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PubMed1994

Localization of NuMA protein isoforms in the nuclear matrix of mammalian cells.

Using a monoclonal antibody 2D3 generated against a kinetochore-enriched human chromosome preparation, we identified a high molecular mass protein with nuclear staining in interphase and polar staining of the pericentriolar region in the mitotic spindle. Initially termed centrophilin, this protein associates with the minus-ends of spindle microtubules (MT) and appears to be important in spindle organization [Tousson et al., 1991: J. Cell Biol. 112:427-440]. Comparison of a partial cDNA sequence obtained for centrophilin with the full length cDNA sequence of nuclear mitotic apparatus protein (NuMA) [Compton et al., 1992: J. Cell Biol. 116:1395-1408; Yang et al., 1992: J. Cell Biol. 116:1303-1317] has indicated that NuMA and centrophilin are the same protein. Using a polyclonal NuMA antibody, we have provided further evidence that NuMA exists as isoforms as shown by peptide mapping and immunoblots. Sequential fractionation experiments along with immunofluorescence, immunoblotting, and EM immunogold labeling have demonstrated that NuMA isoforms are novel components of nuclear core filaments. Thus, NuMA, a long coiled-coil protein, appears to have dual functions in interphase and mitosis during the cell cycle. In interphase, NuMA likely plays a structural role in the nucleoskeleton that may be important in nuclear organization and functions, whereas in mitosis, NuMA appears to be associated with spindle MT organization and chromosome positioning.

Antigens, Nuclear↗
PubMed2004

Proteomic analysis of small heat shock protein isoforms in barley shoots.

The analysis of stress-responsiveness in plants is an important route to the discovery of genes conferring stress tolerance and their use in breeding programs. High temperature is one of the environmental stress factors that can affect the growth and quality characteristics of barley (Hordeum vulgare). In this study a proteomic analysis (2D-PAGE, MS) was used to detect the effects of heat shock on the protein pattern of an abiotic stress-tolerant (Mandolina) and an abiotic stress-susceptible (Jubilant) barley cultivar. Evaluation of two-dimensional gels revealed several proteins to be differentially expressed as a result of heat stress in both cultivars. The protein spots of interest were, after an in-gel tryptic digestion, further investigated by mass spectrometry. For the analysis of the peptide mixture, we both used a matrix-assisted laser desorption/ionization (MALDI) tandem time of flight mass spectrometer (TOF/TOF) and an automated nano-HPLC system coupled to an electrospray ionization-quadrupole linear ion trap (Q-TRAP) instrument. The hyphenation of the latter techniques proved to be a powerful technique as shown by the identification of six isoforms of a 16.9 kDa sHSP in one single spot. We observed that S-adenosylmethionine synthetase (SAM-S) was differentially expressed between the two cultivars. Recent results refer to the role of SAM-S as being involved in abiotic stress tolerance. Furthermore, comparison of the heat shock treated samples also revealed several small heat shock proteins (sHSP), of which distinct isoforms could be characterised.

Electrophoresis, Gel, Two-Dimensional↗
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↗
PubMed1988

Changes in myosin and C-protein isoforms proceed independently of the conversion to singly innervated neuromuscular junctions in developing pectoral muscle.

Changes in contractile protein expression during myogenesis are usually categorized as developmentally programmed or neuronally dependent. Studies on aneurogenic chick embryos indicated that the neuronally dependent phase begins at about Embryonic Day 15, immediately prior to the fetal transition in myosin and C-protein expression. The prime candidate for the neuronal event that induces the fetal transition is the conversion to the adult form of singly innervated neuromuscular junctions (NMJs), which occurs contemporaneously with the fetal transition. Using curare to inhibit the conversion to focal innervation, we find that the fetal transition proceeds unimpaired, demonstrating that there is no causal link between the fetal transition and the conversion to focal innervation. Furthermore, because the doses of curare used inhibit motor activity by more than 80%, the fetal transition can occur in the absence of normal levels of motor activity. These observations show that the fetal transition in ovo is not induced by either a specific change in innervation or use. Rather, the dependence on innervation seems to be a consequence of the need for muscle activity to prevent atrophy, and the fetal transition appears to have characteristics more like the preprogrammed contractile protein transitions that precede it.

Acetylcholinesterase↗
PubMed1993

Regulation of beta-amyloid precursor protein isoform mRNAs by transforming growth factor-beta 1 and interleukin-1 beta in astrocytes.

In cultured astrocytes, all three major transcripts of beta-amyloid precursor protein (APP) were expressed with the ratio for APP695, APP751 and APP770 isoform mRNAs being 1:4:2. In comparison with controls, treatment of astrocytes with transforming growth factor-beta 1 (TGF-beta 1) produced about 6 fold increase in total APP mRNA, while elevation in the interleukin-1 beta (IL-1 beta) treated group was small and may relate to the mitogenic effect of IL-1 beta on astrocytes. Treatment of astrocytes with cytokines also produced marked changes in the upregulation in expression of different APP isoforms. The net increase in mRNAs of KPI-containing isoforms APP751 and APP770 was relatively more than for the APP695 isoform. This phenomenon was mainly related to the differences in the expression of KPI-containing APP isoforms and APP695 isoform in the controls. The present findings provide further evidence for the involvement of astrocytes in a cascade of events leading to the development of senile plaques in Alzheimer's disease and Down's syndrome.

Alzheimer Disease↗
PubMed1998

Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms.

The steady-state levels of many mRNAs are determined in part by their turnover rates. Turnover rates, in turn, are usually controlled by proteins that bind cis-acting sequence elements in mRNAs. One class of cis-acting instability determinants is composed of A + U-rich elements present in the 3'-UTRs of many labile mRNAs. Many A + U-rich elements are bound by the AUF1 family of RNA-binding proteins, which may target these mRNAs for rapid decay. cDNA cloning and immunoblot analyses suggest that the AUF1 family consists of at least four isoforms. Previous genomic cloning combined with FISH and Southern analyses of a panel of monochromosomal mouse/human or hamster/human somatic cell hybrids localized two AUF1 loci to human 4q21.1-q21.2 and Xq12 (B. Wagner et al., 1996, Genomics 34: 219-222). In the present study AUF1 gene organization was examined. The results suggest that the four known AUF1 isoforms are generated by alternative pre-mRNA splicing of a transcript encoded by the chromosome 4 locus. Functionally, this creates isoforms with different RNA-binding affinities and specificities. Thus, alternative pre-mRNA splicing may serve to create functional versatility within the AUF1 family of proteins.

Alternative Splicing↗
PubMed1998

Expression of the genes encoding CCAAT-enhancer binding protein isoforms in the mouse mammary gland during lactation and involution.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the activation of gene expression in the mammary gland during lactation. We have therefore investigated the detailed expression profile of the C/EBP family during lactation and involution of the mouse mammary gland. The expression of C/EBPbeta and C/EBPdelta mRNA was low during lactation, increased dramatically at the beginning of involution and remained constant thereafter. In contrast, C/EBPalpha mRNA expression was relatively high during the early stages of lactation, declined to low levels during the late stages of lactation and at the start of involution, and increased again during involution. Electrophoretic mobility-shift assays showed a close correlation between the expression of the C/EBP genes and the functional C/EBP DNA-binding activity and, additionally, demonstrated the participation of heterodimers, formed from among the three proteins, in DNA-protein interactions. The DNA-binding activity of the activator protein 1 (AP1) family of transcription factors was also induced during involution. These results therefore point to potentially important regulatory roles for both the C/EBP and the AP1 family during lactation and involution of the mammary gland.

Animals↗
PubMed1996

Proteinase-K-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).

Gerstmann-Sträussler-Scheinker (GSS) disease is a cerebral prion protein (PrP) amyloidosis associated with mutations in the PrP gene (PRNP). A GSS disease variant with mutation at codon 198 (F198S) has been studied in a large Indiana kindred. Biochemical investigations showed that the amyloid protein consists of 11 and 7 kDa fragments of PrP. Immunohistochemical studies showed that in addition to amyloid, these patients accumulate PrP deposits which are neither fluorescent nor birefringent when stained with thioflavin S and Congo red. In the present paper, we analyzed proteinase-K (PK)-resistant PrP in 7 patients with GSS F198S disease. Immunoblots of PK-treated brain extracts show prominent bands of ca. 27-29, 18-19, and 8 kDa. Immunohistochemistry and thioflavin-S-fluorescence show that the amyloid deposits are conspicuous in the cerebellum but sparse in the caudate nucleus. However, immunoblot analysis reveals PK-resistant PrP bands of similar intensity in both regions. Treatment with PK and PNGase F generates a pattern similar to that of PK alone. Our findings suggest that brain extracts from GSS F198S disease contain 3 prominent nonglycosylated PK-resistant PrP fragments forming a pattern not previously described in other prion diseases, which may in part explain the pathology of this GSS disease variant.

Adult↗
PubMed2001

Changes in Ca2+ signaling and contractile protein isoforms in smooth muscle cells from guinea pig ileum during culture.

Single smooth muscle cells (SMCs) isolated from guinea pig ileum using collagenase and papain were cultured on laminin-coated dishes in MEM containing fetal calf serum. Temporal changes in intracellular calcium ion concentration in response to carbachol and to ATP were investigated using fluo-3/AM and fluorescence microscopy. It was observed that carbachol caused an increased intracellular calcium ion in freshly isolated single SMCs but a reduced or negative response of cultured SMCs before confluence. On the other hand, ATP was observed to cause an increase in the calcium ion content of SMCs throughout the culture. SDS-PAGE and Western blot analyses revealed changes in the expression of contractile proteins as follows. l-Caldesmon and non-muscle type myosin heavy chain (NMHC) (considered to be marker molecules for dedifferentiation in smooth muscle cells) and non-muscle type tropomyosin were not observed in freshly isolated single SMCs. l-Caldesmon and NMHC appeared in the cultured SMCs within 2 days and the tropomyosin isoform was observed 6 days following seeding. Simultaneously, smooth muscle type myosin heavy chain (SMHC) decreased strikingly and the 41 kDa tropomyosin monomer was lost. The content of alpha-actin decreased gradually to a minimum on day 6 when non-muscle type tropomyosin appeared, and the cells began to proliferate rapidly. These results suggest that the loss of contractility in cultured smooth muscle cells is more closely related to changes in contractile protein profiles than to receptor-mediated signal transduction and that in addition to NMHC and l-caldesmon, non-muscle type tropomyosin may be useful as a marker molecule for de-differentiation of smooth muscle cells.

Actins↗
PubMed1987

Multiple SecA protein isoforms in Escherichia coli.

To define the anti-SecA-LacZ antiserum, immunoprecipitates produced with either whole anti-SecA-LacZ rabbit antiserum or affinity-purified antibodies were used to analyze nondenatured lysates of Escherichia coli. The antiserum contains antibodies that recognize different proteins. Antibody purified by preadsorption to the SecA-LacZ hybrid protein precipitated only the SecA protein from extracts. In contrast, antibody purified from the intact SecA protein precipitated several additional proteins with SecA protein. Ribosomal protein L7L12 is one of the polypeptides coprecipitated with SecA protein by antibody purified by immunoadsorption to the intact SecA protein as well as by unfractionated anti-SecA-LacZ antiserum. Two-dimensional gel electrophoresis of the SecA protein immunoprecipitated by either antiserum or purified antibody indicated that the SecA protein exists in at least two, and probably four, isoforms. Only one of the SecA isoforms is present in a ribosomal preparation.

Antibodies↗
PubMed1998

Differential expression of vascular endothelial growth factor mRNA vs protein isoform expression in human breast cancer and relationship to eIF-4E.

Angiogenesis is the formation of new blood vessels from the existing vasculature. Vascular endothelial growth factor (VEGF) is an endothelium-specific angiogenic factor strongly implicated in pathological angiogenesis. In this study, the mRNA and protein expression of the four alternatively spliced VEGF isoforms (121, 165, 189 and 206 amino acids) were examined in normal and malignant breast tissues. Three VEGF transcripts were detected in both (121>165>189), whereas only VEGF165 protein was detected. The tumours expressed more VEGF mRNA (P = 0.02) and protein (P < 0.0001), with eight-fold more VEGF protein generated per mRNA unit (P = 0.009). To examine this further, the expression of eIF-4E, a translation initiation factor, was examined. Increased eIF-4E mRNA levels were detected in the tumours (P < 0.0001) that correlated with VEGF mRNA (P = 0.0002), implying co-regulation of these genes. VEGF mRNA expression was elevated in tumours expressing the epidermal growth factor receptor (P < 0.01), but there was no difference according to oestrogen receptor status (P = 0.9), node status (P = 0.09) or between differing histologies (P = 0.4). These data suggest that elevated VEGF protein expression, by both enhanced transcription and translation, is a potential means by which tumour angiogenesis is induced in breast carcinomas. VEGF expression is also significantly associated with factors correlating with a poor outcome, implying a role in progression of this disease.

Adult↗
PubMed2001

Differential effects of ischemia and reperfusion on c-Jun N-terminal kinase isoform protein and activity.

Activation of the c-Jun N-terminal (JNK) or stress-activated protein kinases (SAPK) is associated with a wide range of disparate cellular responses to extracellular stimuli, including either induction of or protection from apoptosis. This study investigates the effect of ischemia and reperfusion on JNK isoform activities using a reversible rabbit spinal cord ischemia model. High basal JNK activity, attributed to the p46 JNK1 isoform, was expressed in the CNS of untreated rabbits. JNK activity decreased in the lumbar spinal cord of rabbits occluded for 15-60 min. During reperfusion animals occluded for 15 min recovered neurological function and JNK activity returned to normal levels. In contrast animals occluded for 60 min remained permanently paraplegic and JNK activity was half the control activity after 18 h of reperfusion. In these animals proteolytic fragments of JNK1 and JNK3 were observed and protein levels, but not activity, of JNK isoforms increased in a detergent-insoluble fraction. Two novel c-Jun (and ATF-2) kinase activities increased during reperfusion of animals occluded for 60 min. An activity designated p46(slow) was similar in M(r) to a JNK2 isoform induced in these animals. A second 30-kDa activity associated with the detergent-insoluble fraction co-migrated with a JNK3 N-terminal fragment. The results show that JNK1 is active in the normal CNS and increased activity is not associated with durations of ischemia and reperfusion that induce cell death. However, specific JNK isoform activation may participate in the cell death pathways as increased activity of novel c-Jun (ATF-2) kinase activities was observed in paraplegic animals.

Animals↗
PubMed1996

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.

Alternative Splicing↗
PubMed1994

Differential ontogenetic appearance and regulation of stimulatory G protein isoforms in rat cerebral cortex by thyroid hormone deficiency.

We have examined the effects of perinatal hypothyroidism on the levels of several G protein subunits in developing rat cerebral cortex. Thyroid deficiency significantly reduced the levels of the short form of G alpha s (G alpha s-s) by 70% and 83%, on P17 and P22, respectively, but had no effect on the long form of G alpha s (G alpha s-1), except that the G alpha s-1 levels were moderately increased on P22. Compared with age-matched controls, no significant differences were observed for G alpha i-1, G alpha i-2, G alpha o, G alpha q/11, G beta 1 and G beta 2 immunoreactivity in the cerebral cortex of the 22-day-old hypothyroid pups. These findings suggest that thyroid hormones may play an important role in controlling the pattern of expression of G alpha s isoforms during neonatal brain development and the reduced levels of G alpha s-s may contribute to some of the developmental abnormalities seen with perinatal hypothyroidism by altering signal transduction and intercellular communication.

Aging↗
PubMed1999

Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na+,K+-ATPase activity but unchanged Na+-Ca2+ exchanger protein levels in human heart failure.

BACKGROUND: Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+, K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. METHODS AND RESULTS: Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic alpha1-, alpha2-, alpha3-, and glycoprotein beta1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain-binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase alpha1- (0.62+/-0.06 of control), alpha3- (0.70+/-0.09), and beta1- (0.61+/-0.04) but not alpha2-isoforms were significantly reduced (P<0.01), whereas levels of NCE (0.92+/-0.13 of control) and calsequestrin (0.98+/-0.06) remained unchanged. Both Na+,K+-ATPase activity (NF: 1.9+/-0.29; NYHA class IV: 1.1+/-0.17 micromol ATP/min per milligram of protein) and the 3H-ouabain binding sites (Bmax NF: 15.9+/-1.9 pmol/mg protein; NYHA class IV: 9.7+/-1.5) were reduced in NYHA class IV and correlated significantly to each other (r2=0. 73; P<0.0001), as did beta1-subunit expression. In left ventricular papillary muscle strips from NYHA class IV compared with nonfailing tissue the Na+-channel modulator BDF 9198 exerted an increase in force of contraction with unchanged effectiveness but enhanced potency. CONCLUSIONS: The enhanced sensitivity of failing human myocardium toward cardiac glycosides may be, at least in part, attributed to a reduced protein expression and activity of the sarcolemmal Na+,K+-ATPase without a change in Na+-Ca2+ exchanger protein expression.

Adolescent↗
PubMed1998

The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids.

To investigate the role of acyl carrier protein (ACP) in determining the fate of the acyl moieties linked to it in the course of de-novo fatty acid biosynthesis in higher plants, we carried out in vitro experiments to reconstitute the fatty acid synthase (FAS) reaction in extracts of spinach (Spinacia oleracea L.) leaves, rape (Brassica napus L.) seeds and Cuphea lanceolata Ait. seeds. The action of two major C. lanceolata ACP isoforms (ACP 1 and ACP 2) compared to ACP from Escherichia coli was monitored by saponification of the corresponding FAS products with subsequent analysis of the liberated fatty acids by high-performance liquid chromatography. In a second approach the preference of the medium-chain acyl-ACP-specific thioesterase (EC 3.1.2.14) of C. lanceolata seeds for the hydrolysis of acyl-ACPs prepared from the three ACP types was investigated. Both ACP isoforms from C. lanceolata seeds supported the synthesis of medium-chain fatty acids in a reconstituted FAS reaction of spinach leaf extracts. Compared to the isoform ACP 1, ACP 2 was more effective in supporting the synthesis of such fatty acids in the FAS reaction of rape seed extracts and caused a higher accumulation of FAS products in all experiments. No preference of the medium-chain thioesterase for one specific ACP isoform was observed. The results indicate that the presence of ACP 2 is essential for the synthesis of decanoic acid in C. lanceolata seeds, and its expression in the phase of accumulation of high levels of this fatty acid provides an additional and highly efficient cofactor for stimulating the FAS reaction.

Acyl Carrier Protein↗
PubMed1994

The characterization of a mitochondrial acyl carrier protein isoform isolated from Arabidopsis thaliana.

A cDNA clone was isolated from an Arabidopsis leaf cDNA library that shared a high degree of protein sequence identity with mitochondrial acyl carrier proteins (mtACPs) isolated from Neurospora crassa and bovine heart muscle. The cDNA encoded an 88-amino acid mature protein that was preceded by a putative 35-amino acid presequence. In vitro protein import studies have confirmed that the presequence specifically targets this protein into pea mitochondria but not into chloroplasts. These studies indicated that pea mitochondria were not only able to import and process the precursor protein but also possessed the ability to acylate the mature protein. The mitochondrial localization of this protein, mtACP-1, was confirmed by western blot analysis. Arabidopsis mitochondrial protein extracts contained two cross-reacting bands that comigrated with the mature mtACP-1 and acylated mtACP-1 proteins. The acylated form of mtACP-1 was approximately 4 times more abundant than the unacylated form and appeared to be localized predominantly in the mitochondrial membrane where the unacylated mtACP-1 was present mostly in the matrix fraction. A chloroplast fatty acid synthase system was used, and mtACP-1 was able to function as a cofactor for fatty acid synthesis. However, predominantly short- and medium-chain fatty acids were produced in fatty acid synthase reactions supplemented with mtACP-1, suggesting that mtACP-1 may be causing premature fatty acid chain termination.

Acyl Carrier Protein↗