 9.0, CMC increased from 0.76 to 1.06 μg ml⁻¹, for 27% debenzylated polymer, and from 1.30 to 2.20 μg ml⁻¹, for 50% debenzylated polymers. In contrast, the CMC in polymers without carboxyl group was independent of pH (0.55 μg ml⁻¹). Different changes in micellar size as a function of temperature was observed depending on the degree of debenzylation on the PCBCL block: polymers with 27% degree of debenzylation illustrated a rise in micelle size from ∼38 to 55 nm as the temperature increased above 29 °C, while polymers with 50% debenzylation showed a decrease in micelle size, from ∼52 to 38 nm, with increase in temperature. A similar trend was observed at pH 4.5, 7.0 and 9.0 for polymers containing carboxyl groups on their hydrophobic block. The temperature for the onset of size change and/or the extent of aggregate size change was found to be dependent on the pH of the medium and the polymer concentration. The results point to a potential for the formation of thermo- and pH-responsive micelles from triblock copolymers of PEG and carboxyl substituted caprolactone. The results also imply a potential for the 27% debenzylated PCBCL-b-PEG-b-PCBCL copolymers to form a biodegradable thermoreversible gel with a transition temperature a few degrees below 37 °C.     

The long 3′ non-translated regions of Blackcurrant reversion virus RNAs are highly conserved between virus isolates representing different phenotypes and geographic origins

Summary. Blackcurrant reversion virus (BRV) belongs in the subgroup c of nepoviruses. The 3 NTRs of RNAs 1 and 2 of BRV are 1360 and 1363 nucleotides long, respectively, and highly similar (94.8%). In this study we have compared the sequences of the 3 NTRs of ten BRV isolates, originating from different geographic regions or hosts. All deduced sequences were 94.1–98.8% identical with each other, and with the previously deduced 3 NTR sequences of RNAs1 and 2 of the type isolate. The proceeding 480 nucleotides of the CP coding region were 86.9–97.9% identical between the same isolates.     

Adsorption of albumin,collagen, and fibronectin on the surface of poly(hydroxybutyrate-hydroxyvalerate) (PHB/HV) and of poly(ε-caprolactone) (PCL) films modified by an alkaline hydrolysis and of poly(ethylene terephtalate) (PET) track-etched membranes

The effect of alkaline hydrolysis on several surface properties of poly(hydroxybutyratehydroxyvalerate) (92/8) (PHB/HV) and poly(ε-caprolactone) (PCL) films and of poly(ethylene terephtalate) (PET) track-etched membranes have been characterized, as well as the adsorption of three proteins normally encountered by mammalian cells in vivo, namely albumin, collagen, and fibronectin. The water contact angle decreases and the number of -COOH functions accessible to a chemical reaction at the surface of PCL increases with alkaline hydrolysis. Analysis by atomic force microscopy pictures reveals a change in surface morphology. The modifications of surface properties are correlated with a two times increase of the adsorption of three radiolabelled proteins. The hydrolysis results in a slight increase in the water contact angle of one face of the PHB/HV film and a sharp increase in the number of -COOH functions. Important morphology changes are also induced. The adsorption of the radiolabelled proteins is almost 100 times higher on the hydrolyzed polymer than on the native surface. The increase in hydrophilicity of different PET batches correlates to an increase in the number of -COOH functions. Nevertheless, the surface chemical composition and rugosity are constant and no significant difference in the amount of radiolabelled fibronectin adsorbed on the different surfaces is detectable. In conclusion, the effect of hydrolysis on the surface properties of each of the polyesters studied as well as the proteins adsorption on the different surfaces are different. The results strongly support the hypothesis that, in the system studied, parameters other than hydrophilicity influence protein adsorp     

Effect of γ-ray irradiated poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells

The purpose of this study was to clarify the effects of γ-ray irradiated poly(L-lactide) (PLLA) on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. The PLLA was γ-irradiated at the dose of 10, 25 or 50 kGy, and the molecular weight of irradiated PLLA decreased with increasing irradiation dose. The proliferation and differentiation of MC3T3-E1 cells cultured on irradiated PLLA for 2 weeks were evaluated using micromass culture. The γ-irradiation of PLLA did not affect the proliferation, but stimulated the differentiation of MC3T3-E1 cells cultured on irradiated PLLA. These results suggested that lower change in the molecular weight of PLLA was responsible for stimulation of the differentiation of MC3T3-E1 cells cultured on irradiated PLLA. Furthermore, the proliferation and calcification of MC3T3-E1 cells cultured in the medium containing low molecular weight PLLA for 2 weeks were evaluated. The low molecular weight PLLA also stimulated the calcification of MC3T3-E1 cells with no effect on the proliferation. The γ-irradiation was suitable for PLLA on the differentiation of mouse osteoblasts.     

Nucleotide sequences and mutations of the 5′-nontranslated region (5′NTR) of natural isolates of an epidemic echovirus 11′ (prime)

Summary.  An echovirus 11′ (prime) virus caused an epidemic in Hungary in 1989. The leading clinical form of the diseases was myocarditis. Hemorrhagic hepatitis syndroms were also caused, however, with lethal outcome in 13 new-born babies. Altogether 386 children suffered from registered clinical disease. No accumulation of serous meningitis cases and intrauterine death were observed during the epidemic, and the monovalent oral poliovirus vaccination campaign has prevented the further circulation of the virus. The 5′-nontranslated region (5′-NTR) of 12 natural isolates were sequenced (nucleotides: 260–577). The 5′-NTR was found to be different from that of the prototype Gregory strain (X80059) of EV11 (less than 90% identity), but related to the swine vesicular disease virus (D16364) SVDV and EV9 (X92886) as indicated by the best fitting dendogram. The examination of the variable nucleotides in the internal ribosomal entry site (IRES) revealed, that the nucleotide sequence of a region of the epidemic 5′-NTR was identical to that of coxsackievirus B2. Five of the epidemic isolates were found to carry mutations. Seven EV11′ IRES elements possessed identical sequences indicating, that the virus has evolved before its arrival to Hungary. The comparative examination of the suboptimal secondary structures revealed, that no one of the mutations affected the secondary structure of stem-loop structures IV and V in the IRES elements. Although it has been shown previously, that the echovirus group is genetically coherent and related to coxsackie B viruses the sequence differences in the epidemic isolates resulted in profound modification of the central stem (residues 477–529) of stem-loop structure No.V known to be affecting neurovirulence of polioviruses. Two alternate cloverleaf (stem-loop) structures were also recognised (nucleotides 376 to 460 and 540 to 565) which seem to mask both regions of the IRES element complementary to the 3′-end of the 18 S rRNA (460 to 466 and 561 to 570), thus probably diminishing initiation of translation. The possible biological importance of the alternative cloverleaf structures is supported by the fact that neither the 17 variable nucleotides nor the two mutations of epidemic isolates within the regions seem to modify the predicted alternative secondary structures in EV11, SVDV and CBV1-4. Accepted May 31, 2000 Received July 21, 1999