Neuropsychological effects of interferon beta-1a in relapsing multiple sclerosis. Multiple Sclerosis Collaborative Research Group.

Cognitive dysfunction is common in multiple sclerosis (MS), yet few studies have examined effects of treatment on neuropsychological (NP) performance. To evaluate the effects of interferon beta-1a (IFNbeta-1a, 30 microg administered intramuscularly once weekly [Avonex]) on cognitive function, a Comprehensive NP Battery was administered at baseline and week 104 to relapsing MS patients in the phase III study, 166 of whom completed both assessments. A Brief NP Battery was also administered at 6-month intervals. The primary NP outcome measure was 2-year change on the Comprehensive NP Battery, grouped into domains of information processing and learning/memory (set A), visuospatial abilities and problem solving (set B), and verbal abilities and attention span (set C). NP effects were most pronounced in cognitive domains vulnerable to MS: IFNbeta-1a had a significant beneficial effect on the set A composite, with a favorable trend evident on set B. Secondary outcome analyses revealed significant between-group differences in slopes for Brief NP Battery performance and time to sustained deterioration in a Paced Auditory Serial Addition Test processing rate, favoring the IFNbeta-1a group. These results support and extend previous observations of significant beneficial effects of IFNbeta-1a for relapsing MS.

The Drosophila Fos-related AP-1 protein is a developmentally regulated transcription factor.

Drosophila AP-1 consists of two proteins (dFRA and dJRA) that have functional and structural properties in common with mammalian Fos and Jun proto-oncogene products. Here, we report the isolation and characterization of cDNAs encoding the full-length dFRA and dJRA proteins. The predicted amino acid sequences reveal that both proteins contain a bipartite DNA-binding domain consisting of a leucine repeat and an adjacent basic region, which are characteristic of members of the AP-1 family. By using protein translated in vitro or expressed in Escherichia coli, we demonstrate that dFRA, in contrast to the mammalian cFos proteins, recognizes the AP-1 site on its own and activates transcription in vitro in the absence of dJRA or Jun. Heteromeric complexes formed between dFRA and dJRA bind the AP-1 site better than either protein alone, and the two proteins activate transcription synergistically in vitro. In the developing embryo, dFRA mRNA is first expressed in a limited set of cells in the head and is later restricted to a subset of peripheral neurons, several epidermal cells near the muscle attachment sites, and a portion of the gut. In contrast, dJRA appears to be uniformly expressed at a low level in all cell types. These results indicate that dFRA is a developmentally regulated transcription factor and suggest that its potential interplay with dJRA plays an important role in cell-type-specific transcription during Drosophila embryonic development.

In vitro analysis of the Antennapedia P2 promoter: identification of a new Drosophila transcription factor.

We have investigated the transcriptional regulation of the Antennapedia P2 (Antp P2) promoter using nuclear extracts prepared from Drosophila embryos. Transcriptional analysis of deletion mutants reveals the presence of multiple cis-regulatory elements located both upstream and downstream of the start site. One of the elements appears to mediate negative regulation, since deletion of this element results in higher levels of transcription. Several factors that interact with these control elements have been detected and isolated. One DNA-binding protein, Drosophila Transcription Factor-1 (DTF-1), specifically recognizes the consensus sequence GCAACAT/CG/C that is reiterated four times within an upstream activating element. DTF-1 was purified by sequence-specific DNA affinity chromatography and identified as a doublet of approximately 50 kD. Purified DTF-1 enhances transcription from the Antp P2 promoter 7- to 15-fold in a binding site-dependent manner.

Novel Jun- and Fos-related proteins in Drosophila are functionally homologous to enhancer factor AP-1.

A homolog of mammalian enhancer binding factor AP-1 was detected in Drosophila and was purified from embryo nuclear extracts by sequence-specific DNA affinity chromatography. The purified fraction, dAP-1, displays the sequence specificity as well as transcriptional activation properties of mammalian AP-1 and consists of two major proteins of mol. wts 40 and 70 kd. Antibody cross-reactivity experiments suggest that these proteins are Drosophila homologs of proto-oncogene products, Jun and Fos. The Drosophila Jun- and Fos-related antigens, when separated, are individually capable of sequence-specific DNA binding, and the Jun-related antigen activates transcription in vitro.

In vitro formation of a lariat structure containing a G2'-5'G linkage.

We have examined the effects of base changes at the lariat branch site of a modified adenovirus major late precursor mRNA (pre-mRNA). Replacement of the A residue at the lariat attachment site with a G residue was studied. Incubation of this altered pre-mRNA with nuclear extracts of HeLa cells yielded less spliced mRNA (10-fold) than similar reactions with the wild type pre-mRNA. The intron lariat formed during the reaction with the mutant transcript contained the predominant branch (2'-5' phosphodiester linkage) to an upstream A residue. In contrast, the intron/exon 2 lariat contained the predominant branch to the substituted G residue. These results indicated that detectable spliced RNA was formed when the lariat was attached at the A residue but not when the lariat was attached to the substituted G residue. A second mutation was introduced into the transcript by substituting an additional G residue at the alternative A branch site. When transcript derived from this plasmid was incubated with nuclear extract, cleavage occurred at the 5' splice site, and an intron/exon 2 lariat was produced, but spliced RNA was not detected. T1 RNase digestion and primer extension analyses of this intron/exon 2 lariat revealed that all of the lariat formed on the G residue at the normal attachment site.

RNA splicing products formed with isolated fractions from HeLa cells are associated with fast-sedimenting complexes.

Three fractions (designated Ia, Ib, and II) have been isolated from HeLa cell nuclear extracts that are required for splicing of adenovirus and human beta-globin RNA transcripts in vitro. The incubation of two of the fractions (Ib and II) in the presence of ATP resulted in cleavage of precursor mRNA at the 5' splice site and formation of the intron-exon lariat. Addition of fraction Ia to the combination of Ib and II resulted in the formation of spliced RNA and the intron lariat. When fraction II was incubated with precursor RNA in the presence of ATP and the resulting products were sedimented through sucrose gradients, a 30S complex was detected that contained precursor RNA. The combination of fractions Ib and II resulted in the production of a 55S complex that contained the 5' exon as a prominent RNA species. The combination of fractions I (containing Ia and Ib) and II resulted in the formation of the 55S complex and material sedimenting between 40 S and 20 S, in which the predominant RNA species was spliced RNA.

Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro.

A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro. Both fractions are heat labile and sensitive to N-ethylmaleimide. The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment. This abolition indicates an essential nucleic acid component. Fractions I and II are required for the in vitro splicing of human beta-globin and adenovirus transcripts.

Isolation and characterization of an RNA ligase from HeLa cells.

An RNA ligase has been purified from HeLa cells, which catalyzes the intra- and intermolecular ligation of linear RNA substrates possessing 5'-hydroxyl and 2',3'-cyclic phosphate termini in the presence of ATP or dATP. In this reaction, the 2',3'-cyclic phosphate is incorporated into a 3'-5'-phosphodiester bond, in agreement with the findings of Filipowicz et al. [Filipowicz, W., Konarska, M., Gross, H. J. & Shatkin, A. J. (1983) Nucleic Acids Res. 11, 1405-1418]. The activity of the purified enzyme is dependent on the addition of ATP or dATP, a divalent cation (Mg2+), and 5'-hydroxyl, 2',3'-cyclic phosphate-terminated RNA substrates. No ligation occurs with the substrates OH(Up)10G(3')p or OH(Up)10G(2')p or with 5'-phosphate, 2',3'-cyclic phosphate-terminated oligoribonucleotides.

Ozone Degrades into Hydroxyl Radical under Physiological Conditions : A Spin Trapping Study.

Defining the reactants is a critical step towards elucidating the mechanism of ozone toxicity to biomembranes. To document ozone-induced HO.radicals, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide was used and the resulting spin adduct was monitored with electron spin resonance spectroscopy. Chelexed potassium phosphate buffer (10 millimolar and 0.2 molar) at pH 7.2 and 7.8 was exposed to ozone (1-40 microliters per liter) by directing a stream of ozone over the surface for 60 seconds. Under these conditions, no HO. was detected. Using 0.5 x 10(-4) molar caffeic acid in phosphate buffer, strong DMPO.OH electron spin resonance signals were obtained, indicating HO. production. Air controls yielded no signal. High pH (7.8) enhanced signal strength. Furthermore, with sorbitol (0.4 osmolal final concentration), a net HO. signal loss of 28% was observed, while a carbon-centered sorbitol radical adduct appeared. Although HO. radicals were produced, no breakage of Daucus carota protoplast plasma membranes was observed nor were differences in membrane fluidity observed as determined by 5-doxyl stearic acid.