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PubMed1997

Muscle fibre stress in response to exercise: synthesis, accumulation and isoform transitions of 70-kDa heat-shock proteins.

Heat-shock or stress proteins (HSPs) are considered to play an essential role in protecting cells from stress and preparing them to survive new environmental challenges. This study investigates the induction kinetics of synthesis and accumulation of 70-kDa stress proteins in the soleus and extensor digitorum longus (EDL) muscles of the rat following exercise, as well as the isoform transitions that take place during the post-exercise period. Relative synthesis rates (referred to constitutively expressed stress protein HSP73) of the 70-kDa heat-shock proteins were greatly enhanced after a single bout of exercise in both muscles. They peaked early in the post-exercise period and returned to resting levels after approximately 5-6 h. The levels of the inducible stress protein HSP72 in the EDL rose only transiently following exercise, while its accumulation in the soleus was more continuous and stable. The amount of HSP73 increased only transiently in both muscle types after exercise. The constitutive expression of the stress protein HSP72 in the soleus muscle was much higher than in the EDL and other tissues, while that of HSP73 was relatively constant among tissues. Rat skeletal muscle HSP72 and HSP73 were made up of at least three isoforms of the same molecular mass and very close isoelectric points, although only one radiolabelled isoform was detected. The relative proportion of the most abundant isoforms of HSP72, isoforms 1 and 2, as well as their ratio (isoform 2/isoform 1), increased during the post-exercise period. Since isoform 2 of HSP72 partially disappeared after incubating soleus muscle extracts of exercised rats with alkaline phosphatase, these data indicate that phosphorylation of HSP72 is an early event in the stress response of skeletal muscle to exercise stress.

Animals↗
PubMed1997

Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms -- ecdysteroid-dependent transcription in cultured anterior silk glands.

Previously, we isolated a cDNA clone for the ecdysone receptor B1 isoform of the silkworm, Bombyx mori (BmEcR-B1). Here we report the cloning of a cDNA that encodes the Bombyx ecdysone receptor A isoform (BmEcR-A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval-pupal transformation, mRNA expression of BmEcR-B1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR-A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR-A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR-A and BmEcR-B1 was induced dose dependently by more than 5 ng/ml 20-hydroxyecdysone. BmEcR-A and BmEcR-B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20-hydroxyecdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.

Amino Acid Sequence↗
PubMed1989

Kinetics of carboxylmethylation of the charge isoforms of myelin basic protein by protein methyltransferase II.

The charge isoforms (C1-C5) of bovine myelin basic protein (MBP) were used as substrates for the rat brain enzyme protein carboxylmethyltransferase (PM II). The objective of these experiments was to ascertain whether the kinetic behavior of the MBP isoforms reflected differences in the structures of this molecular family. Initial velocity plots as a function of the MBP-isoform concentration showed significant differences (p less than 0.05) among the assayed isoforms except for isoforms C2 and C4. Under the conditions of our experiment all the curves exhibited a consistent sigmoidicity. The kinetic data were best fitted by a model, previously described for the enzyme D-beta-hydroxybutyrate dehydrogenase, in which two independent sites must be randomly occupied before any catalytic activity can occur. This mechanism is substantially different from that proposed by other investigators for similar PM II enzymes and other substrates. The differences in the rates of isoform carboxylmethylation are largely accounted for by the different apparent dissociation constants Ks and is explained on the basis of inherent structural differences among the charge isoforms.

Animals↗
PubMed2005

Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigra.

Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, alpha, beta, gamma and delta, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT-PCR analyses revealed that the gamma and delta isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were gammaA, gammaC, delta1 and delta3. An immunohistochemical study also confirmed the preferential localization of gamma and delta isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKIIdelta1-4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of gamma isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKIIdelta3 isoform is involved in the expression of BDNF in the SN.

Animals↗
PubMed1993

A new approach to quantitate carbohydrate-deficient transferrin isoforms in alcohol abusers: partial iron saturation in isoelectric focusing/immunoblotting and laser densitometry.

Carbohydrate-deficient transferrin (Tf) represents a significant advance over previous markers of alcohol abuse. Isoelectric focusing (IEF) analysis of affinity-purified Tf, under conditions of total iron saturation, identifies a major isoform at pI 5.4 in both normal consumers and alcohol abusers; three additional Tf isoforms (pI 5.6, 5.7, and 5.8) are associated with alcohol abuse. Under conditions of partial iron saturation, IEF analysis of affinity-purified Tf reveals up to seven isoforms (pI range 5.3-6.0) common to normal consumers and alcohol abusers; three additional transferrin isoforms (pI range 6.1-6.3) are present in 68% (15/22) of the alcohol abuser specimens, but in only 8% (1/12) of the specimens from normal consumers and in none of the three specimens from abstainers. These three diagnostic bands comigrate with a set of defined Tf isoforms: human iron-free Tf containing two sialic acid residues, human sialic acid-free Tf with one iron molecule, and human sialic acid-free, iron-free Tf. Serum specimens from normal consumers and alcohol abusers, analyzed for Tf isoforms by an IEF-immunoblot method under conditions of partial iron saturation, expressed Tf isoforms similar to those found using affinity-purified Tf in standard IEF. Visual examination of the immunoblots reveals the diagnostic bands in 67% (32/48) of patients with histories of sustained alcohol abuse compared with only 17% (8/48) of the normal consumers. Scanning densitometry and volume integration analysis of the immunoblots representative of normal consumer and alcohol abuser populations results in mean (+/- SE) values of 4.1 +/- 0.8 and 19.3 +/- 3.6 units, respectively (p < 0.0002).

Adult↗
PubMed1994

Myosin heavy chain isoforms in single fibres from m. vastus lateralis of soccer players: effects of strength-training.

The myosin heavy chain (MHC) composition of single fibres (n = 2171) was analysed with an electrophoretic technique in biopsy material from m. vastus lateralis of two groups of soccer players before and after a 3-month period of either strength- (n = 8) or non-training (control) (n = 6). Traditional myofibrillar ATPase histochemistry demonstrated a decrease in type IIA fibres with strength-training (35.4 +/- 2.1 vs. 26.7 +/- 2.4% (P < 0.05)). This was not observed in the non-training group (25.7 +/- 4.6 vs. 23.8 +/- 1.7%). One-dimensional electrophoresis on muscle homogenates showed no significant change in the amount of MHC isoforms in either of the two groups. The MHC isoform IIB was undetectable in all but three samples. No changes in the proportions of fibres containing any of the MHC isoforms were observed. Fibres containing only MHC isoform IIB were found in very small numbers (only 11 out of 2171). Before the experimental period, between 6 and 10% histochemical type IIB fibres were found in both groups. This was identical with the proportion of fibres showing co-existence of MHC isoforms IIA and IIB, but in contrast to the very few fibres containing only MHC isoform IIB. This suggests that nearly all histochemical type IIB fibres of the soccer players display co-existence of both MHC isoform IIA and IIB. No major change in the muscle fibre area of the two groups was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

Expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase slow (SERCA2) isoform in regenerating rat soleus skeletal muscle depends on nerve impulses.

We have examined the influence of innervation on the expression of different isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in regenerating rat slow twitch muscle. The process of degeneration/regeneration was induced by injection of bupivacaine into rat soleus muscle under four different conditions: (1) in the presence of intact motor nerves, (2) after surgical denervation, (3) with nerve impulse conduction blocked by the Na(+)-channel blocker tetrodotoxin (TTX), and (4) with the axoplasmic flow blocked by vinblastine. Expression of SERCA isoforms was visualized by immunohistochemical and Western blot analysis. In regenerating innervated muscle, SERCA1, the isoform normally expressed in fast twitch fibres, was present after 5 days and was then progressively replaced by SERCA2, the isoform typical of slow twitch fibres. The maximum Ca(2+) uptake, measured in single skinned fibres regenerating for 10-21 days, was similar to that of slow adult fibres and significantly lower than that of fast adult fibres. Denervation or TTX treatment prevented the expression of the SERCA2 isoform. Conversely, vinblastine did not affect the expression of SERCA isoforms. These data indicate that nerve impulses play a determinant role in the expression of the SERCA2 isoform.

Anesthetics, Local↗
PubMed2003

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat.

20-Hydroxyeicosatetraenoic acid (20-HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20-HETE-synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, -4A2 and -4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, -4A2 and -4A3 reduced 20-HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nomega-L-nitromethyl arginine ester (L-NAME, 25 mg kg(-1)), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Delta = +18 to 26 mmHg), reduced renal blood flow (RBF, Delta = -1.8 to 2.9 ml min(-1)), increased renal vascular resistance (RVR, Delta = +47 to 54 mmHg ml(-1) min(-1)), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and -4A2 but not -4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not -4A1 or -4A3 blunted the increase in MABP by L-NAME (38 +/- 9 %, P < 0.05). ASODN to CYP4A1, -4A2 and -4A3 attenuated the reduction in RBF and the consequent increase in RVR by L-NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and -4A2 but not -4A3 attenuated L-NAME-induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L-NAME-induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, -4A2, and -4A3 isoforms in the renal vasculature and the nephron.

Animals↗
PubMed2002

Catalytic specificity of CYP2D isoforms in rat and human.

In rats, six cytochrome P450 (P450) 2D isoforms have been genetically identified. Nonetheless, there is little evidence of catalytic properties of each CYP2D isoform. In this study, using recombinant CYP2D isoforms (rat CYP2D1, CYP2D2, CYP2D3, and CYP2D4 and human CYP2D6) or hepatic microsomes, we investigated the catalytic specificity toward bufuralol, debrisoquine, and propranolol, which are frequently used as CYP2D substrates. Bufuralol was oxidized to three metabolites by rat and human hepatic microsomes. 1'-Hydroxybufuralol was the major metabolite. 1'2'-Ethenylbufuralol, one of the others, was identified as a novel metabolite. The formation of 1'-hydroxybufuralol and 1'2'-ethenylbufuralol in hepatic microsomes was inhibited by anti-CYP2D antibody, suggesting that these metabolites were formed by CYP2D isoforms. All rat and human recombinant CYP2D isoforms possessed activity for the 1'-hydroxylation of bufuralol, indicating that this catalytic property was common to all CYP2D isoforms. However, the 1'2'-ethenylation of bufuralol was catalyzed only by rat CYP2D4 and human CYP2D6. Debrisoquine was oxidized to two metabolites, 3-hydroxydebrisoquine, and 4-hydroxydebrisoquine, by hepatic microsomes. Recombinant CYP2D2 and CYP2D6 had very high levels of activity for the 4-hydroxylation of debrisoquine with low K(m) values. Only CYP2D1 had a higher level of 3-hydroxylation than 4-hydroxylation activity. Propranolol 4-hydroxylation was catalyzed by CYP2D2, CYP2D4, and CYP2D6. The 7-hydroxylation of propranolol was catalyzed only by CYP2D2. In conclusion, in rats, bufuralol 1'2'-ethenylation activity was specific to CYP2D4 and debrisoquine 4-hydroxylation and propranolol 7-hydroxylation activities were specific to CYP2D2. These catalytic activities are useful as a probe for rat CYP2D isoforms.

Alcohol Oxidoreductases↗
PubMed2003

Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes.

Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity. The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

Animals↗
PubMed2006

GABAB1 receptor subunit isoforms exert a differential influence on baseline but not GABAB receptor agonist-induced changes in mice.

GABA(B) receptor agonists produce hypothermia and motor incoordination. Two GABA(B(1)) receptor subunit isoforms exist, but because of lack of specific molecular or pharmacological tools, the relevance of these isoforms in controlling basal body temperature, locomotor activity, or in vivo responses to GABA(B) receptor agonists has been unknown. Here, we used mice deficient in the GABA(B(1a)) and GABA(B(1b)) subunit isoforms to examine the influence of these isoforms on both baseline motor behavior and body temperature and on the motor-incoordinating and hypothermic responses to the GABA(B) receptor agonists l-baclofen and gamma-hydroxybutyrate (GHB). GABA(B(1b))(-/-) mice were hyperactive in a novel environment and showed slower habituation than either GABA(B(1a))(-/-) or wild-type mice. GABA(B(1b))(-/-) mice were hyperactive throughout the circadian dark phase. Hypothermia in response to l-baclofen (6 and 12 mg/kg) or GHB (1 g/kg), baclofen-induced ataxia as determined on the fixed-speed Rotarod, and GHB-induced hypolocomotion were significantly, but for the most part similarly, attenuated in both GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice. We conclude that l-baclofen and GHB are nonselective for either GABA(B(1)) receptor isoform in terms of in vivo responses. However, GABA(B(1)) receptor isoforms have distinct and different roles in mediating locomotor behavioral responses to a novel environment. Therefore, GABA(B(1a)) and GABA(B(1b)) isoforms are functionally relevant molecular variants of the GABA(B(1)) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors.

Animals↗
PubMed2006

Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.

Animals↗
PubMed1998

Etoposide targets topoisomerase IIalpha and IIbeta in leukemic cells: isoform-specific cleavable complexes visualized and quantified in situ by a novel immunofluorescence technique.

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.

Antigens, Neoplasm↗
PubMed1996

Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response.

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

Amino Acid Sequence↗
PubMed1998

Cytoplasmic dynein intermediate-chain isoforms with different targeting properties created by tissue-specific alternative splicing.

The intermediate chains (ICs) are the subunits of the cytoplasmic dynein that provide binding of the complex to cargo organelles through interaction of their N termini with dynactin. We present evidence that in Drosophila, the IC subunits are represented by at least 10 structural isoforms, created by the alternative splicing of transcripts from a unique Cdic gene. The splicing pattern is tissue specific. A constitutive set of four IC isoforms is expressed in all tissues tested; in addition, tissue-specific isoforms are found in the ovaries and nervous tissue. The structural variations between isoforms are limited to the N terminus of the IC molecule, where the interaction with dynactin takes place. This suggests differences in the dynactin-mediated organelle binding by IC isoforms. Accordingly, when transiently expressed in Drosophila Schneider-3 cells, the IC isoforms differ in their intracellular targeting properties from each other. A mechanism is proposed for the regulation of dynein binding to organelles through the changes in the content of the IC isoform pool.

Alternative Splicing↗
PubMed1994

Creatine kinase isoform electrophoresis for the early confirmation of myocardial infarction detected by timed sequential CK slope analysis.

Creatine kinase (CK)-MM and -MB isoforms were evaluated for the early diagnosis of myocardial infarction in patients aged over 65 years admitted to a district general hospital with acute chest pain. Samples were collected for standard cardiac enzymes, timed CK slope analysis, and CKMM and CKMB isoform analysis from 48 patients admitted with acute chest pain. CKMM and CKMB isoform analyses were conducted using a Helena Rep electrophoresis system under standard conditions supplied by the company. In addition to the results of the biochemical tests the discharge diagnosis of the patients were also recorded. CKMM isoform analysis resulted in three false-negative classifications of patients and one false-positive. The predictive value of this test was 100% for a positive result and 94% for a negative result. CKMB isoform analysis was less accurate and there were six false-negative results and five false-positive results. The predictive value of a positive result was 75% and 85% for a negative result. CK isoform analysis became unreliable when mean total CK levels in serum were 210 IU/l (+/- 171). CK isoform analysis may be of use in the investigation of patients whose samples have a total CK concentration greater than the reference range but was no better than timed CK slope analysis for the detection of myocardial infarction in patients aged more than 65 years.

Aged↗
PubMed1990

Stability of Na(+)-K(+)-ATPase alpha-subunit isoforms in evolution.

Encoding DNA for alpha 2- and alpha 3-isoforms of the alpha-subunit of the chicken Na(+)-K(+)-ATPase have been cloned, and their nucleotide sequences and deduced amino acid sequences are reported. Comparisons between these data and comparable data for the rat alpha-subunit isoforms make possible an assessment of alpha-subunit isoform diversity among vertebrates. There is approximately twice as much amino acid sequence difference between alpha-isoforms within a single species as there is difference between corresponding alpha-isoforms of bird and mammal. These data are consistent with triplication of the alpha-subunit gene and evolution of substantially different alpha-subunit isoforms before the separation of avian and mammalian lineages over 200 million years ago and then retention of the majority of these structural differences through subsequent evolution. The implications of this conversation of isoform-specific structural features are discussed in terms of transport functions and bioregulation of the Na(+)-K(+)-ATPase.

Amino Acid Sequence↗
PubMed1997

Myosin isoform expression and force generation in cultured resistance arteries.

Organ culture of mesenteric resistance arteries results in a loss of force-generating ability, which is prevented by 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We have tested the hypothesis that the culture-induced decrease in active stress is associated with altered myosin isoform expression. Rat mesenteric resistance arteries were studied immediately (fresh) or after incubation at 37 degrees C for 48 h in culture medium (control), with 300 pg/ml 1,25(OH)2D3, or with 5 microg/ml insulin. Isometric force was measured by myography; myosin heavy chain (MHC) and regulatory myosin light chain isoform (MLC) contents were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal active stress to 100 mM K+ (mN/mm2) was greater for fresh (147.8 +/- 4.9) than control (109.2 +/- 4.6, P = 0.001) or insulin (79.6 +/- 8.6, P < 0.001) but not 1,25(OH)2D3 (137.4 +/- 9.5, P = 0.197). Organ culture did not alter MLC or MHC smooth muscle (SM)-1 isoform content. MHC SM-2 content (nmol/mg protein) was greater in fresh (0.038 +/- 0.003) than control (0.026 +/- 0.003, P = 0.012) and insulin (0.027 +/- 0.002, P = 0.018) but not 1,25(OH)2D3 (0.036 +/- 0.003, P = 0.693); nonmuscle MHC (NMM) was observed in insulin. The maximal active stress response to K+ significantly correlated with SM-2 MHC isoform content (r2 = 0.483, P < 0.001). We conclude that 1) arterial organ culture alters MHC isoform content, 2) SM-2 MHC isoform content positively correlates with active stress generation, 3) 1,25(OH)2D3 maintains force-generating capacity by preventing the shift of MHC isoform expression, and 4) insulin impairs force-generating ability by lowering MHC SM-2 content and stimulating NMM expression.

Animals↗