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PubMedDate not supplied

Expression of p63 mRNA isoforms in normal human tissue.

This report describes the presence of different p63 isoforms in normal human tissue. The p63, expressed by basal cells of stratified epithelia and myoepithelial cells, is characterized by different mRNA isoforms formed by alternative splicing. Three isoforms were shown to act pro-apoptotically, while the remaining three isoforms are anti-apoptotic. Therefore a shift in a balance between different p63 isoforms might promote or inhibit normal and malignant growth. Our results show that in bone marrow, brain, colon, skeletal muscle, spleen and testis only pro-apoptotic p63 isoforms are expressed, whereas only anti-apoptotic isoforms are present in lung, placenta and salivary gland. In kidney, prostate, skin, thymus, trachea and uterus both variants are present. Presented results will allow in the future to compare p63 isoforms in various pathological processes with normal tissue pattern in order to evaluate the contribution of p63 isoform imbalance to the pathophysiology of various malignant diseases.

Alternative Splicing↗
PubMed2001

Vesicle-associated membrane protein isoforms in the tiger salamander retina.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.

Ambystoma↗
PubMed1988

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle.

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.

Actins↗
PubMed1993

In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants.

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.

Actins↗
PubMed1999

Altered microtubule-associated tau messenger RNA isoform expression in livers of griseofulvin- and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-treated mice.

Tau proteins belong to the family of microtubule-associated proteins (MAPs), which so far have been mostly detected in neuronal cells. Different domains on the protein serve different functions. By alternative splicing, several mRNAs and tau isoforms are created from one gene, which contain these functionally important domains to various degrees, and thus differ in their microtubule-related properties. In the present article, several novel observations are reported. Tau mRNA and proteins have been identified and further characterized in mouse liver. It is shown on the basis of mRNA determinations that at least three tau isoforms differing particularly with respect to their amino-terminal domains are present in mouse liver. The major and predominant isoform (isoform 1) lacks portions encoded by exons 2 and 3, which are responsible for cross-talk of microtubules with their environment ("projection domain"). Moreover, mRNA encoding tau protein with four repeats of the microtubule binding domain predominate in embryonal as well as adult mouse liver in contrast to brain, in which a shift from the predominant three-repeat isoform to the four-repeat isoform characterizes the transition from the embryonic to the adult stage. Intoxication with griseofulvin (GF) or 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) significantly affects in a reversible manner the levels of tau mRNA as well as isoform ratios in mouse liver, but not in mouse brain. Tau mRNAs are significantly increased in intoxicated mouse livers. Moreover, a shift to isoform 1 lacking exons 2 and 3 occurs. However, the increase in liver tau protein was less than expected from increased mRNA levels, which could be the result of translational or posttranslational regulation. The consequences on microtubular function are as yet unclear, but impairment can be expected because the overexpressed tau mRNA isoform lacks the domain that mediates interaction of microtubules with their environment. On the other hand, the ratio of polymerized (microtubules) to nonpolymerized tubulin remained unaffected.

Animals↗
PubMed1994

Simultaneous automated measurement of serum total CK and CK-MM isoform ratio in serum.

We automated a two-step kinetic procedure for determining serum CK-MM isoform ratio using an immunoinhibition method. By measuring the total CK activity and the residual CK activity (serum CK-MM isoform) remaining after the inhibition by tissue CK-MM isoform specific monoclonal antibody reagent (CK-M01) the CKMM isoform ratio is calculated using the difference between total CK and residual CK activities divided by the residual CK activity. Linearities of total CK and residual CK assays were < or = 7750 U/L and 2,500 U/L, respectively; within-run CVs of isoform ratio (N = 10) were 2.8 and 7.0% (mean 0.14 and 0.60), respectively. The MM3/MM1 isoform ratio obtained with the proposed method (X) correlated well with the results of electrophoretic method (Y) according to the equation: Y = 0.98X-0.3, r = 0.988. The normal reference range of isoform ratios obtained by assaying 1,222 serum samples from healthy subjects was 0.09-0.75. The isoform ratio increased after onset of chest pain, peaking at 2-6 hr thereafter. A mean isoform ratio of 1.86 was obtained with serum sample from 86 patients diagnosed as having an acute myocardial infarction (AMI). This method is accurate and highly sensitive, as the detection and early diagnosis of AMI can be completed in 10 min.

Adolescent↗
PubMed1995

Evaluation of a new automated system for the determination of CK-MB isoforms.

We evaluated a new analyzer (Cardio REP) specifically designed for cardiac CK-MB isoenzyme and isoforms activity, with a performance time of 24 minutes. Ten AMI patients, with times elapsed between the onset of chest pain and admission to hospital ranging from 30 minutes to 4 hours, were monitored every 3-4 hours until the 16th hour of hospitalization. In each serum sample, in addition to total CK-MB and CK-MB isoforms measured by the Cardio REP analyzer, we also assayed total CK activity, CK-MB activity by immunoinhibition method, CK-MB mass concentration, CK-MB isoforms by REP method, troponin T, and myoglobin. The precision study demonstrated acceptable within assay and between assay CVs% for total CK-MB (8.1 and 10.4), MB1 (9.1 and 14.2), and MB2 (9.1 and 8.2) isoforms. The method was found to be linear up to 371 U/L for MB2 isoform fraction and up to 516 U/L for total CK-MB. Results for CK-MB obtained with the Cardio REP correlated well with those for CK-MB activity obtained with the immunoinhibition method (r = 0.869) and those of CK-MB mass concentration (r = 0.923). The sensitivity of the Cardio REP CK isoforms method was found to be greater than that of the REP CK isoforms method. Time to first increased value of MB2/MB1 ratio and MB2 isoform was earlier in comparison to that for CK-MB mass concentration and similar to that for myoglobin, a marker that, however, lacks specificity. The diagnostic efficiency of CK-MB isoforms and the availability of a real-time, fully automated method for their measurement suggest that utilization of this biochemical marker in emergency for the early diagnosis of AMI.

Blood Protein Electrophoresis↗
PubMed1989

Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover.

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform-specificity, the A/B-type (binds all three isoforms) and the B-type (binds only PDGF-BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF-AB and PDGF-BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF-AA binding sites (A/B-receptors) per cell, whereas human fibroblasts express only 20,000 A/B-receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF-BB and PDGF-AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose-dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform-specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.

Animals↗
PubMed1990

Developmental regulation of type II calcium/calmodulin-dependent kinase isoforms in rat cerebellum.

Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.

Animals↗
PubMed1998

Differential motogenic and biosynthetic response of fetal and adult skin fibroblasts to TGF-beta isoforms.

Data are presented in this communication comparing fetal and adult fibroblasts with respect to the effects of transforming growth factor beta (TGF-beta) isoforms (-beta 1, -beta 2 and -beta 3) on cell migration and hyaluronan (HA) synthesis. Cell migration was assessed on three-dimensional native type I collagen substrata. Fetal and adult cells differed in terms of their motogenic response to the three TGF-beta isoforms in a manner which was modulated by cell density, i.e.: (1) the migration of subconfluent fetal cells was unaffected by TGF-beta 1 and -beta 2, but inhibited by TGF-beta 3, whilst the migration of subconfluent adult cells was inhibited by all three isoforms, and (2) the migration of confluent fetal cells was inhibited by all three TGF-beta isoforms, whilst the migration of confluent adult cells was unaffected by TGF-beta 1 and -beta 2, but stimulated by TGF-beta 3. This diverse pattern of motogenic response to the three TGF-beta isoforms was paralleled by similar effects on HA synthesis (i.e. inhibition, no effect or stimulation). Linear regression analysis revealed a significant correlation between cell migration and total HA synthesis (r2 = 0.861; P < 0.0001). Gel filtration chromatography of cell-produced HA indicated that the effects of TGF-beta isoforms on total HA synthesis reflected alterations in the relative production of high molecular mass species (M(r) > 10(6)). Taken together with previously published data, these observations indicate that (1) fetal and adult fibroblasts exhibit distinct responses to the three TGF-beta isoforms with respect to both cell migration and HA synthesis, (2) cellular response to the TGF-beta isoforms is modulated by cell density, and (3) TGF-beta 3 is the only isoform which stimulated cell migration and HA synthesis (with confluent adult cells).

Adult↗
PubMed1997

Increased expression of fibronectin isoforms after myocardial infarction in rats.

Fibronectin is a known chemoattractant for several cell types which play a role in the wound healing process, like fibroblasts, endothelial cells and macrophages. In addition, fibronectin generates a scaffold to which other extracellular matrix components can attach. The possible involvement of fibronectin in the wound healing process after myocardial infarction (MI) was investigated by studying the expression of fibronectin isoforms after induction of a MI in the rat. Deposition of plasma (pFN) and cellular fibronectin (cFN) protein was determined immunohistochemically, using monoclonal antibodies specific for cFN and polyclonal anti-human total FN (tFN antibodies). Expression of the mRNAs of total cFN and the embryonic isoforms EIIIA and EIIIB was investigated, using in situ hybridization (ISH). The ratio between EIIIA containing fibronectin (EIIIA+-FN) mRNA and total cFN mRNA was determined using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). cFN protein was present from day 4 until day 35 after infarction and was located around the area of infarction, in the epi- and endomyocardium and in the wall of larger vessels. pFN was found in the infarcted cardiomyocytes 1 day after the induction of the MI. From day 4 on pFN protein deposition was found in the border zone of the infarction and in the wall of larger vessels. pFN immunoreactivity remained present at high levels around the area of infarction and in the vessel wall throughout the entire period of investigation (90 days). From day 35 after the infarction pFN protein was detected in cardiomyocytes of the right ventricle and septum. cFN mRNA, determined by in situ hybridization, was present in the border zone of the infarcted area as early as 1 day after MI, and its expression peaked at 4 days after MI. Four days after MI the mRNA's coding for both the embryonic isoforms EIIIA and EIIIB could also be detected in the same area. Because expression of the EIIIA isoform was more abundant than the EIIIB isoform we only determined the percentage of the EIIIIA containing isoform from total FN. EIIIA+ mRNA was elevated 1 day after MI. We conclude that various fibronectin isoforms including the embryonic isoforms accumulate in the heart after MI. This suggests that these isoforms may play a role in the wound healing process in the left ventricle of the infarcted heart.

Animals↗
PubMed1993

Forced expression and assembly of rat cardiac troponin T isoforms in cultured muscle and nonmuscle cells.

Cardiac troponin T (cTnT), a tropomyosin (TM)-binding subunit of the troponin complex, undergoes a developmentally regulated isoform switch from embryonic form to adult form in the rat heart. To investigate the in vivo assembly of cTnT isoforms, we transiently transfected cDNA clones of either rat cTnT isoform into nonmuscle CHO cells and chick embryo myogenic (CEM) cells. As determined by Western blotting, both isoforms can be expressed in CHO and CEM cells. The expressed proteins had the same mobility as native rat cTnT proteins on SDS polyacrylamide gels and were recognized by anti-TnT antibodies. Conventional and confocal microscopy of transfected cells, double-labelled with antibodies against cTnT and against TM, revealed that neither isoform appears to associate with the nonmuscle TM in CHO cells, although both are able to colocalize with muscle TM-containing microfilament bundles in the myogenic CEM cells. There was no appreciable cTnT isoform-related difference in association with TM, suggesting that the functional significance of isoform variability in rat cTnT does not correspond to an assembly advantage for the maturing cardiac thin filament. To help determine whether cTnT nonassembly in CHO environment is primarily due to the nonmuscle nature of the endogenous TM, or if it involves the absence of other factors specific to muscle, we have isolated several stably-transfected clones of skeletal beta TM-expressing CHO cells which incorporate this muscle TM onto stress fibres. When either isoform of cTnT was transiently expressed in these beta TM-CHO cells, the strictly filamentous beta TM staining pattern was no longer observed. Instead, beta TM codistributed with cTnT in brightly staining aggregates not associated with the intact stress fibres. This suggests that both isoforms of cTnT are interacting with the beta TM in the nonmuscle environment and that other muscle-specific proteins may indeed be required for stable assembly of cTnT onto microfilaments. It also suggests that the interaction between cTnT and muscle TM is stronger than that between muscle TM and nonmuscle microfilaments.

Actin Cytoskeleton↗
PubMed1996

Myosin isoform transitions in four rabbit muscles during postnatal growth.

Four rabbit muscles (i.e. semimembranosus proprius, psoas major, biceps femoris and longissimus lumborum), differing in their fibre type composition in the adult, were investigated during postnatal development. Muscle samples were taken at 1, 7, 14, 21, 28, 35, 49 and 77 days of age. Complementary techniques were used to characterize myosin heavy chain (MHC) isoform transitions, i.e. SDS-PAGE, immunocytochemistry and conventional histochemistry. Good accordance was found between electrophoretic and immunocytochemical techniques. Our results show that rabbit muscles were phenotypically immature at birth. At 1 day of age, perinatal isoform represented 70-90% of the total isoform content of the muscles. Two generations of myofibres could be observed on the basis of their morphology and reaction to specific antibodies. In all muscles, primary fibres expressed slow MHC. In contrast, secondary generation of fibres never expressed slow MHC in future fast muscles, while half of them expressed slow MHC in the future slow-twitch muscle, the semimembranosus proprius. During the postnatal period, all muscles displayed a transition from embryonic to perinatal MHC isoforms, followed by a transition from perinatal to adult MHC isoforms. These transitions occured mainly during the first postnatal month. The embryonic isoform was no longer expressed after 14 days, except in longissimus where it disappeared after 28 days. On the contrary, large differences were found in the timing of disappearance of the perinatal isoform between the four muscles. The perinatal isoform disappeared between 28 and 35 days in semimembranosus proprius and 35 and 49 days in psoas and biceps femoris. Interestingly, the perinatal isoform was still present in 6% of the fibres in longissimus at 77 days, the commercial slaughter age, denoting a great delay in the maturation. Fate of each generation of fibres differed between muscles.

Aging↗
PubMed1990

Different phosphorylation behaviour of regulatory subunit isoforms of type II cAMP-dependent protein kinase from bovine heart.

Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII55 and RII52) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII55 isoform migrated more slowly (RII55/57) while the migration of RII52 isoform did not shift. Both isoforms showed different affinity for cAMP. The RII55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strength whereas the RII52 isoform required cAMP, plus 2M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8 125I-peptides patterns of both isoforms are quite similar. Incubation of partially purified holoenzyme with 10 nM [gamma-32P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII55/57 isoform. The incubation, however, at 20 microM [gamma-32P]ATP yielded two phosphobands corresponding to both RII55/57 and RII52 isoforms. The phosphorylation of RII52 took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII55/57.

Adenosine Triphosphate↗
PubMed1989

Identification and pattern of expression of a developmental isoform of troponin I in chicken and rat cardiac muscle.

A monoclonal antibody that reacts with all known isoforms of troponin I detected a single isoform of cardiac troponin I in both atrial and ventricular chambers of adult chicken and rat hearts in an immunoblotting analysis. Another isoform of troponin I in addition to the adult cardiac form, however, was present in all chambers of the heart during early development in both species. This developmental isoform appeared to have the same electrophoretic mobility on SDS tris glycine polyacrylamide gels as that observed for the adult slow skeletal muscle isoform. In the rat, only the developmental isoform of troponin I was present in the early foetal heart and small amounts of the adult cardiac isoform were not apparent until late in gestation, whereas the developmental and adult isoforms were expressed in approximately equal amounts throughout embryonic development in the chicken. The level of developmental isoform of troponin I in both the chicken and the rat hearts gradually decreased so that only small amounts of this variant were detectable two weeks after birth or post hatching.

Aging↗
PubMed1993

Detection of reperfusion 30 and 60 minutes after coronary recanalization by a rapid new assay of creatine kinase isoforms in acute myocardial infarction.

We measured creatine kinase (CK) isoforms by a new immunoinhibition method to evaluate their usefulness in detecting early coronary reperfusion. Blood samples were collected at 15-minute intervals from 50 patients with acute myocardial infarction. CK isoforms were determined by a 10-minute immunoinhibition method with an autoanalyzer. Values for inhibited isoforms (MM3, MM2/2, and MB2/2) were divided by those of noninhibited isoforms (MM1, MM2/2, MB1, MB2/2, and BB) to calculate the isoform ratio. In the reperfused group the increase in the isoform ratio was 2.69 +/- 1.80 (SD) 30 minutes after reperfusion and 2.41 +/- 2.01 at 60 minutes, which was significantly higher than the corresponding values in the nonreperfused group (0.17 +/- 0.16 and 0.32 +/- 0.26, respectively). When an increase of 0.70 or more in the isoform ratio was used as the criterion for reperfusion, the sensitivity and specificity were 92% and 100% at 30 minutes and 100% and 100% at 60 minutes after recanalization, respectively. We conclude that the isoform ratio obtained by the new 10-minute assay of CK isoforms is useful for the noninvasive detection of reperfusion 30 and 60 minutes after recanalization in acute myocardial infarction.

Angioplasty, Balloon, Coronary↗
PubMed1994

Elevation of the creatine kinase myocardial isoform following otherwise successful directional coronary atherectomy and stenting.

Moderate elevation of creatine kinase (CK) MB isoform is common following otherwise successful percutaneous coronary revascularization, and is frequently interpreted as evidence of a non-Q-wave myocardial infarction. It is not clear, however, whether elevation of CK MB isoform carries sufficient adverse clinical impact to be categorized as a "major" complication. We therefore explored the incidence and clinical consequence of elevation of CK MB isoform in a consecutive series of 565 patients who had otherwise successful directional coronary atherectomy (n = 274) or stenting (n = 291), and were followed for a mean of 2 years. Of this cohort, 11.5% had postprocedure elevation of the CK MB isoform above normal (10 IU/liter). These patients tended to be older and to have undergone atherectomy of a de novo lesion with adverse morphology (thrombus, calcification, eccentricity). Patients with elevation of CK MB isoform following otherwise successful revascularization generally showed no adverse long-term sequelae (death, recurrent myocardial infarction, repeat revascularization) compared with patients without elevation of CK MB isoform. Only 2.3% of the patients who had CK MB isoform release > 50 IU/liter demonstrated a trend (p = 0.08) toward decreased late survival, compared with patients without CK MB isoform elevation. While minor CK MB isoform elevation is common (11.5%) after successful coronary stenting or directional atherectomy, it generally has no adverse clinical consequences, and should not be considered a major complication.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1984

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms.

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.

Amino Acids↗