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At least 793 records · Page 44Linked to original sources
PubMed1995

Acetylcholinesterase of human intestinal tissue affected by Hirschsprung's disease: effect of magnesium chloride on isoforms.

Acetylcholinesterase (AChE) molecular isoforms from anglionic and adjacent unaffected (control) colonic tissue in patients with Hirschsprung's disease (HD) were analysed by density gradient centrifugation in order to determine the major AChE isoforms and the effect of a reported MgCl2 inactivation assay method upon them, with a view to improving the AChE assay used in the diagnosis of Hirschsprung's disease. The total AChE level was greater in the HD-affected colonic tissue than the control tissue (HD: 9.0 vs. control: 7.3 units/g tissue) and this was due to a consistently greater elevation of the globular tetrameric form, G4 (HD: 48.8% vs. control: 35.5% of total AChE). The inactivation of whole tissue homogenates by brief exposure to 4 mol/l MgCl2 followed the same pattern (HD: 48.4% vs. control: 28.7% inactivation). The detergent-extractable G4 is inhibited to a greater extent than the low salt-soluble G4 by exposure to 4 mol/l MgCl2 (83.8% vs. 51.1%). These results imply that the elevated AChE levels in HD are mainly due to increases of the hydrophobic globular tetrameric form of AChE of the same type that is found in differentiating nervous tissue before synapse formation. The monomeric globular isoform G1 is not inhibited but the asymmetric A4, A8 and A12 isoforms are completely inhibited by exposure to 4 mol/l MgCl2. All isoforms of Torpedo (electric ray) electroplax and human erythrocyte AChE, mainly amphiphilic G2, are almost completely inhibited. The inhibition by 4 mol/l MgCl2 of the main G4 isoform in HD-affected intestine accentuates the difference between aganglionic and unaffected intestine in fractionated samples, but does not provide a sufficiently specific G4 isoform assay. The use of 2-4 mol/l MgCl2 in histochemical AChE staining reduces the activity slightly but does not differentiate the tetrameric AChE isoform that is increased in Hirschsprung's disease but does reduce contaminating erythrocyte AChE levels that can obscure the result in blood-stained biopsies. A specific immunochemical stain for hydrophobic AChE tetramers or the associated 20 kDa membrane associated subunit is therefore needed to provide specificity.

Acetylcholinesterase↗
PubMed1992

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.

Animals↗
PubMed1996

The relative electrophoretic mobility of apo(a) isoforms depends on the gel system: proposal of a nomenclature for apo(a) phenotypes.

Genetic apo(a) isoforms were originally defined according to their relative mobility in SDS-PAGE compared to apoB-100 and were designated as F, B or S1-S4 isotypes. This widely accepted nomenclature does not accommodate the broad spectrum of apo(a) isoforms (> 30) detected by high resolution SDS-agarose gel electrophoresis. Moreover we here show that the relative mobilities of apo(a) isoforms depend on the SDS-gel system used. Comparison of the SDS-PAGE system originally used for phenotyping with SDS-agarose gel electrophoresis and two commercial SDS-PAGE systems (PhastGel, Pharmacia, Sweden and NOVEX, USA) demonstrated marked differences in resolving power and resulted in very different Rf values for identical isoforms. Hence phenotyping results from laboratories using different systems are not comparable. We therefore propose a nomenclature of apo(a) isoforms which reports the number of kringle IV repeats in the apo(a) allele (e.g. apo(a) K-IV20 would designate an isoform with 20 K-IV repeats). This is achieved by using standards in which the number of kringle IV repeats has been determined by pulsed field gel electrophoresis of genomic DNA. The proposed nomenclature (i) accounts for the increased resolution of apo(a) phenotyping methods: (ii) is flexible to the introduction of smaller or larger isoforms; (iii) allows to report data from systems with lower resolution as 'binned' isoform categories; (iv) allows the comparison of phenotyping results between different investigators; and (v) can be applied on DNA as well as on protein based apo(a) phenotyping.

Apolipoproteins A↗
PubMed1990

Ability of high-affinity heparin fractions with decreasing affinity for antithrombin III to activate ATIII isoforms.

Previous studies investigated the effect of heparin fractions on the rates of thrombin inhibition by naturally occurring antithrombin III (ATIII) isoforms differing in affinity for heparin. Heparin with low-affinity for ATIII increased the rate of thrombin inhibition by the higher affinity isoform about 10-fold more effectively than by the other isoform. This paper reports on the effect of a series of high-affinity heparin fractions with decreasing affinity for ATIII. As affinity decreased, the ability of the heparin fractions to increase the rate of the ATIII-thrombin reactions decreased, and these fractions slightly more effectively increased the rate of thrombin inhibition by the higher-affinity ATIII isoform. The effect of the heparin fractions on the ATIII-factor Xa reactions was also investigated. The activity of the fractions in this reaction also showed a dependence on ATIII-affinity. Studies on the competition of isoforms for immobilized heparin showed that the isoform with higher affinity for ATIII effectively competes with its congener for binding to heparin. The results indicate that heterogeneity in high-affinity heparin results in heterogeneity in affinity for ATIII that is significantly correlated with the ability of the heparin to potentiate ATIII-protease reactions. In spite of about equal activation of the ATIII isoforms by high-affinity heparin, the importance of the higher-affinity isoform is indicated by its ability to compete effectively for these heparin species.

Antithrombin III↗
PubMed1993

The structure and expression of the FGF receptor-1 mRNA isoforms in rat tissues.

In this paper, we describe the structure of rat FGF receptor-1 mRNA isoforms and their expression in a variety of rat tissues. The rat FGFR-1 has the characteristics of FGFR-1 as well as mouse, human and chicken homologs. FGFR-1 mRNA was detectable in all the tissues examined by Northern analysis or polymerase chain reaction, indicating that FGFR-1 mRNA is widely expressed in rat tissues. The rat FGFR-1 mRNA has isoforms in both the extracellular and intracellular regions. The extracellular isoforms which have two or three immunoglobulin-like domains, are expressed almost equally in the tissues except for brain. However, the large form is a major form in the brain. Furthermore, in the brain, half of FGFR-1 mRNAs have the six nucleotides, which encode a potential serine-threonine kinase phosphorylation site in the intracellular juxta-membrane region, deleted. In contrast to the brain, the deletion isoform is a minor form in the other tissues. The tissue-specific expression of the isoforms indicates that they have different physiological functions. Although other isoforms of FGFR-1 mRNA in tumor cell lines have been reported, the isoforms were undetectable in all rat tissues examined, indicating the isoforms are products of abnormal alternative splicing in tumor cell lines.

Amino Acid Sequence↗
PubMed1990

Features of the beta-amylase isoform system in dry and germinating seeds of alfalfa (Medicago sativa L.).

Five isoforms of beta-amylase were purified to homogeneity from alfalfa seeds (Medicago sativa L.) by chromatofocusing and cation-exchange chromatography. These isoforms were identified as beta-amylase based on their catalytic mode to the substrates. These isoforms of beta-amylase were also found in germinating seeds of alfalfa. All the isoforms existed in free form, because they could be extracted without reducing agent. The five isoforms had different isoelectric points (5.05, 4.97, 4.85, 4.82 and 4.77), but their Mr was the same (61 kDa) on SDS-polyacrylamide gels. The amino acid compositions were similar, but not identical, to each other. An antiserum raised against one of the five isoforms cross-reacted with all of other isoforms, but did not recognize the component 2 of soybean beta-amylase. The amounts of five isoforms increased during seed germination, which was responsible for significant increase of the beta-amylase activity in germinating seeds.

Amino Acids↗
PubMed1996

Regulation of UDPG-pyrophosphorylase isoforms in Saccharomyces cerevisiae and their roles in trehalose metabolism.

UDPG-pyrophosphorylase (EC 2.7.7.9) from Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophosphorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex: in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cells collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II, and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a MAL4C allele rendered isoform II constitutive. Interestingly, a gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.

Hot Temperature↗
PubMed1993

Analysis of the subunits, isoforms and substrate specificity of mouse liver alpha-L-fucosidase.

1. SDS-PAGE indicates the presence of two major protein bands (57 and 62 kDa) for mouse fucosidase and Western blotting indicates that both bands are immunoreactive with polyclonal antibodies (PAbs) and/or monoclonal antibodies (MAbs) raised against human liver fucosidase. The lectins SNA and GNA recognized both mouse protein bands, indicating that both subunits are glycosylated and contain sialic acid residues. 2. Polyacrylamide gel-isoelectric focusing (PAG-IEF) indicated that mouse liver fucosidase contains at least seven isoforms, with three isoforms above pI 6.0, which were not detected in human liver fucosidase. Blotting indicates that the PAbs recognized seven mouse fucosidase isoforms (pIs 3.6-6.8) whereas the four MAbs did not appear to recognize any of the mouse isoforms. 3. The subunit composition of the separated isoforms of mouse alpha-L-fucosidase was investigated by SDS-PAGE. One-to-two closely-spaced protein bands are found in each isoform with a trend of increasing relative amounts of the high-M(r) band in the more acidic isoforms relative to the more neutral isoforms. 4. Human and mouse liver alpha-L-fucosidases hydrolyze L-Fuc from oligosaccharides and glycolipids at comparable rates, with the exception of ganglioside Fuc-GMI which was hydrolyzed by human, but not by mouse, alpha-L-fucosidase.

Animals↗
PubMed1993

Analysis of mammalian metallothionein isoforms by high-resolution SDS-gel electrophoresis.

Metallothioneins (MTs) are cysteine-rich heavy metal-binding proteins, whose possible functions are thought to be the protection against toxic metals as well as the regulation of essential metals. It is known that there are several MT isoforms, but the biological roles of the individual isoforms have not been elucidated. To facilitate the functional analysis of these isoforms, we improved an analytical method of MTs developed previously, which is based on a denaturing gel electrophoresis of chemically modified MTs. The established technique makes it possible not only to separate MT isoforms with a high resolution, but to estimate the levels of the individual isoforms by analyzing directly crude cell extracts. By this method, six MT isoforms were identified in the extracts of Cd-exposed human cells. It was also revealed that there is an apparent heterogeneity of the rat liver MT; five isoforms were identified in the liver extracts of Cd-injected rats. The present method will be useful in the functional analysis of the MT isoforms, as well as in a variety of aspects of the MT studies.

Animals↗
PubMed1994

Na,K-ATPase alpha and beta subunit isoform distribution in the rat cochlear and vestibular tissues.

The distribution of five Na,K-ATPase subunit isoforms (alpha 1, alpha 2, alpha 3, beta 1 and beta 2) in rat cochlear and vestibular tissues was determined by immunocytochemical techniques using subunit isoform specific polyclonal antibodies. The expression of Na,K-ATPase alpha and beta subunit isoforms varied among different cell regions of the inner ear. The alpha 1 subunit isoform was more extensively distributed in all inner ear tissues than the alpha 2 or alpha 3 subunit isoforms. The beta 1 subunit isoform was distributed primarily in spiral ligament and inner hair cells of the cochlea, and in crista ampullaris and macula of the saccule. The beta 2 subunit isoform was most abundant in the stria vascularis, dark cells of the ampullae and utricle. The alpha 1 beta 1 subunit combination of Na,K-ATPase was most commonly found in the spiral ligament, while the alpha 1 beta 2 combination was most abundant in the stria vascularis. Similarly, alpha 1 beta 2 was confined more to the dark cells of the ampullae and utricle. The alpha 3 beta 1 subunit combination of Na,K-ATPase was identified in the inner hair cells of the cochlea and the sensory regions of the vestibular end organs. These observations may reflect functional diversity of Na,K-ATPase in the individual inner ear regions and may provide insight into the differences between fluid and ion transport in the inner ear and that of other transporting tissues. Overall, the distribution pattern further indicates that the different isoform combinations have specific roles.

Animals↗
PubMed1996

Differential expression of tumor necrosis factor-alpha isoforms from lipopolysaccharide- and cytokine-stimulated mouse macrophages.

Tumor necrosis factor-alpha (TNF alpha) is a biologically active cytokine with a wide range of functions, which is primarily expressed by macrophages. It is produced as a biologically active propeptide that becomes processed to the mature form of secreted protein. Previous studies used a mouse macrophage cell line and showed that after stimulation with lipopolysaccharide, TNF alpha propeptide is expressed as multiple isoforms with approximate molecular masses of 26, 29 and 32 kDa. However, little is known of the production of TNF alpha isoforms from normal macrophages or of the effects of cytokines on TNF alpha production by macrophages in the absence of co-stimulation by lipopolysaccharide. We have compared the TNF alpha isoforms produced by cytokine-and lipopolysaccharide-stimulated bone marrow-derived macrophages from mice that normally respond to lipopolysaccharide (C3H/HeN) and mice that are hyporesponsive (C3H/HeJ). We found that the pattern of immunoprecipitated TNF alpha propeptide isoforms expressed depended on the stimulus: lipopolysaccharide, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor. Lipopolysaccharide induced three isoforms of 25, 29 and 35 kDa, supporting previous studies. However, macrophage and granulocyte-macrophage colony-stimulating factors also stimulated cells to express the 24 and 27 kDa isoforms, but not the 35 kDa isoform. In addition, cells stimulated with granulocyte-macrophage colony-stimulating factor expressed a novel 20 kDa propeptide. The results show that granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and lipopolysaccharide differently regulate TNF alpha protein expression and suggest that different isoforms may have different functions.

Animals↗
PubMed2006

Anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma-isotope dilution-time-of-flight mass spectrometry for speciation analysis of metal complexes with metallothionein isoforms in gibel carp (Carassius auratus gibelio) exposed to environmental metal pollution.

The capability of post-column isotope dilution (ID) combined with anion-exchange HPLC-ICP-time-of-flight (TOF)-MS was for the first time investigated for environmental quality assessment through metal speciation analysis of metallothionein (MT) isoforms in cytosols of gibel carp (Carassius auratus gibelio), used as biomarkers for environmental metal exposure. A full spectral scanning of the biological sample (with 50 microl injection volume) using ICP-TOF-MS in transient mode allowed fast multi-isotope screening of cytosolic metal-containing fractions and to investigate the presence of matrix-induced interferences. The MT cytosolic fraction of liver and kidney of the carp, sampled at three different sampling sites in Belgium, was partially purified using size-exclusion (SE) HPLC. Quantification of the elements Cd (toxic) and Zn and Cu (essential) associated with MT isoforms in this fraction was addressed using an hybrid approach based on post-column addition of the enriched isotopes 65Cu, 67Zn, 106Cd and monitoring on-line the isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd by ICP-MS with a time of flight instrument, which was coupled to anion-exchange HPLC. With this separation method, baseline separation of up to five MT isoforms, which is required for quantitative metal speciation by HPLC-ICP-IDMS, was achieved within a run of 15 min. The MT fraction of the cytosols was also analysed for the total metal content using IDMS with size-exclusion HPLC-ICP-MS and species-unspecific calibration. Results showed significant differences between speciation results and total MT concentrations of control fish and fish from the most contaminated sampling sites, revealing the potential of gibel carp MT for sequestering excess intracellular free-ions (essential and toxic elements) and for its protection against metal toxicity. Preferences for metal sequestration of metal complexes with MT isoforms were also found to be tissue-specific: excess of Cd was found preferably bound to a major MT isoform (tR = 8.0 min) in kidney, whereas excess intracellular Zn appeared to be mostly sequestered by four MT isoforms (tR=7.3, 8.0, 12.2 and 14.4 min) in liver, the MT form with tR = 8.0 min being the main Zn scavenger form. Such kind of quantitative speciation information on the preferences of MT isoforms in different fish organs for sequestering heavy metals, reported here for the first time, is important to elucidate the role of isoform-specific induction of vertebrate fish MT in metal detoxification and the use of MT as biomarker.

Animals↗
PubMed2006

α2 isoform-specific activation of 5'adenosine monophosphate-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside at a physiological level activates glucose transport and increases glucose transporter 4 in mouse skeletal muscle.

5'Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the alpha1 and alpha2 isoforms of AMPK, the alpha2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of alpha2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the alpha2 isoform, we used 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the alpha2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the alpha2 isoform for 90 minutes, but not the alpha1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the alpha2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in alpha2 activity paralleled the phosphorylation of Thr(172), the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase beta, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the alpha2 isoform of AMPK.

AMP-Activated Protein Kinases↗
PubMed2000

Electrophoretic study of tartrate-resistant acid phosphatase isoforms in endstage renal disease and rheumatoid arthritis.

The objective of this study was to identify the isoform, type-5a or type-5b, responsible for increased tartrate-resistant acid phosphatase (TRAP) activity in endstage renal disease (ESRD) and TRAP protein in rheumatoid arthritis (RA). We studied 24 sera each from healthy, ESRD and RA subjects. Type-5 TRAP activity and protein were quantitated by immunoassays. Isoform expression was determined by computerized imaging of non-denaturing polyacrylamide gels (PAGE) stained for TRAP activity. Other biochemical markers included: intact parathyroid hormone (iPTH), total and bone-specific alkaline phosphatase (TAP, BAP), N-telopeptides of type-I collagen (NTx), and free pyridinoline (Pyd). Isoform 5a was normal in both ESRD and RA. Isoform 5b was elevated in ESRD only. Serum TRAP activity correlated with both isoforms 5a and 5b in RA, but only with 5b in ESRD. TRAP protein assays did not correlate with PAGE assays for 5a or 5b. TRAP activity, but not protein, correlated with BAP and NTx in RA sera. Both TRAP activity and protein correlated with iPTH, TAP and Pyd in ESRD sera. Increased TRAP activity in ESRD was due to increased osteoclastic isoform 5b and related to bone turnover. Increased TRAP protein in RA was suspected, but not proven, to be isoform 5a and not related to bone turnover. Heterogeneity of serum TRAP and preferential expression of isoforms has clinical significance in different diseases including ESRD and RA.

Acid Phosphatase↗
PubMed1999

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning.

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.

Animals↗
PubMed1998

Expression of the sarco/endoplasmic reticulum Ca(2+)-transport ATPase protein isoforms during regeneration from notexin-induced necrosis of rat soleus muscle.

Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and "housekeeping" (SERCA2b) isoforms of the sarcoplasmic reticulum Ca(2+)-transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the house-keeping SERCA2b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.

Animals↗
PubMed1997

Tropomyosin isoforms in nonmuscle cells.

Vertebrate nonmuscle cells, such as human and rat fibroblasts, express multiple isoforms of tropomyosin, which are generated from four different genes and a combination of alternative promoter activities and alternative splicing. The amino acid variability among these isoforms is primarily restricted to three alternatively spliced exon regions; an amino-terminal region, an internal exon, and a carboxyl-terminal exon. Recent evidence reveals that these variable exon regions encode amino acid sequences that may dictate isoform-specific functions. The differential expression of tropomyosin isoforms found in cell transformation and cell differentiation, as well as the differential localization of tropomyosin isoforms in some types of culture cells and developing neurons suggest a differential isoform function in vivo. Tropomyosin in striated muscle works together with the troponin complex to regulate muscle contraction in a Ca(2+)-dependent fashion. Both in vitro and in vivo evidence suggest that multiple isoforms of tropomyosin in nonmuscle cells may be required for regulating actin filament stability, intracellular granule movement, cell shape determination, and cytokinesis. Tropomyosin-binding proteins such as caldesmon, tropomodulin, and other unidentified proteins may be required for some of these functions. Strong evidence for the distinct functions carried out by different tropomyosin isoforms has been generated from genetic analysis of yeast and Drosophila tropomyosin mutants.

Animals↗
PubMed1998

Expression of CD45 isoforms lacking exons 7, 8 and 10.

The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous. All Th lines produced mRNA encoding this isoform, but in only three of the Tc lines was the 89 isoform detectable by RT/PCR. RNase protection assays with RNA isolated from normal CD8+ splenic cells demonstrated the 89 splice product was present in low abundance. The relative abundance of the different isoforms in the thymic lymphoma, BW5147, was determined through RNase protection analysis. The 789 isoform predominates, representing approximately 75% of the CD45 mRNA whereas the 89 form constitutes about 24%. In addition, an isoform lacking exons 4-8 (isoform 9) also was detected and comprises approximately 1% of the total CD45 mRNA in this cell line. Finally, these studies demonstrate that exon 10 is also used as an alternatively spliced exon.

Alternative Splicing↗