Spectrofluorometric determination of ibuprofen in pharmaceutical formulations.

The spectrofluorometric determination of ibuprofen in pharmaceutical tablets, creams and syrup is described. It involves excitation at 263 nm and emission at 288 nm. the linear range is 2-73 mg L(-1). Other drugs or excipients present in the different formulations do not interfere, except in the case of chlorzoxazone containing tablets. Due to its strong absorbance in the spectral range the chlorzoxazone does interfere, so that in this case the proposed method can't be applied.

Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes.

Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14 degrees C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.

Rainbow trout sleeping disease virus is an atypical alphavirus.

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented. To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV). Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing. Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length. Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined. Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins. Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases. But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined.

Fish rhabdovirus cell entry is mediated by fibronectin.

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.

Comparison of European systemic piscine and amphibian iridoviruses with epizootic haematopoietic necrosis virus and frog virus 3.

Iridovirus-like agents isolated from systemic infected fish (Silurus glanis, SFIR; Ictalurus melas, CFIR I, CFIR II, CFIR III) and from frogs (Rana esculenta, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Perca fluviatilis), and Frog Virus 3 (FV 3) isolated from frogs (Rana pipiens) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. All virus isolates proved to be similar in morphology and in size and reacted with EHNV polyclonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates cleaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3. Southern-blot analysis of viral BamH I digested DNA using an EHNV DNA probe revealed cross-hybridization with DNA of the investigated iridoviruses. Using a set of primers designed for an open reading frame of the EHNV genome, PCR products of about 250 bp were obtained with the DNA of systemic piscine and amphibian iridoviruses. The data suggest that the systemic piscine and amphibian iridoviruses should be regarded as members of the the genus Ranavirus within the family Iridoviridae.

Spectrofluorometric determination of piroxicam.

The spectrofluorometric determination of piroxicam [4-hydroxy-2-methyl-N-(2-pyridyl)-2II-1,2-benzothiazine-3-carboxam ide-1, 1-dioxide] in pharmaceutical tablets is described. It involves excitation at 330 nm of an acid solution (HNO3 0.5 M) of the drug, and measurement of the fluorescence intensity at 440 nm. The linear range is 0.01-1.25 micrograms ml-1.

Investigación retrospectiva de relaciones familiares en pacientes con colagenopatías / Retrocpectives investigation of the familiars relations with collagen diseases patients

n pacientes portadores de enfermedades del colágeno se detectaron familiares con patologías relacionadas. Es muy probable que existan factores genéticos predisponentes en LES, esclerodermia, dermatomiositis y artritis reumatoiodea.Se estudiaron 118 pacientes portadores de colagenopatías; a los cuales se les realizaron examen clínico-dermatológico y microcirculación cutánea por capilaroscopía. Se detectaron 8 pacientes índice con familiares, padres, hermanos e hijos con enfermedades del colágeno

The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice.

Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.

The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice.

The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.

Eighteen years of vaccination against viral haemorrhagic septicaemia in France.

Viral haemorrhagic septicaemia (VHS) has been considered for many years to be a major cause of loss in the French trout industry. The high prevalence of VHS in certain geographic areas made a control strategy based on control policy unfeasible. This provided the impetus for immunoprophylaxis development that resulted in 3 successive types of vaccines: inactivated, live attenuated and recombinant vaccines. When delivered by intraperitoneal injection, the 2 propiolactone-inactivated VHS virus was immunogenic and/or protective for trout all of sizes, but it was not suitable for the practical immunization of alevin, the trout life stage that is the most sensitive to VHS. A carp cell-passed, attenuated variant of the VHS virus was effective after both immersion or injection delivery and met the practical requirements of juvenile vaccination. However, this vaccine was discarded because it retained some virulence that discouraged the launching of its commercialization. Then came the era of genetically engineered vaccines. The recombinant glycoprotein of VHSV produced in Escherichia coli or in Saccharomyces cerevisiae failed to protect fish whatever the route of delivery. A recombinant baculovirus vaccine was found to be immunogenic and protective against VHS, but only when delivered by injection. Due to its cost and route of delivery, the latter vaccine was not licensed. Simultaneously, the sudden occurrence of another rhabdovirosis, infectious haematopoietic necrosis (IHN), in France, rendered vaccination against VHS questionable. Indeed, no cross-protection between these 2 rhabdoviroses exists. If vaccination is still believed to be an effective control method for VHS, it should be based in the future upon an autoreplicative vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

The glycoprotein of viral hemorrhagic septicemia virus (VHSV): antigenicity and role in virulence.

In order to study the antigenic structure of the G protein of VHSV, we produced several anti-G monoclonal antibodies (MAbs) and used 4 neutralizing MAbs (NMAbs) to select resistant (MAR) mutants. Each MAR mutant was confronted with the 4 NMAbs in a neutralization test, and also with our panel of MAbs in surface plasmon resonance (SPR) analysis to determine the extent of their relatedness. Determination of the sequence of the entire G gene of representative MAR mutants allowed us to map the mutations responsible for the resistant phenotypes. We identified several locations on the G protein sequence, which represent, most probably, critical positions within the binding sites of the neutralizing MAbs. In addition, the MAR mutants selected with a cross-reactive MAb exhibited a reduced pathogenicity for fish. This indicated that the regions bearing the point mutations selected with MAb C10 were probably involved in the determination of the virulent phenotype.

Gene expression following transfection of fish cells.

Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.

Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I.

An antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I (VHSV I) was developed. The assay employs two monoclonal antibodies (mAb) directed against distinct epitopes of the viral envelope glycoprotein (Gp). The antigen bound by the capture mAb (A17) was detected by addition of a second mAb (L7) conjugated to horseradish peroxidase, followed by addition of the enzyme substrate. The technique is highly sensitive, enabling detection of the virus at a protein concentration as low as 1 ng/ml of total proteins (1.5 fmol of envelope Gp per ml) in purified preparations. VHSV I was also detected in culture supernatants (5 x 10(5) PFU/ml) and in extracts of kidney and spleen of rainbow trout infected experimentally (5 x 10(5) PFU/ml). The assay was highly specific: infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus, spring viraemia of carp virus, pike fry rhabdovirus, eel rhabdovirus and perch rhabdovirus could not be detected by the antigen-capture ELISA for VHSV I.