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At least 307 records · Page 17Linked to original sources

Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE).

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.

Amino Acid Sequence↗

Short-term change of optic nerve head topography after trabeculectomy in adult glaucoma patients as measured by Heidelberg retina tomograph.

Using Heidelberg Retina Tomograph (HRT, Heidelberg Engineering, Heidelberg, Germany) we investigated whether reversal of glaucomatous optic disc cupping occurs in adult glaucoma patients following intraocular pressure (IOP) reduction by trabeculectomy, and whether topographic change in the optic disc differs according to region. Thirteen eyes of 13 glaucoma patients in whom IOP reduction was 25% or more at postoperative follow-up of 2 months were enrolled in the study. Cup/disc area ratio had decreased significantly, while rim area and rim volume showed significant increases (p < 0.05, Wilcoxon signed rank test). Among the optic disc sectors, rim area in the superior and inferior quadrants, especially in the superotemporal and inferotemporal octant, had increased significantly. The decrease in maximum cup death correlated significantly with the recent IOP reduction (r = 0.698, p = 0.016). Reversal of glaucomatous disc cupping after IOP reduction following trabeculectomy in adult patients as confirmed by HRT. Furthermore, the regions of selective reversal in the superior and inferior quadrants coincided with those parts of the optic nerve head in which early functional and structural glaucomatous damage has been known to occur.

Adult↗

Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast.

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.

1-Methyl-3-isobutylxanthine↗

Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain.

A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.

Amino Acid Sequence↗

Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.

We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.

Culture Media, Conditioned↗

Performance of discrete heat engines and heat pumps in finite time

The performance in finite time of a discrete heat engine with internal friction is analyzed. The working fluid of the engine is composed of an ensemble of noninteracting two level systems. External work is applied by changing the external field and thus the internal energy levels. The friction induces a minimal cycle time. The power output of the engine is optimized with respect to time allocation between the contact time with the hot and cold baths as well as the adiabats. The engine's performance is also optimized with respect to the external fields. By reversing the cycle of operation a heat pump is constructed. The performance of the engine as a heat pump is also optimized. By varying the time allocation between the adiabats and the contact time with the reservoir a universal behavior can be identified. The optimal performance of the engine when the cold bath is approaching absolute zero is studied. It is found that the optimal cooling rate converges linearly to zero when the temperature approaches absolute zero.

Journal Article↗

Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strain produced under a quality system.

Human influenza vaccine reference strains are prepared as required when an antigenically new strain is recommended by WHO for inclusion in the vaccine. Currently, for influenza A, these strains are produced by a double infection of embryonated hens' eggs using the recommended strain and the laboratory strain PR8 which grows to high titre in eggs, in order to produce a high growth reassortant (HGR). HGRs are provided by WHO reference laboratories to the vaccine manufacturing industry which use them to prepare seed virus for vaccine production. The use of reverse genetics in preparing vaccine reference strains offers several advantages over the traditional method: (i) the reverse genetics approach is a direct rational approach compared with the potentially hit-or-miss traditional approach; (ii) reverse genetics will decontaminate a wild type virus that may have been derived in a non-validated system, e.g. a cell line not validated for vaccine purposes, or that may contain additional pathogens; (iii) at the plasmid stage, the HA can be engineered to remove pathogenic traits. The use of reverse genetics in deriving HGRs has been demonstrated by several laboratories, including its use in deriving a non-pathogenic reassortant strain from a highly pathogenic virus. In this report, we have advanced the use of reverse genetics by making use of a cell line acceptable for human vaccine production, by demonstrating directly the short time frame in which a reassortant virus can be derived, and by deriving a non-pathogenic pandemic vaccine reference virus in cells validated for vaccine production and under quality controlled conditions.

Animals↗

Exhaust emissions from engines of the Detroit Diesel Corporation in transit buses: a decade of trends.

In the U.S.A., exhaust emissions from city buses fueled by diesel are not characterized well because current emission standards require engine tests rather than tests of whole vehicles. Two transportable chassis dynamometer laboratories developed and operated by West Virginia University (WVU) have been used extensively to gather realistic emission data from heavy-duty vehicles, including buses, tested in simulated driving conditions. A subset of these data has been utilized for a comprehensive introspection into the trends of regulated emissions from transit buses over the last 7 years, which has been prompted by continuously tightening restrictions on one hand, along with remarkable technological progress, on the other hand. Two widely used models of diesel engines manufactured by the Detroit Diesel Corporation (DDC) have been selected as a case-study for such an overview, based on full-scale, on-site testing of actual city buses, driven in accordance with the SAE J1376 standard of a Commercial Business District (CBD) cycle. The results provide solid, quantitative evidence that most regulated emissions from engines produced by DDC have declined over the years, especially with the transition from the 6V-92TA to the Series 50 models. This improvement is remarkable mainly for the emissions of particulate matter (PM), that are lower by over 70%, on average, for the Series 50 engines, though the emissions of nitrogen oxides (NOx) exhibit a reversed trend, showing a degradation of about 6%, on average, with the transition from 6V-92TA to the Series 50 engines. The expected trend of decreasing emission levels with the model year of the engine is clear and consistent for particulate matter (PM), hydrocarbons (HC), carbon monoxide (CO), and nitrogen oxides (NOx), starting with the 1990 models, although it is not conclusive for carbon dioxide (CO2) emissions.

Air Pollution↗

Effects of exposure to 16.7 Hz magnetic fields on urinary 6-hydroxymelatonin sulfate excretion of Swiss railway workers.

The aim of our study was to examine the effects of 16.7 Hz electromagnetic-field exposure on pineal melatonin production in healthy humans. The study was based on comparing urinary 6-hydroxymelatonin sulfate (6-OHMS) levels of 108 male railway workers between leisure periods and days following the start of service on electrically powered engines (66 engineers) or working beneath transmission lines (42 railway employees such as train attendants and station managers; controls). A repeated measures design was used, i.e., each volunteer served as his own control. The exposure averaged 20 muTesla in the most exposed workers and around 1 muTesla in the least exposed. Apart from magnetic exposure the workers were subject to a shift work schedule with daily advances between 15 min and 1 hr. Melatonin was assessed by sampling urinary 6-OHMS both in the morning and the early evening. Evening 6-OHMS values appeared to be lowered by a factor of 0.81 (95%CI: 0.73-0.90) during work days compared to leisure days among engine drivers, but not in the controls. The lowering was not confined to certain types of shift work such as early, normal, or late shifts. During subsequent leisure periods evening values recovered significantly, mean ratio = 1.27 (95%CI: 1.03-1.56), i.e., the effects appeared to be reversible. In contrast, morning 6-OHMS samples of engineers and controls did not differ much between work and leisure days. There was, however, a tendency for a rebound of morning values in a leisure period following a work period both for engineers and controls. The observed pattern appears to be in line with predictions of the "phase response curve." No evidence for a dose-response relation was found. The results support the hypothesis that 16.7 Hz magnetic fields alter 6-OHMS excretion in humans exposed to magnetic fields. An alternative explanation that cannot be excluded in this study is that the difference between engineers and controls is due to differential exposure to day light at work.

Adult↗

Tissue-engineered human vascular media with a functional endothelin system.

BACKGROUND: Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor's hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel. METHODS AND RESULTS: Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ET(A) receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ET(A) but not the ET(B) receptor was present in these human tissue-engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media. CONCLUSIONS: Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ET(A) receptors and endothelin-converting enzyme.

Aspartic Acid Endopeptidases↗

A new engine-driven canal preparation system with electronic canal measuring capability.

A new cordless engine-driven root canal preparation system has been developed that electronically monitors the location of the file tip and the torque applied to the file during all instrumentation procedures. The Root ZX is mounted inside the handpiece to measure the canal length. The engine is driven with a rechargeable battery, and it works more than 40 minutes without a recharge. Nickel-titanium files are used for this preparation system. The file is rotated at 240 to 280 rpms. When the file tip reaches the apical constriction, the revolution of the file can be automatically reversed (Auto-apical-reverse mechanism). If there is too much torque, the rotation is automatically reversed (Auto-torque-reverse mechanism). These automatic functions promise a safer engine preparation of the canal with nickel-titanium files.

Dental Alloys↗

Expression of human recombinant cAMP phosphodiesterase isozyme IV reverses growth arrest phenotypes in phosphodiesterase-deficient yeast.

The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters. We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV). We have engineered the inducible expression of this human PDEase in yeast. Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases. PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source. We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S. cerevisiae reverses these aberrant phenotypes. Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo. This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.

3',5'-Cyclic-AMP Phosphodiesterases↗

Recombinant soluble human complement receptor type 1 inhibits inflammation in the reversed passive arthus reaction in rats.

The human CR1 was genetically engineered by site directed mutagenesis into a truncated form which was secreted from transfected Chinese hamster ovary cells. This soluble recombinant CR1 (sCR1) was purified from the supernatants of the Chinese hamster ovary cells cultured in a hollow fiber bioreactor. sCR1 inhibits the C3 and C5 convertases of the classical and the alternative pathways in vitro. The ability of sCR1 to inhibit the immune complex-mediated inflammation in vivo was tested in a rat reversed passive Arthus reaction model. Administration of sCR1 at the dermal sites reduced the Arthus vasculitis in a dose-dependent manner as judged by both gross and microscopic examination, as well as by immunohistologic localization of C3 and C5b-9 neoantigen deposits. These data suggest that sCR1 inhibits the Arthus reaction by interrupting the activation of the C cascade, hence limiting the detrimental immune complex-induced tissue damage in vivo.

Animals↗

The retinoblastoma protein--from bench to bedside.

The retinoblastoma tumour suppressor protein (Rb) has come a long way since its initial discovery in 1986. Encoded by the first candidate tumour suppressor gene it has emerged a versatile and context-dependent modulator of cell behaviour. Its activity is managed by signalling networks sensing intra- and extracellular cues. These cues are relayed to hold or permit inactivation of Rb by phosphorylation. Loss or mutation of the retinoblastoma gene is rare in sporadic cancers but defects in the pathways that license inactivation of Rb are found in the majority of them, suggesting that loss of Rb control is central to tumour development and arguing that its reinstatement might reverse tumour formation. Furthermore, mouse models with engineered defects in the Rb-phosphorylating kinases provide evidence that moderation of Rb inactivation may be a strategy for the prevention of tumour formation. The rationale behind these arguments, their underlying molecular concepts and strategies towards therapeutic application will be discussed.

Animals↗

Replication strategies of rabies virus.

Rabies virus (RV) is a prototype neurotropic virus that causes fatal disease in human and animals. RV infects hosts at the periphery, enters motoneurons or sensory nerves and moves to the central nervous system (CNS) via retrograde axonal transport. At later stages, there is also centrifugal spread to major exit portals, such as the salivary glands. Transmission to other hosts is facilitated by behavioral changes related to the CNS infection. Successful accomplishment of the RV infectious cycle depends on multiple functions of the virus, and of individual virus proteins, all together defining the typical pathogenicity and virulence, i.e. the biological fitness of this virus. In particular, it appears important for RV to sneak into the host without causing pronounced host responses and to preserve, at least for some time, the integrity of infected cells and of the neuronal network. The availability of reverse genetics systems that allow generation of engineered recombinant RV has provided tools for a more detailed analysis of viral functions relevant to the typical RV pathogenesis. Novel developments such as tracking of live fluorescent RV are further increasing the opportunities to decipher RV pathogenicity factors. In this review, we describe different aspects of the molecular biology of RV that are relevant to pathogenesis, with a particular emphasis on the accurate control of RV transcription, gene expression, and replication. In addition, the role of individual virus proteins in maintaining host cell integrity and supporting retrograde transport is discussed. The potential of recombinant RVs with single or multiple pathogenicity factors eliminated is being discussed in terms of vaccine and virus vector development.

Amino Acid Sequence↗

Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

Amino Acid Sequence↗

Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.

3T3 Cells↗

Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro.

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

Animals↗