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Biomedical subjects

J Sperling

Publications and source records attributed to J Sperling.

At least 37 records · Page 2Linked to original sources
PubMed1995

Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles.

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.

Animals↗
PubMed1994

Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles.

The effect of heat shock on the packaging and splicing of nuclear CAD pre-mRNA, a transcript expressed constitutively from a non heat-inducible promoter, was studied in vivo in Syrian hamster cells. While mild heat shock did not affect significantly the packaging of CAD RNA in 200S InRNP particles, it caused perturbation to splicing. First, the heat shock inhibited splicing of CAD pre-mRNA. Second, it affected 5' splice site selection by activating cleavage at a cryptic 5' splice site; yet ligation of the cryptic exon to the downstream proximal exon was not observed. Base complementarities of the cryptic site with U1, U5, or U6 snRNAs are comparable, or even better, than those with the neighboring normal site. Hence, the exclusion of the cryptic site under normal growth conditions cannot be attributed to weaker base pairing with these snRNAs. On the other hand, these results imply the involvement of a heat labile factor in the selection of the 5' cleavage site. The exclusion of the cryptic site at 37 degrees C and the aborted splicing at this site after heat shock may also be explained by a proposed nuclear checking mechanism that detects in-frame stop codons upstream of the 5' splice site, and aborts splicing at such sites to prevent the production of a defective message.

Animals↗
PubMed1994

Splicing components are excluded from the transcriptionally inactive XY body in male meiotic nuclei.

The study of the effect of programmed cessation of transcription in a large nuclear domain, on the distribution of elements of the pre-mRNA splicing machinery, is the main aim of this paper. To this end, we took advantage of the nuclear partitioning of mouse spermatocytes early in meiosis into autosomal transcribing and XY nontranscribing compartments. This system also allows to extend this study to stages in sperm differentiation that are accompanied by reduction and eventual cessation of transcription. We show by indirect immunofluorescence in spermatogenetic cells that 1) fluorescent signals of the pre-mRNA splicing factors SF53/4 and SC35, of the Sm antigens, and of RNA polymerase II, are largely absent from the nontranscribing, X-inactivated compartment, but are abundantly present in the transcribing autosomal compartment and 2) the presence, gradual reduction, and absence of transcriptive activity in nuclei undergoing the sperm formation sequence are positively correlated with the fluorescence patterns of the antibodies against SF53/4, SC35, and the Sm antigens. These data suggest that cessation of transcription during spermatogenesis is accompanied by exclusion of the splicing machinery from nontranscribing chromatin to its vicinity.

Animals↗
PubMed1994

An autoantibody derived from mice with experimental systemic lupus erythematosus is directed against the essential splicing factor SF53/4--a possible role for large nuclear ribonucleoprotein particles in autoimmune disorders.

We have previously shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in a form of large nuclear ribonucleoprotein (InRNP) particles. These particles, which invariably sediment at the 200S region in sucrose gradient, contain all U small nuclear RNPs required for pre-mRNA splicing and a multitude of heterogeneous nuclear RNP proteins. From a panel of mAbs raised against the InRNP particles, a specific mAb (53/4) identified a nuclear protein of 88 kDa as an essential splicing factor (SF53/4). In a parallel independent study, mAbs were established in mice with experimental systemic lupus erythematosus (SLE), that had been induced by immunization with a murine mAb against a human anti-DNA mAb bearing the common 16/6 idiotype. One of the produced mAbs (2C5/3) recognized an 88 kDa RNP protein. In the present study we have used the following criteria to demonstrate that mAb 2C5/3 and mAb 53/4 recognize the same protein. First, mAb 2C5/3 inhibited splicing by direct addition. Second, the 88 kDa polypeptide that had been immunodepleted from HeLa cells nuclear extract by mAb 2C5/3 was recognized by mAb 53/4 in protein blots. Third, the HeLa nuclear extract depleted by mAb 2C5/3 was devoid of splicing activity and could not assemble into splicing complexes with exogenous pre-mRNA; however, splicing and spliceosome assembly activities were restored to such a defective extract by adding back the 88 kDa protein that had been purified by immunoaffinity binding to immobilized mAb 53/4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Importance of secondary TXA2 release in mediating of endothelin-1 induced bronchoconstriction and vasopressin in the guinea-pig.

The i.v. bolus application of 1 nmol/kg endothelin-1 (ET-1) in anaesthetized and ventilated guinea-pigs caused an increase in mean arterial blood pressure (BP) and a bronchoconstriction. Both have been reduced or abolished by COX inhibition (ASA) or substances specifically reducing the thromboxane A2 (TXA2) generation (HOE 944) or receptor binding (BM 13177). The significant increase in TXB2 concentrations in plasma and bronchoalveolar lavage (BAL) after ET-1 challenge (15-fold and 4-fold, respectively) have been reduced or abolished by ASA as well as HOE 944 and not altered by BM 13177.

Animals↗
PubMed1991

Possible involvement of (2'5')oligoadenylate synthetase activity in pre-mRNA splicing.

The first step in splicing of pre-mRNA involves an intermediate lariat structure, in which a 2'5' phosphodiester bond between the 5' terminal guanosine residue of the intron and a specific adenosine residue near the 3' end of the intron is formed. A mammalian enzyme that generates 2'-5' phosphodiester bonds is (2'-5')oligoadenylate synthetase [(2'-5')OASE]. Although the expression of this enzyme is induced by interferon, low constitutive levels can be detected in untreated cells and tissues. The structural similarity between the lariat branch point and the 2'-5' phosphodiester bond generated by (2'-5')OASE prompted the experiments described here which suggest that this enzyme is involved in pre-mRNA splicing. (i) We show that a (2'-5')OASE activity is associated with 60S spliceosomes in an ATP- and RNA-dependent manner and that it can be indirectly immunoprecipitated by anti-Sm antibodies. (ii) Antibodies against (2'-5')OASE inhibit the lariat formation in the first step of splicing when added directly to a splicing reaction in vitro. (iii) HeLa cell nuclear extracts immunodepleted of (2'-5')OASE activity were also deficient in splicing activity.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

A novel splicing factor is an integral component of 200S large nuclear ribonucleoprotein (InRNP) particles.

In previous studies we have shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in the form of large nuclear ribonucleoprotein (InRNP) particles. By electron microscopy, these particles appeared as compact composite structures, 50 nm in diameter, which invariably sedimented at the 200S region in sucrose gradients. In order to identify putative protein splicing factors associated with the 200S InRNP particles, a panel of monoclonal antibodies directed against these particles were screened for their ability to inhibit splicing of pre-mRNA in vitro. In this study we have focused on a nuclear protein of 88 kd in molecular weight, which is an integral component of the InRNP complex and is recognized by monoclonal antibodies from a specific clone. This protein has been identified here as a novel splicing factor by, (i) antibody inhibition of splicing in vitro and (ii) depletion of splicing activity from HeLa cell nuclear extract after removing the 88 kd polypeptide by immunoadsorption, and complementation of the depleted activity with an affinity-purified 88 kd antigen. This splicing factor has further been shown to be required for the assembly of an active splicing complex.

Animals↗
PubMed1990

Monoclonal anti-La antibody derived from a mouse with experimental SLE is similar to human anti-La antibodies.

The La antigen is a highly conserved protein, originally defined by sera of patients with Sjögren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.

Animals↗
PubMed1990

Induction of experimental systemic lupus erythematosus in mice by immunization with a monoclonal anti-La autoantibody.

Experimental systemic lupus erythematosus (SLE) in mice can be induced by immunization either with a human monoclonal anti-DNA antibody bearing the 16/6 idiotype (16/6 Id) or with a mouse monoclonal anti-idiotypic antibody specific for the 16/6 Id. In the present report we investigated the pathogenic role of a monoclonal anti-La autoantibody in the induction and mediation of experimental SLE in mice. The monoclonal anti-La antibody was derived from a mouse in which experimental SLE was induced by immunization with the monoclonal anti-16/6 Id antibody. Following immunization with the anti-La antibody the mice produced antibodies to double-stranded DNA, single-stranded DNA, Sm, SS-A/Ro, SS-B/La, and ribonucleoprotein. Furthermore, even though the anti-La antibody does not express nor react with the 16/6 Id, the immunized mice produced high titers of anti-16/6 Id antibodies as well as 16/6 Id bearing antibodies. Four months following immunization the mice exhibited significant proteinuria, and kidney sections revealed immune complex deposits on the basement membrane of the glomeruli. These results suggest that anti-La autoantibodies are involved in the induction and mediation of SLE in mice.

Animals↗
PubMed1989

Autoantibodies against a nuclear 56 kDa protein: a marker for inflammatory muscle disease.

Antibodies to a 56 kDa nuclear protein have been found in the sera of 85% of patients with myositis using an immunoblotting technique. This auto-antibody appears to be relatively disease-specific though more frequently detectable in both adult and childhood onset dermatomyositis. In adult patients with myositis the anti-56 kDa antibody level reflects disease activity. This antibody is thus much more frequently found than previously described disease-specific autoantibodies in patients with myositis such as the Jo-1 antibody, and may represent a useful diagnostic aid in patients with muscle weakness.

Animals↗
PubMed1989

Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs.

We have previously shown that nuclear transcripts of the multifunctional enzyme, carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydroorotase RNA can be released from nuclei of Syrian hamster cells as compact ribonucleoprotein (RNP) particles that sediment at the 200S region in a sucrose gradient. The 200S nuclear RNP particles contain U1, U2, and U6 small nuclear RNPs, which are known to be required for splicing of pre-mRNA, as integral components of the particles. In this study we demonstrate that nuclear transcripts of dihydrofolate reductase in Syrian hamster cells and of beta-actin in both Syrian hamster and human cells are also released from the respective nuclei as 200S particles--despite the difference in length of these RNAs. Electron microscopy of the 200S particles revealed discrete compact composite structures with a cross section of approximately 50 nm. Finding that two more nuclear RNAs from two different cell types and two different species are released as 200S RNP particles suggests a general mode for packaging of heterogeneous nuclear RNA in large compact RNP particles the size of which is independent of the RNA length.

Actins↗
PubMed1988

Hypoxanthine-DNA glycosylase from Escherichia coli. Partial purification and properties.

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.

Ammonium Sulfate↗
PubMed1987

Autoantibodies against a specific nuclear RNP protein in sera of patients with autoimmune rheumatic diseases associated with myositis.

Polymyositis is an autoimmune, inflammatory disease affecting human skeletal muscle. In the presence of concomitant vasculitis in the skin, the term dermatomyositis is used. In contrast, systemic lupus erythematosus (SLE) is a multisystem disease in which involvement of the skin, kidneys, joints, brain, and other organs may be found. The clinical manifestations vary according to the organ/system involved. It is clinical and therapeutic importance to define which organ/system is involved during the course of the disease. We approached this problem by studying the specificity of autoantibodies that are generated in patients with SLE and polymyositis/dermatomyositis. Among such antibodies are those directed against nuclear components including a variety of ribonucleoprotein (RNP) complexes. We have utilized mammalian nuclear preparations enriched with RNP particles as the antigenic source for immunoblotting studies to identify specific antigenic polypeptides. In the study reported here, sera from five groups of patients were examined: 10 patients with dermatomyositis/polymyositis; six patients with SLE and myositis; 12 lupus patients with cerebral and/or renal disease; eight patients with SLE but no myositis, renal, or cerebral disease; and 5) 11 patients with muscle weakness or muscle disease not due to myositis. In the first two groups of patients with myositis, antibodies against a nuclear RNP protein of 56 KD was identified in 12 of 16 sera. In contrast, such antibodies were found in the serum of only two of 20 patients with SLE but without muscle involvement (groups 3 and 4), and were not found at all in patients with other muscle diseases. This study has identified a new marker, antibodies against a nuclear RNP protein of 56 KD for detecting muscle involvement among the autoimmune rheumatic diseases.

Animals↗
PubMed1987

Clinical features of 100 human immunodeficiency virus antibody-positive individuals from an alternate test site.

We evaluated 100 human immunodeficiency virus (HIV) antibody-positive persons from the only alternate test site in Los Angeles. Thirty-five subjects complained of systemic symptoms suggestive of HIV infection and 65 were completely asymptomatic. Irrespective of symptoms, the group as a whole demonstrated clinical and laboratory evidence of immunodeficiency. Eighty had generalized lymphadenopathy, 16 onychomycosis, six oral candidiasis, and two biopsy-proved Kaposi's sarcoma. Seventy-seven were anergic to seven intradermal antigens. Despite normal white blood cell counts in most subjects, the T-helper-cell count was less than 300/mm3 in 48% of asymptomatic and 46% of symptomatic subjects. The degree of immune depression was less severe but approximated that of patients with acquired immunodeficiency syndrome after Pneumocystis carinii pneumonia. We believe these findings justify the need for comprehensive medical evaluation and follow-up care for seropositive persons from alternate test sites.

Adolescent↗
PubMed1986

'Key paper' for covalent binding of proteins and its uses.

A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed 'Key paper' is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

Antibodies↗