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L Roux

Publications and source records attributed to L Roux.

At least 19 recordsLinked to original sources
PubMed2001

"Rule of six": how does the Sendai virus RNA polymerase keep count?

The "rule of six" stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3'-OH end (3'-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3'-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.

DNA-Directed RNA Polymerases↗
PubMed2001

Scattering by a slab containing randomly located cylinders: comparison between radiative transfer and electromagnetic simulation.

This study is devoted to the examination of scattering of waves by a slab containing randomly located cylinders. For the first time to our knowledge, the complete transmission problem has been solved numerically. We have compared the radiative transfer theory with a numerical solution of the wave equation. We discuss the coherent effects, such as forward-scattering dip and backscattering enhancement. It is seen that the radiative transfer equation can be used with great accuracy even for optically thin systems whose geometric thickness is comparable with the wavelength. We have also shown the presence of dependent scattering.

Journal Article↗
PubMed2000

Structure-function analysis of the Sendai virus F and HN cytoplasmic domain: different role for the two proteins in the production of virus particle.

The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glycoproteins in the process of virus assembly and budding are evaluated. Recombinant Sendai virus (rSeV) mutants are generated carrying modifications in the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state (35)[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cell supernatants are measured relative to the extent of the infection, assessed by the intracellular N protein signal. For both the F and HN cytd truncation mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutants, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this threshold is reached up appears to depend on the nature of the F cytd. A minimal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd truncations result in significant reduction of virion production. On the other hand, a normal virion production can take place with an underrepresentation of or, even, an undetectable HN in the particles. The HN uptake in virion is confirmed to depend on the previously proposed cytd SYWST signal (T. Takimoto, T. Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Virol. 72, 9747-9754.).

Amino Acid Sequence↗
PubMed2000

Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.

Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.

Alleles↗
PubMed2000

Nadroparin in the prevention of deep vein thrombosis in acute decompensated COPD. The Association of Non-University Affiliated Intensive Care Specialist Physicians of France.

Low molecular weight heparins are as effective as unfractionated heparin in deep-vein thrombosis (DVT) prophylaxis for major surgery. However, there is no evidence nor consensus for prophylaxis in medical patients. We compared the efficacy and safety of nadroparin calcium (nadroparin) with placebo in medical patients at high risk of DVT. A total of 223 patients mechanically ventilated for acute, decompensated chronic obstructive pulmonary disease, were randomized to treatment with subcutaneous nadroparin adjusted for body weight (0.4 ml, i.e., 3,800 AXa IU, or 0.6 ml, i.e., 5,700 AXa IU) or placebo. The average duration of treatment was 11 d. The incidence of DVT in patients receiving nadroparin was significantly lower than that in patients receiving placebo (15.5 versus 28.2%; p = 0.045). Although the incidence of adverse events was high in both groups, there were no significant differences between nadroparin and placebo for total adverse events (46.3 versus 39.8%; p = 0.33), serious adverse events (25.0 versus 19.5%; p = 0.32), or those resulting in early permanent discontinuation of treatment (12.0 versus 8.8%; p = 0.44). The most common adverse event was hemorrhage. There was the same number of deaths in both treatment groups. Subcutaneous nadroparin resulted in 45% decrease in incidence of DVT compared with placebo.

Aged↗
PubMed1998

Comparison of PCR-heteroduplex characterization by automated DNA sequencing and line probe assay for the detection of rifampicin resistance in Mycobacterium tuberculosis isolates from KwaZulu-Natal, South Africa.

Progress in understanding the basis of resistance to rifampicin (RifR) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of RifR by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RifR strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed. Most isolates (7/8) harboring Leu533 --> Pro codon mutation required minimum inhibitory concentrations (MICs) of < or = 8 microg/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser531 --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.

Antibiotics, Antitubercular↗
PubMed1998

The activity of Sendai virus genomic and antigenomic promoters requires a second element past the leader template regions: a motif (GNNNNN)3 is essential for replication.

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of approximately 15 kb, contains six transcription units flanked at the 3' and 5' ends by a short (approximately 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3' end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3'-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5'-3' positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.

Binding Sites↗
PubMed1998

[Intracameral lidocaine and phacoemulsification under topical anesthesia. Apropos of 80 operations].

PURPOSE: To evaluate the advantage of intracameral unpreserved lidocaine for patient comfort during phacoemulsification under topical anesthesia. METHODS: In this prospective study, we performed 80 phacoemulsifications under topical anesthesia, with tetracaine 1% drops, 10 minutes before and at the start of surgery: 40 patients received 0.3 cc balanced salt solution (BSS) intracameral injection; 40 patients received 0.3 cc unpreserved lidocaine 1% intracameral injection. The same surgical procedure was performed in both groups: 3.2 mm temporal corneal self-sealing incision, capsulorhexis, foldable polyHEMA IOL implantation into the capsular bag. There was no intravenous sedation. RESULTS: Forty-eight percent (19) in the BSS group and 70% (28) in the lidocaine group felt no pain. 10% (4) in the BSS group reported sharp pain during phacoemulsification. During IOL insertion, no pain was reported by 48% (19) in the BSS group, and 75% (30) in the lidocaine group; 10% (4) in the BSS group felt severe pain (significant difference: p < 0.05). Endothelial cell loss was 6% in the BSS group, and 6.4% in the lidocaine group (non significant difference). CONCLUSION: Intracameral lidocaine is safe and effective in decreasing discomfort among patients undergoing phacoemulsification under topical anesthesia.

Administration, Topical↗
PubMed1997

Inhibition of Sendai virus genome replication due to promoter-increased selectivity: a possible role for the accessory C proteins.

The role of the negative-stranded virus accessory C proteins is difficult to assess because they appear sometimes as nonessential and thereby of no function. On the other hand, when a function is found, as in the case of Sendai virus, it represents an enigma, in that the C proteins inhibit replication under conditions where the infection follows an exponential course. Furthermore, this inhibitory function is exerted differentially: in contrast to the replication of internal deletion defective interfering (DI) RNAs, that of copy-back DI RNAs appears to escape inhibition, under certain experimental conditions (in vivo assay). In a reexamination of the C effect by the reverse genetics approach, it was found that copy-back RNA replication is inhibited by C in vivo as well, under conditions where the ratio of C to copy-back template is increased. This effect can be reversed by an increase in P but not L protein. The "rule of six" was differentially observed in the presence or absence of C. Finally, a difference in the ability of the replicating complex to tolerate promoter modifications in RNA synthesis initiation was shown to occur in the presence or the absence of C as well. We propose that C acts by increasing the selectivity of the replicating complex for the promoter cis-acting elements governing its activity. The inhibitory effect of C becomes the price to pay for this increased selectivity.

DNA-Directed RNA Polymerases↗
PubMed1996

The efficiency of Sendai virus genome replication: the importance of the RNA primary sequence independent of terminal complementarity.

From the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3' end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3'/5' end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identify cis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1-31 of the antigenome 3' end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing.

Animals↗
PubMed1996

A Sendai virus vector leading to the efficient expression of mutant M proteins interfering with virus particle budding.

A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.

Animals↗
PubMed1996

The Sendai paramyxovirus accessory C proteins inhibit viral genome amplification in a promoter-specific fashion.

Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.

Gene Amplification↗
PubMed1996

Polyol pathway and osmoregulation in JS1 Schwann cells grown in hyperglycemic and hyperosmotic conditions.

To test the osmoregulatory rules of Schwann cell aldose reductase (AR) and myo-inositol, JS1 Schwann cells were grown under control and hyperosmotic conditions with and without excess glucose or galactose. JS1 cells cultured in control conditions possessed AR protein and activity that were not altered by the inclusion of 25 mM glucose or galactose. Following culture with 100 mM NaCl, there was a decline in cell number accompanied by an increase in AR activity, both of which were attenuated by the addition of 25 mM glucose or galactose. Sorbitol was not detected in JS1 Schwann cells following culture in control, glucose-supplemented, or hyperosmotic medium, and dulcitol accumulated only following culture with galactose. However, both polyols were dramatically increased in JS1 cells cultured in hyperosmotic medium supplemented with 25 mM glucose or galactose. In contrast, myo-inositol levels were elevated only during hyperosmotic exposure but decreased when glucose or galactose was also present. These data are consistent with the use of polyol formation by JS1 Schwann cells as a means of responding to osmotic stress.

Aldehyde Reductase↗
PubMed1995

A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.

Animals↗
PubMed1995

Unusual anionic N-linked oligosaccharides from bovine lung.

We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

Acetone↗
PubMed1995

Functional characterisation of the genomic and antigenomic promoters of Sendai virus.

A natural Sendai virus internal deletion defective interfering (DI) RNA, previously shown to encode a truncated NP protein and previously cloned under the control of the T7 RNA polymerase promoter, was expressed from plasmid and shown to replicate in cell tissue culture when the viral proteins NP, P, and L were coexpressed from cloned genes. The efficient replication was dependent on the total length of the RNA to be a multiple of 6 nucleotides, showing that the "rule of six" applied for a DI RNA that has conserved the end sequences of the nondefective viral RNA. Compared to the copy-back H4 DI RNA, the replication efficiency of the internal deletion DI RNA was reproducibly 20-fold lower. Reciprocal exchanges between the minus-strand 3'-end primary sequences of the two DI RNAs showed that the replication efficiency of the derivatives obtained directly correlated with the origin and the extent of the primary sequence. Moreover, some of the derivatives exhibited a replication efficiency comparable to that of the copy-back DI RNA with, however, the ability to transcribe a functional mRNA similar to the internal deletion DI RNA. This indicated that the transcription ability of a viral RNA was not sufficient to explain a low replication efficiency.

Animals↗