Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family.

The multiple sclerosis-associated retrovirus (MSRV) isolated from plasma of MS patients was found to be phylogenetically and experimentally related to human endogenous retroviruses (HERVs). To characterize the MSRV-related HERV family and to test the hypothesis of a replication-competent HERV, we have investigated the expression of MSRV-related sequences in healthy tissues. The expression of MSRV-related transcripts restricted to the placenta led to the isolation of overlapping cDNA clones from a cDNA library. These cDNAs spanned a 7.6-kb region containing gag, pol, and env genes; RU5 and U3R flanking sequences; a polypurine tract; and a primer binding site (PBS). As this PBS showed similarity to avian retrovirus PBSs used by tRNATrp, this new HERV family was named HERV-W. Several genomic elements were identified, one of them containing a complete HERV-W unit, spanning all cDNA clones. Elements of this multicopy family were not replication competent, as gag and pol open reading frames (ORFs) were interrupted by frameshifts and stop codons. A complete ORF putatively coding for an envelope protein was found both on the HERV-W DNA prototype and within an RU5-env-U3R polyadenylated cDNA clone. Placental expression of 8-, 3.1-, and 1.3-kb transcripts was observed, and a putative splicing strategy was described. The apparently tissue-restricted HERV-W long terminal repeat expression is discussed with respect to physiological and pathological contexts.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis.

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

Development of a pan-retrovirus detection system for multiple sclerosis studies.

INTRODUCTION: Although recent claims implicating HTLV-1 in multiple sclerosis (MS) have been refuted, several reports suggest that another, hitherto uncharacterised, retrovirus may be involved. We have developed and applied a novel PCR-based strategy to explore this possibility. METHODS: Degenerate oligonucleotides were used in a semi-nested format to amplify, from reverse-transcribed RNA, a region of the pol gene which is well conserved amongst all known retroviruses. RESULTS: The 'pan-retrovirus' detection system was shown to be capable of detecting diverse retroviruses including human lentivirus, human oncovirus, simian D-type virus and murine oncovirus. The 'pan-retrovirus' technique identified a novel retroviral sequence, designated MSRV-cpol, in the serum of an MS patient and also in purified virions from MS patient-derived tissue cultures. Sequence comparisons suggest that in the pol gene MSRV is related (approximately 75% homology) to the endogenous retroviral element ERV9. CONCLUSION: These findings lend further support to the concept of retroviral involvement in MS.

Cell cultures and associated retroviruses in multiple sclerosis. Collaborative Research Group on MS.

Retroviral particles associated with reverse transcriptase (RT) activity in cell-cultures from MS patients have been reported by different groups. Cell-cultures have been used for the study and characterization of the corresponding retroviral genome which we have shown is related to ERV9 in the pol region. Previously unpublished details of a study with monocyte cultures are presented together with observations on leptomeningeal and choroid-plexus cultures. The generation of self-transformed cultures after inhibition of interferon, followed by the loss of retroviral expression and recurrent apoptosis, is analyzed. Retroviral particles with RT-activity are produced in monocyte cultures with an apparent correlation with MS disease activity. However, though leptomeningeal and choroid plexus cells from MS can be passaged for a limited period, their evolution in vitro is not compatible with stable retroviral expression. These culture limitations greatly hampered progress on the elucidation of the retroviral genome sequence.

Rat liver cytosol oxysterol-binding protein. Characterization and comparison with the HTC cell protein.

A cytosol protein that specifically binds cholesterol derivatives oxygenated on the side chain has been demonstrated in rat liver and transformed HTC cells. This protein, of which the sedimentation coefficient is about 8 S, was saturable and showed a high binding affinity (Kd about 5 X 10(-9) M) for 25-hydroxycholesterol. Its molecular mass is about 160 kDa. The physicochemical characteristics of this protein were identical whether the model was normal or transformed. This oxysterol-binding protein differs from the well-known sterol carrier proteins.

Characterization of oxysterol-binding protein in rat embryo fibroblasts and variations as a function of the cell cycle.

A cell-free system assay involving cell freeze-thawing and protein fractionation by ammonium sulfate precipitation was developed to characterize a cytosol binding protein specific for oxysterols in rat embryo fibroblasts. This protein shared common characteristics with the oxysterol-binding protein described in L cells and in normal human lymphocytes: 8 S sedimentation coefficient, sterol-protein complex of Mr 160 600, saturability, high affinity (Kd in the range of 10(-9) M) and specificity for cholesterol derivatives oxidized on the side chain. These compounds were better inhibitors of DNA synthesis than the compounds oxidized on the nucleus, whereas no difference was found between sterols oxygenated either on the side chain or on the nucleus, as far as inhibition of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) was concerned. Macromolecular components capable of specifically binding 25-hydroxycholesterol were also detected in the fibroblast nucleus. The cytosol oxysterol-binding protein showed a sharp increase (5-fold) in the G2M phase of the cell cycle and in exponentially growing cells (maximal binding site number/cell: 43 500, versus 8850 in confluent cells). Neither the affinity nor the sedimentation coefficient of the protein changed in either situation. The quantitative (but not qualitative) variations of oxysterol-binding protein could be related to the inhibitory effect of 25-hydroxycholesterol on DNA synthesis, which becomes critical when this sterol is added in the G2M phase of the cell cycle.

[Anti-inflammatory activity of 5 copper complexes]. / Activité anti-inflammatoire de cinq complexes du cuivre.

The anti-inflammatory activity of five copper complexes is shown in the cotton wad granuloma test in Rats. The activity due to copper seems to be only modulated by the ligands in the complexes studied.

Evaluation of EMIT-TOX Enzyme Immunoassay for the analysis of benzodiazepines in serum: usefulness and limitations in an emergency laboratory.

The EMIT-TOX Enzyme Immunoassay for benzodiazepines was evaluated. Reproducibility, linearity, accuracy, sensitivity, and interferences were tested and found to be in good agreement with the manufacturer's specifications. Furthermore, the reactivity of 15 benzodiazepines were studied. According their differential reactivity, the 15 benzodiazepines can be classified into three groups: good reactivity similar to diazepam (potassium clorazepate, prazepam, estazolam, medazepam, flunitrazepam, nitrazepam); medium reactivity (clobazam, clonazepam, bromazepam, chlordiazepoxide, triazolam); and low reactivity (oxazepam, ethyl loflazepate, lorazepam). A possible structure/reactivity relationship is discussed. It is concluded that this kit is well adapted for the rapid detection of most benzodiazepines, but in no way can the EMIT technique permit quantitative results without clinical information.

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol. Effects of high salt concentrations and molybdate.

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to 'activate' the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KCl) but the resulting 4.3 S complex dissociates easily whereas the 'native' 8.3 S form does not. Moreover, molybdate did not prevent the 8.3----4.3 S transformation induced by KCl and neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors.