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PubMed2004

Differential expression of calcineurin A isoforms in the diabetic kidney.

Calcineurin is an important signaling molecule in mesangial cells in vitro and is involved in some manifestations of diabetic nephropathy in vivo. However, calcineurin acts in a cell-specific and tissue-specific manner in the kidney, and mechanisms of specificity are unknown. Three closely related isoforms of the calcineurin A (CnA) subunit are expressed in a tissue-specific manner. This study was undertaken to determine if specificity of calcineurin action is linked to regulation of CnA isoforms in the diabetic kidney. After induction of diabetes with streptozotocin, expression of all three CnA isoforms rapidly increased, primarily in the thick ascending limb of Henle (TAL). After prolonged diabetes, increase specifically of the alpha isoform was observed in collecting ducts (CD) and in endothelial cells of glomeruli. Aquaporin 2 (AQP2), a putative substrate of calcineurin phosphatase in the kidney, is also involved in diabetic nephropathy. Co-localization of CnA isoforms with AQP2 revealed that CnA-alpha is the predominant isoform that associates with AQP2 in the diabetic kidney. Furthermore, inhibition of calcineurin with cyclosporin A (CsA) alters AQP2 localization and phosphorylation in principal cells of CD. Alterations in subcellular localization of AQP2 were parallel with CnA-alpha. Similarly, CsA treatment results in a further increase in urine output compared with diabetes alone, suggesting a functional consequence of inhibiting calcineurin-mediated regulation of AQP2. In conclusion, all three isoforms of CnA are upregulated in the diabetic kidney. Increased expression of CnA-alpha, in particular, is observed in glomeruli and CD and participates in regulation of AQP2 expression, phosphorylation, and function.

Animals↗
PubMed2005

The UNC-73/Trio RhoGEF-2 domain is required in separate isoforms for the regulation of pharynx pumping and normal neurotransmission in C. elegans.

In both Caenorhabditis elegans and Drosophila, UNC-73/Trio functions in axon guidance by signaling through the Rac GTPase to regulate cytoskeletal rearrangements necessary for growth cone migrations. Here, we show that the complex C. elegans unc-73 gene encodes at least eight differentially expressed UNC-73 intracellular protein isoforms. Previously reported mutations affecting UNC-73 isoforms encoding the Rac-specific RhoGEF-1 domain cause uncoordinated movement, correlating with defects in axon guidance. Mutations in isoforms encoding the Rho-specific RhoGEF-2 domain, which we describe here, result in L1 stage larval lethality with no associated axon guidance defects. Isoform-specific rescue experiments reveal separate functions for the various RhoGEF-2-containing UNC-73 isoforms, which would not likely be discovered by conventional genetic screening. UNC-73 D1 and D2 appear to function redundantly in pharynx muscle to regulate the rate and strength of pharynx pumping, and in the HSN neurons and vulval muscles to control egg laying. Isoforms C1, C2, E, and F act redundantly within the nervous system to regulate the speed of locomotion. The multiple UNC-73 isoforms containing Rac- and Rho-specific RhoGEF domains therefore have distinct physiological functions. In addition to its previously identified role involving RhoGEF-1 in migrating cells and growth cones, our data indicate that UNC-73 signals through RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission.

Animals↗
PubMed2003

Identification and differential expression of two thioredoxin h isoforms in germinating seeds from pea.

The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.

Amino Acid Sequence↗
PubMed2005

Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans.

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni-1, tni-2/unc-27 and tni-3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni-4 was expressed solely in the pharynx. Expressing tni-1 and -2 gene RNAi caused motility defects similar to unc-27 (e155) mutant, a tni-2 null allele. The tni-3 RNAi expression produced egg laying defects while the tni-4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI-4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

Amino Acid Sequence↗
PubMed2006

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth.

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.

Adenosine Triphosphatases↗
PubMed2005

Differential agonist-mediated internalization of the human 5-hydroxytryptamine 7 receptor isoforms.

The human 5-hydroxytryptamine 7 (5-HT(7)) serotonin receptor is a class A G-protein coupled receptor that has three isoforms, 5-HT(7(a)), 5-HT(7(b)), and 5-HT(7(d)), which are produced by alternative splicing. The 5-HT(7) receptors are expressed in discrete areas of the brain and in both vascular and gastrointestinal smooth muscle. Central nervous system 5-HT(7) receptors may play a role in mood and sleep disorders. 5-HT(7) receptors show high affinity for a number of antidepressants and typical and atypical antipsychotics. We report here that the human 5-HT(7(d)) isoform expressed in human embryonic kidney (HEK) 293 cells exhibits a pattern of receptor trafficking in response to agonist that differ from 5-HT(7(a)) or 5-HT(7(b)) isoforms. We employed a modification of a live cell-labeling technique to demonstrate that surface 5-HT(7(d)) receptors are constitutively internalized in the absence of agonist. This is in contrast to 5-HT(7(a)) and 5-HT(7(b)) isoforms, which do not show this profound agonist-independent internalization. Indeed, the 5-HT(7(d)) isoform displays this internalization in the presence of a 5-HT(7) -specific antagonist. In addition, the human 5-HT(7) isoform shows a diminished efficacy in stimulation of cAMP-responsive reporter gene activity in transfected cells compared with 5-HT(7(a)) or 5-HT(7(b)) receptors expressed at comparable levels. Thus, the carboxy-terminal tail of 5-HT(7(d)), which is the longest among known human 5-HT(7) isoforms, may contain a motif that interacts with cellular transport mechanisms that is distinct from 5-HT(7(a)) and 5-HT(7(b)).

Biological Transport↗
PubMed2004

An apamin- and scyllatoxin-insensitive isoform of the human SK3 channel.

We have isolated an hSK3 isoform from a human embryonic cDNA library that we have named hSK3_ex4. This isoform contains a 15 amino acid insertion within the S5 to P-loop segment. Transcripts encoding hSK3_ex4 are coexpressed at lower levels with hSK3 in neuronal as well as in non-neuronal tissues. To investigate the pharmacokinetic properties of hSK3_ex4, we expressed the isoforms hSK3 and hSK3_ex4 in tsA cells. Both isoforms were similarly activated by cytosolic Ca2+ (hSK3, EC50=0.91 +/- 0.4 microM; hSK3_ex4, EC50=0.78 +/- 0.2 microM) and by 1-ethyl-2-benzimidazolinone (hSK3, EC50=0.17 mM; hSK3_ex4, 0.19 mM). They were both blocked by tetraethylammonium (hSK3, Kd=2.2 mM; hSK3_ex4, 2.6 mM) and showed similar permeabilities relative to K+ for Cs+ (hSK3, 0.17 +/- 0.04, n=3; hSK3_ex4, 0.17 +/- 0.05, n=3) and Rb+ (hSK3, 0.79 +/- 0.04, n=3; hSK3_ex4, 0.8 +/- 0.07, n=3). Ba2+ blocked both isoforms, and in both cases, the block was strongest at hyperpolarizing membrane potentials. However, the voltage-dependence of hSK3 was stronger than that of hSK3_ex4. The most obvious distinguishing feature of this new isoform was that whereas hSK3 was blocked by apamin (Kd=0.8 nM), scyllatoxin (Kd=2.1 nM), and d-tubocurarine (Kd=33.4 microM), hSK3_ex4 was not affected by apamin up to 100 nM, scyllatoxin up to 500 nM, and d-tubocurarine up to 500 microM. So far, isoform hSK3_ex4 forms the only small-conductance calcium-activated potassium (SK) channels, which are insensitive to the classic SK blockers.

Alternative Splicing↗
PubMed2006

Differential role of Sp100 isoforms in interferon-mediated repression of herpes simplex virus type 1 immediate-early protein expression.

Nuclear domains called ND10 or PML nuclear bodies contain interferon (IFN)-upregulated proteins like PML and Sp100. Paradoxically, herpes simplex virus 1 (HSV-1) begins its transcriptional cascade at aggregates of ND10-associated proteins, which in turn are destroyed by the HSV-1 immediate-early protein ICP0. While PML is essential in the formation of ND10, the function of Sp100 in the cells' defense against viral infection is unknown. In this study we investigated the potential antiviral effect of IFN-beta-induced Sp100. We found that IFN-beta treatment leads to a differential accumulation of four Sp100 isoforms in different cell lines. Using an HEK293 cell line derivative, 293-S, producing no detectable amounts of Sp100 even after IFN exposure, we analyzed individual Sp100 isoforms for their effect on HSV-1 infection. Sp100 isoforms B, C, and HMG, but not Sp100A, suppressed ICP0 and ICP4 early after infection. Isoforms B, C, and HMG suppressed expression from the ICP0 promoter in transient transfection, whereas Sp100A enhanced expression. Moreover, Sp100A localized in ND10, whereas the repressive isoforms were either dispersed within the nucleus or, at unphysiologically higher expression levels, formed new aggregates. The repressive activity was dependent on an intact SAND domain, since Sp100B bearing a W655Q mutation in the SAND domain lost this repressive activity and accumulated in ND10. Using RNA interference to knock down the repressive Sp100 isoforms B, C, and HMG, we find that they are an essential part of the IFN-beta-mediated suppression of ICP0 expression. These data suggest that repression by the Sp100 isoforms B, C, and HMG takes place outside of ND10 and raise the possibility that viral genomes at Sp100A accumulations are more likely to start their transcription program because of a more permissive local environment.

Antigens, Nuclear↗
PubMed2006

A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization.

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5' and 3' rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3' long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus.

Alternative Splicing↗
PubMed2006

Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops.

Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75(MDM2) is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75(MDM2) doublets and p90(MDM2), which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90(MDM2). The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90(MDM2) promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.

Animals↗
PubMed2006

Specific isoforms of translation initiation factor 4GI show differences in translational activity.

The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2alpha phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

Amino Acid Sequence↗
PubMed1999

C/EBPalpha regulates generation of C/EBPbeta isoforms through activation of specific proteolytic cleavage.

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP.

Animals↗
PubMed1999

Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms.

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

Alternative Splicing↗
PubMed2002

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons.

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

5' Untranslated Regions↗
PubMed2003

Selective degradation of AU-rich mRNAs promoted by the p37 AUF1 protein isoform.

An AU-rich element (ARE) consisting of repeated canonical AUUUA motifs confers rapid degradation to many cytokine mRNAs when present in the 3' untranslated region. Destabilization of mRNAs with AREs (ARE-mRNAs) is consistent with the interaction of ARE-binding proteins such as tristetraprolin and the four AUF1 isoforms. However, the association of the AUF1-mRNA interaction with decreased ARE-mRNA stability is correlative and has not been directly tested. We therefore determined whether overexpression of AUF1 isoforms promotes ARE-mRNA destabilization and whether AUF1 isoforms are limiting components for ARE-mRNA decay. We show that the p37 AUF1 isoform and, to a lesser extent, the p40 isoform possess ARE-mRNA-destabilizing activity when overexpressed. Surprisingly, overexpressed p37 AUF1 also destabilized reporter mRNAs containing a noncanonical but AU-rich 3' untranslated region. Since overexpressed p37 AUF1 could interact in vivo with the AU-rich reporter mRNA, AUF1 may be involved in rapid turnover of mRNAs that lack canonical AREs. Moreover, overexpression of p37 AUF1 restored the ability of cells to rapidly degrade ARE-mRNAs when that ability was saturated and inhibited by overexpression of ARE-mRNAs. Finally, activation of ARE-mRNA decay often involves a translation-dependent step, which was eliminated by overexpression of p37 AUF1. These data indicate that the p37 AUF1 isoform and, to some extent, the p40 isoform are limiting factors that facilitate rapid decay of AU-rich mRNAs.

Animals↗
PubMed2002

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells.

In smooth muscle cells (SMCs) isolated from rabbit carotid, femoral, and saphenous arteries, relative myosin isoform mRNA levels were measured in RT-PCR to test for correlations between myosin isoform expression and unloaded shortening velocity. Unloaded shortening velocity and percent smooth muscle myosin heavy chain 2 (SM2) and myosin light chain 17b (MLC(17b)) mRNA levels were not significantly different in single SMCs isolated from the luminal and adluminal regions of the carotid media. Saphenous artery SMCs shortened significantly faster (P < 0.05) than femoral SMCs and had more SM2 mRNA (P < 0.05) than carotid SMCs and less MLC(17b) mRNA (P < 0.001) and higher tissue levels of SMB mRNA (P < 0.05) than carotid and femoral SMCs. No correlations were found between percent SM2 and percent MLC(17b) mRNA levels and unloaded shortening velocity in SMCs from these arteries. We have previously shown that myosin heavy chain (MHC) SM1/SM2 and SMA/SMB and MLC(17a)/MLC(17b) isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression and unloaded shortening velocity do not vary with distance from the lumen of the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/or MLC(17a)/MLC(17b) isoform expression does not correlate with unloaded shortening velocity, and 3) intracellular expression of the MHC SM1/SM2 and MLC(17a)/MLC(17b) isoforms is not coregulated.

Animals↗
PubMed2005

Three-dimensional compartmentalization of myosin heavy chain and myosin light chain isoforms in dog thyroarytenoid muscle.

The thyroarytenoid muscle, a vocal fold adductor, has important roles in airway protection (e.g., prevention of aspiration) and phonation. Isoform expression of myosin heavy chain (MHC), a major determinant of muscle-shortening velocity, has been reported to be heterogeneous in this muscle in several mammals, differing markedly between the medial and lateral divisions. The objective was to determine the isoform expression patterns of both MHC and myosin light chain (MLC), with the latter having a modulatory role in determining shortening velocity, to further test whether the expression of both myosin subunits differs in multiple specific sites within the divisions of the dog thyroarytenoid muscle, potentially revealing even greater compartmentalization in this muscle. Our results indicate the existence of large gradients in the relative levels of individual MHC isoforms in the craniocaudal axis along the medial layer (i.e., airflow axis), where levels of MHC-I and MHC-IIA are low at both ends of the axis and high in the middle and MHC-IIB has a reciprocal distribution. The lateral layer is more uniform, with high levels of MHC-IIB throughout. The level of MHC-IID is relatively constant along the axis in both layers. Large differences exist in the distribution of MHC isoforms among single fibers isolated from sites along the craniocaudal axis, especially in the lateral layer. Systematic regional variations are apparent in the MLC isoform composition of single fibers as well, including some MLC isoform combinations that are not observed in dog limb muscles. Variations of MHC and MLC isoform expression in the dog thyroarytenoid muscle are greater than previously recognized and suggest an even broader range of contractile properties within this multifunctional muscle.

Animals↗
PubMed2001

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms.

The cardiac Na(+)/Ca(2+) exchanger (NCX), an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7 degrees C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents (I(NCX)) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I(NCX) of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 degrees C using the giant excised patch technique. The I(NCX) for both isoforms exhibited Na(+)-dependent inactivation and Ca(2+)-dependent positive regulation. The I(NCX) of NCX1.1 exhibited typical mammalian temperature sensitivities with Q(10) values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I(NCX) of NCX-TR1.0 was relatively temperature insensitive with Q(10) values of 1.2 and 1.1 for peak and steady-state currents, respectively. I(NCX) current decay was fit with a single exponential, and the resultant rate constant of inactivation (lambda) was determined as a function of temperature. As expected, lambda decreased monotonically with temperature for both isoforms. Although lambda was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on lambda was not different between the two isoforms. These data suggest that the disparities in I(NCX) temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after alpha-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I(NCX) temperature dependence between the two isoforms are not due to potential disparities in either the I(NCX) regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.

Animals↗