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J Sperling

Publications and source records attributed to J Sperling.

At least 19 recordsLinked to original sources
PubMed2000

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.

Animals↗
PubMed2000

Cardiac performance after deep hypothermic circulatory arrest in chronically cyanotic neonatal lambs.

OBJECTIVES: It is controversial whether immature cyanotic hearts are more susceptible to ischemic injury than normoxemic hearts. Acutely induced alveolar hypoxic stress before cardiopulmonary bypass has been used as a model of cyanosis and is reported to worsen recovery of immature hearts after subsequent ischemic insult by means of a free radical injury mechanism. Because of concerns about the relevance of acute alveolar repair to the chronic cyanosis encountered clinically, we assessed the effects of chronic cyanosis without alveolar hypoxia, acute alveolar hypoxia, and normoxemia on recovery of cardiac function after deep hypothermic circulatory arrest. METHODS: A chronic cyanosis model was created in 8 lambs by an anastomosis between the pulmonary artery and the left atrium (cyanosis group). Eight lambs underwent sham operation (control). One week later, the animals underwent cardiopulmonary bypass with 90 minutes of deep hypothermic circulatory arrest at 18 degrees C. Another 8 lambs underwent 45 minutes of hypoxic ventilation before bypass, with arterial oxygen tension being maintained at 30 mm Hg (acute hypoxia group). Cardiac index, preload recruitable stroke work, and tau were measured. Malondialdehyde and nitrate-nitrite, nitric oxide metabolites, were also measured in the coronary sinus. Myocardial antioxidant reserve capacity at 2 hours of reperfusion was assessed by measuring lipid peroxidation in left ventricular tissue samples incubated with t-butylhydroperoxide at 37 degrees C. RESULTS: Oxygen tension was 35 +/- 3 mm Hg in the acute hypoxia group versus 93 +/- 7 mm Hg in the control group. In the acute hypoxia group the recovery of cardiac index, preload recruitable stroke work, and tau were significantly worse than that found in both the control and cyanosis groups. Preload recruitable stroke work at 2 hours of reperfusion was slightly but significantly lower in the cyanosis group than in the control group. The postischemic level of nitric oxide metabolites was significantly lower in the acute hypoxia group than in the cyanosis and control groups. However, malondialdehyde levels in the coronary sinus and myocardial antioxidant reserve capacity were not significantly different among the groups. CONCLUSION: Recovery of left ventricular function after deep hypothermic circulatory arrest in neonatal lambs with chronic cyanosis was slightly worse than that found in acyanotic animals. Acute hypoxia before bypass was associated with significantly worse recovery of left ventricular function, and the mechanism of injury may be related to an impairment of nitric oxide production. Free radical injury does not appear to explain any differences among cyanotic, acyanotic, and acutely hypoxic animals in recovery of left ventricular function after ischemia.

Analysis of Variance↗
PubMed2000

Heparinless partial cardiopulmonary bypass for the repair of aortic trauma.

OBJECTIVE: We hypothesized that partial cardiopulmonary bypass with a heparin-bonded system would be a technically simple, effective adjunct for reducing paraplegia during repair of traumatic aortic rupture. It avoids the risk of heparin, but, unlike left atrial-arterial bypass, it can heat, cool, oxygenate, and rapidly infuse volume if needed. METHODS: A retrospective review was conducted of patients admitted for aortic trauma from July 1994 to December 1999. Bypass consisted of femoral venous (right atrial) cannulation, a centrifugal pump, and an oxygenator-heater/cooler. Arterial return was to the femoral artery or distal aorta. The entire system was heparin-bonded and no systemic heparin was given. RESULTS: Heparin-bonded partial bypass was established in 50 patients (mean age 43 +/- 17 years). Crossclamp time was 32 +/- 11 minutes (range 14-70 minutes), mean flow 3.0 +/- 0.8 L/min, and bypass time 64 +/- 43 minutes. During repair, 58% of patients received volume through the system (mean 1.1 +/- 1.9 L). Core temperature rose slightly (35.9 degrees C +/- 0.7 degrees C to 36.3 degrees C +/- 0.8 degrees C). Three of the 15 patients who underwent percutaneous femoral arterial and venous cannulation concomitant with their angiograms had vessel injury, with one limb loss, and this procedure was discontinued. Thirty-five patients underwent percutaneous femoral vein and direct distal aortic cannulation without event. The mortality rate for patients supported by bypass was 10%, and all deaths were due to other injuries. There were no new cases of paraplegia and no worsening of intracranial or pulmonary injuries. CONCLUSIONS: Heparin-bonded bypass is technically simple to use and avoids the risk of anticoagulation. Paraplegia was avoided. The ability to correct hypothermia, oxygenate, and rapidly infuse volume may simplify management and improve outcomes.

Adult↗
PubMed1999

Gold-tagged RNA-A probe for macromolecular assemblies.

Ribonucleic acids (RNAs) play a key role in many fundamental life processes. These polymers are often found complexed with proteins in extremely large particles whose molecular mass may reach several millions of daltons (e.g., ribosomes, spliceosomes, and viruses). Structural studies of such RNA-protein complexes should help elucidate their mode of action. For the structural analyses of many macromolecular assemblies, electron microscopy (EM) has served an instrumental role. However, localization by EM of RNA within biological complexes is not yet a straightforward undertaking. Here we describe a methodology for the covalent tagging of RNA molecules with gold clusters, thereby enabling their direct visualization by microscopical methods. Our strategy involves transcription in vitro of RNAs that carry free thiol groups, using ribonucleoside triphosphate analogs containing a substituent with a terminal thiol group on their heterocyclic ring. This synthesis is followed by coupling of gold clusters to the thiolated transcript through a maleimido group. Visualization of such gold-tagged RNAs by transmission electron microscopy showed spots of gold clusters, with a diameter of 1-2 nm, arranged at nearly regular distances on an imaginary curve that presumably corresponds to the RNA chain. This assignment was corroborated by atomic force microscopy that exhibited images of RNA chains in which knob-like structures, whose height corresponds to the diameter of the gold clusters, were clearly seen. This study demonstrates the potential use of nucleic acids that are covalently labeled with gold clusters for the structural characterization of protein-RNA complexes.

Globins↗
PubMed1999

Effects of cyanosis and hypothermic circulatory arrest on lung function in neonatal lambs.

BACKGROUND: Lung function is often impaired after cardiac surgery and cardiopulmonary bypass (CPB), particularly in chronically cyanotic patients. This study aimed to evaluate lung function in a surgically created chronic cyanotic neonatal lamb model after CPB and deep hypothermic circulatory arrest (DHCA) and to assess the role of nitric oxide (NO) in the pathogenesis of increased pulmonary vascular resistance. METHODS: A chronic cyanosis model was surgically created in 7 lambs (4.7+/-0.8 days old) by anastomosing the pulmonary artery (PA) to the left atrium (LA). Another 7 lambs underwent a sham operation (control). One week later, the animals underwent shunt takedown and CPB with 90 minutes of DHCA at 18 degrees C. Cardiac index (CI), pulmonary vascular resistance index (PVRI), lung dynamic compliance (Cdyn), alveolar-arterial oxygen difference (AaDO2), left atrial plasma nitrate/nitrite (NO metabolites) levels, and pulmonary cGMP production (concentration difference between LA and PA) were measured before CPB and at 1 and 2 hours after reperfusion. RESULTS: The cyanosis model consistently produced significantly lower arterial oxygen tension (34.8+/-2.3 vs 93.1+/-8.8 torr in control, p < 0.001) and Qp/Qs (0.6+/-0.1 vs 1.0+/-0.0 in control, p < 0.001) than controls. Postoperative PVRI was significantly lower in the cyanosis group than in controls, although CPB with DHCA significantly elevated PVR in both cyanotic and control animals. There were no significant differences in AaDO2 and Cdyn after CPB between groups. The level of NO metabolites did not change before or after CPB in either cyanotic or acyanotic animals. NO metabolite levels tended to be higher in the cyanotic animals (p = 0.08). There was no significant difference in pulmonary cGMP production between both groups. CONCLUSIONS: These findings suggest that CPB with DHCA, per se, does not affect NO production in cyanotic or acyanotic neonatal lambs but causes increased PVR in both groups. Chronic cyanosis does not result in reduced pulmonary function after CPB with DHCA, and is associated with lower PVR. The mechanism may involve an increased NO production in cyanotic animals.

Animals↗
PubMed1998

A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy.

Pre-mRNA splicing is an important regulatory step in the expression of most eukaryotic genes. In vitro studies have shown splicing to occur within 50-60 S multi-component ribonucleoprotein (RNP) complexes termed spliceosomes. Studies of mammalian cell nuclei have revealed larger complexes that sediment at 200 S in sucrose gradients, termed large nuclear RNP (lnRNP) particles. These particles contain all factors required for pre-mRNA splicing, including the spliceosomal U snRNPs and protein splicing factors. Electron microscopy has shown them to consist of four apparently similar substructures. In this study, mass measurements by scanning transmission electron microscopy of freeze-dried mammalian lnRNP preparations, both confirm the similarity between the lnRNP particles and reveal the mass uniformity of their subunits. Thus, the tetrameric lnRNP particle has a mass of 21.1(+/-1.6) MDa, while each repeating subunit has a mass of 4.8(+/-0.5) MDa, which is close to the estimated mass of the fully assembled 60 S spliceosome. The 1.9 MDa discrepancy between the lnRNP particle's mass and the cumulative masses of its four subunits may be attributed to an additional domain frequently observed in the micrographs. Notably, strands and loops of RNA were often seen emanating from lnRNP particles positively stained with uranyl formate. Our results support the idea that the nuclear splicing machine is a supraspliceosome complex. For clarity, we define spliceosomes devoid of pre-mRNA as spliceosome cores, and propose that the supraspliceosome is constructed from one pre-mRNA, four spliceosome cores, each composed mainly of U snRNPs, and additional proteins. In this way a frame is provided to juxtapose exons about to be spliced.

Mass Spectrometry↗
PubMed1998

Does child care involvement increase or decrease distress among dual-earner couples?

This study addressed how parents' relative involvement in child care is related to marital-role quality and psychological distress. These relationships were examined in a random sample of 133 mothers and fathers in dual-earner couples. Regression analyses employing hierarchical linear modeling techniques indicated that the more fathers participated in child care relative to their wives, the lower the father's distress. For mothers, the effect of child care involvement was complex: Although there were psychological benefits to spending proportionally more time involved in child care (lowered distress), these benefits were offset by a decrease in marital-role quality, which in turn increased distress. These findings indicate that the relation between child care involvement and the psychological health of both women and men in dual-earner couples is intertwined and complex.

Adult↗
PubMed1997

Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography.

Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are packaged in multi-component large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200 S region in sucrose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factors, including U2AF and the SR proteins. Electron microscopy of lnRNP particles revealed a large compact structure of 50 nm in diameter. In this study we employed automated computed tomography from electron micrographs for the three-dimensional (3D) image reconstruction of individual lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed according to the back projection principle. For rendering, real time display and comparison of the reconstructed particles, interactive computer graphics was employed. The reconstructed 3D images show a compact structure composed of four major subunits connected to each other. Comparison of the reconstructed lnRNP particles revealed morphological similarity of the individual particles, as well as similarity among the sub-structures. Based on these observations we propose a model for the packaging of nuclear pre-mRNAs in lnRNP particles where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multi-intronic pre-mRNAs, and with the fact that the splicing of multi-intronic pre-mRNAs does not occur in a sequential manner.

Animals↗
PubMed1997

PACT: cloning and characterization of a cellular p53 binding protein that interacts with Rb.

Cellular functions of tumor suppressor proteins can be mediated by protein-protein interactions. Using p53 as a probe to screen an expression library, a cDNA encoding a 250 kDa protein was isolated. Recombinant forms of this protein, designated PACT, bind to wild type p53 while two different mutations abolish this interaction. PACT protein can also interfere with p53 specific DNA binding. PACT contains a serine/arginine (SR) rich region and a C' terminal lysine rich domain. The 250 kDa PACT protein can be precipitated from cell lysates by a method specific for SR proteins. snRNPs can be co-immunoprecipitated from cells with anti-PACT antibodies. These antibodies stain cell nuclei in a speckled pattern reminiscent of the distribution of known splicing factors. Recently, RBQ1, a truncated human homologue of PACT was identified by virtue of Rb binding. We show that RBQ1 is truncated as a result of a possible mutational event. PACT can interact with both cellular Rb and p53.

Alternative Splicing↗
PubMed1997

Automated electron tomography of large nuclear RNP (InRNP) particles--the naturally assembled complexes of precursor messenger RNA and splicing factors.

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to the cytoplasm to function in protein synthesis. Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA, are packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200S region in sucrose gradients, contain all U snRNPs required for pre-mRNA splicing and several protein splicing factors, including U2AF and the SR proteins and can thus be viewed as naturally assembled complexes of pre-mRNA and splicing factors. We have previously reconstructed the three-dimensional image of negatively stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, composed of four major subunits. Here we further analyzed the reconstructed models and the apparent connectivity between the subunits using a new rendering technique. The uniformity of the lnRNP particles was substantiated by measurement of the volume engulfed by their surface. This study further supports the model proposed for the packaging of nuclear pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner.

Animals↗
PubMed1996

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

Animals↗
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↗