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PubMed1996

Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.

We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.

Aging↗

PubMed1989

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle.

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.

Age Factors↗

PubMed1991

Multiple isoforms of the mouse retinoic acid receptor alpha are generated by alternative splicing and differential induction by retinoic acid.

Together with the previously described mouse retinoic acid receptor alpha-1 (mRAR-alpha 1, formerly mRAR-alpha 0), we have isolated and characterized here a total of seven mRAR-alpha cDNA isoforms (mRAR-alpha 1 to alpha 7). These isoforms are generated from mRAR-alpha primary transcript(s) of a single gene by alternative splicing of at least eight different exons with the exon which encodes the amino acid sequence of their common B region. All of these isoforms differ in their 5'-untranslated regions (5'-UTRs) and, in the case of mRAR-alpha 1 and alpha 2, also in the sequences encoding the N-terminal A region which is known to be important for differential trans-activation by other members of the nuclear receptor superfamily. In addition, the sequences encoding the open reading frames (ORFs) of mRAR-alpha 3 and alpha 4 cDNA isoforms remain open to their very 5' ends, which suggests that these two isoforms may also encode RAR-alpha s with unique A region amino acid sequences. The two predominant isoforms, mRAR-alpha 1 and alpha 2, were found to be differentially expressed in mouse adult and fetal tissues, as well as in P19 and F9 embryonal carcinoma (EC) cell lines. Interestingly, the expression of mRAR-alpha 2, in contrast to that of the mRAR-alpha 1 isoform, was induced by retinoic acid (RA) in EC cells, thus suggesting the presence of two promoters in the 5' region of the mRAR-alpha gene, which differ in their response to RA. The conservation between mouse and human RAR-alpha 1 and alpha 2 cDNA isoform sequences, as seen by cross-hybridization in Southern blots or by DNA sequence analysis, together with their differential patterns of expression, strongly suggests that they perform specific functions during embryogenesis and in the adult.

Amino Acid Sequence↗

PubMed1994

Expression of activin receptor II and IIB mRNA isoforms in mouse reproductive organs and oocytes.

Activins, the dimeric polypeptides of inhibin beta-subunits, exhibit paracrine effects on cell proliferation, differentiation, and various other cell functions. The complex biological response to activin appears to involve multiple receptors. In the present study, we examined the isoform mRNA expression of both activin receptor type II (ActR-II) and type IIB (ActR-IIB) genes in mouse reproductive organs, cumulus-oocyte complexes (COCs), and ovulated oocytes. Northern blot analyses of female and male reproductive organs with single-stranded ActR-II cDNA probes revealed that mouse ovaries expressed high levels of the 6.0 kilobase (kb) mRNA, whereas the 3.0 kb transcript was the major mRNA species found in the testis. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that both COCs and oocytes contained ActR-II mRNA. To examine the expression of ActR-IIB gene, primer selection was made outside the two alternative splicing sites in order to amplify the cDNAs of all four distinct receptor isoforms. The results of RT-PCR demonstrated that isoforms IIB2 and IIB4 were the major mRNA species expressed in both female and male gonads and extragonal reproductive tissues. The ovary expressed all four mRNA isoforms, whereas the testes expressed only three isoforms. whereas the testes expressed only three isoforms. Furthermore, COCs and oocytes contained only the ActR-IIB2 isoform. The differential expression of both activin receptor mRNA isoforms in the reproductive organs suggests that distinct alternative splicing mechanisms are involved in activin receptor gene expression in male and female gonads, and that each of the activin receptors may have its own biological function in reproduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Activin Receptors↗

PubMed1994

The plasma membrane calcium pump: functional domains, regulation of the activity, and tissue specificity of isoform expression.

The plasma membrane Ca2+ pump is responsible for the fine regulation of the intracellular Ca2+ level and is thus involved in the control of several cellular processes. The activity of the pump is regulated by a multiplicity of mechanisms, among which are calmodulin, acidic phospholipids, kinase-mediated phosphorylation, or an oligomerization process. The C-terminal part of the molecule interacts with the region of the pump close to the active site, leading to the decrease of the activity in the resting state. Four genes coding for different isoforms of the plasma membrane Ca2+ ATPase are known in humans. Isoform 1 and 4 represent housekeeping isoforms, whereas isoforms 2 and 3 are only present in specialized tissues. The variability of the protein is further increased by alternative RNA splicing at two sites (A, C). Alternative splicing occurs within (splice site C) or near (splice site A) regions coding for regulatory domains of the protein. In all isoforms a corresponding splice form exists at both splice sites. These common splice forms are present in all tissues, whereas isoform unique splice forms are normally only present in specialized tissues. In neuronal tissues all isoforms and almost the complete set of splice forms are found. The transcripts of the different isoforms are distributed in a region-specific manner in neuronal tissues.

Animals↗

PubMed1997

Parallel detection of Na,K-ATPase alpha subunit isoforms by pan-specific monoclonal mAb 9A7.

While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEnery et al., 1991, Proc. Natl. Acad. Sci. 88, 11095-11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.

Animals↗

PubMed1996

A switch in broad-complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs.

The Broad-Complex (BR-C) is a key member of the 20-hydroxyecdysone regulatory hierarchy that coordinates changes in gene expression during Drosophila metamorphosis. The family of transcription factors encoded by the BR-C share a common amino-terminal domain which is fused by alternative splicing to one of four pairs of C2H2 zinc-finger domains (Z1, Z2, Z3, and Z4). In this study, we examine the temporal expression of transcripts encoding each BR-C zinc-finger isoform-including the newly discovered fourth zinc-finger domain-during the metamorphosis of imaginal discs which form the integumental structures of the adult head and thorax. We find that all BR-C zinc-finger RNA isoforms are induced as a primary response to 20-hydroxyecdysone. However, induced BR-C RNA isoforms exhibit two divergent expression profiles. The Z2, Z3, and Z4 RNA isoforms accumulate to high levels at the beginning of the ecdysone response and abruptly disappear after several hours. In contrast, the Z1 RNA isoform continues to accumulate while the others decline, resulting in a switch in relative isoform levels. Using probes specific to different regions of the BR-C, we show that the switch in BR-C RNA isoform expression appears to be posttranscriptionally regulated, presumably by ecdysone-responsive factors. We propose that this switch results from a change in splice acceptor site choice. Finally, we present a model describing how this temporal switch in isoform expression could mediate changes in BR-C function, from transcriptional activation to repression and vice versa, that are critical for coordinate downstream target gene expression.

Amino Acid Sequence↗

PubMed1996

Effect of phorbol 12, 13-dibutyrate on ligand binding, enzyme activity and translocation of protein kinase C isoforms in the alpha T3-1 gonadotrope-derived cell line.

The effect of incubating alpha T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly alpha, epsilon and zeta isoforms) was assessed by immunoblotting, enzyme activity assay and [3H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC alpha and PKC epsilon had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC zeta was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca2+) indicated that translocation of PKC alpha and epsilon was very rapid-detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC zeta in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight ("authentic') PKC zeta immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC zeta secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC epsilon moved more rapidly than PKC zeta which appeared to translocate more quickly than PKC alpha. After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC zeta was not translocated by LHRH.

Animals↗

PubMed1994

CD44 variant isoforms are preferentially expressed in basal epithelial of non-malignant human fetal and adult tissues.

CD44 is a transmembrane glycoprotein, which can exist in a multitude of isoforms due to alternative splicing of the pre-mRNA. We have generated monoclonal antibodies to several of these variant regions, which are encoded by 10 additional exons in the extracellular part of the molecule. CD44 variant isoforms have been reported to be involved in the malignant progression of rat and human tumours. The precise localization of CD44 variant isoforms in normal developmental and morphogenetic processes is essential for diagnostic studies of human tumorigenesis. Therefore, we have analysed a large number of different human tissues by immunohistochemistry for the expression of CD44 isoforms containing either exons 4v, 6v or 9v. Expression of exon 9v-isoforms was detected in almost all epithelia analysed, with a few exceptions. Exon 6v isoforms are expressed only in squamous and glandular epithelial, e.g. skin epidermis, sweat and sebaceous glands, oesophagus, ducts of the mammary gland, salivary and prostate glands. Detection of exon 4v-encoded isoforms was restricted to the epidermis and the oesophagus. Similar tissue distributions of CD44 variant isoforms were observed in 10-week-old fetal tissues. Since one of the ligands of CD44 is hyaluronic acid (HA), we also analysed the tissue distribution of HA synthetase. HA synthetase was detected in all tissues analysed, showing good correlation with the expression of the standard form of CD44, CD44s.

Adult↗

PubMed1992

Histological distribution and developmental changes of tropomyosin isoforms in three chicken digestive organs.

Histological localization of tropomyosin isoforms in three digestive organs from embryonic and adult chickens was performed by using rabbit antisera against chicken skeletal muscle tropomyosin and against low-Mr-type tropomyosin from chicken small intestine mucosa. The former antiserum (named TM-SH) reacted with alpha, beta, and high-Mr-type isoforms, and the latter (named TM-HL) reacted with alpha, beta, high-Mr-type and low-Mr-type isoforms, alpha and beta Isoforms were detected in muscle cells of the muscular layer and the muscularis mucosa. Low-Mr-type isoforms, however, were detected along the cell membrane and cytoplasm of almost all nonmuscle cells, especially in terminal webs of epithelial cells. Developmental changes of tropomyosin isoforms in digestive organs were studied by two-dimensional gel electrophoresis and image analysis. The relative amounts of alpha and beta isoforms increased in the course of development, but those of low-Mr-type and high-Mr-type isoforms decreased.

Animals↗

PubMed1992

Na,K-ATPase: isoform structure, function, and expression.

An interesting feature of the Na,K-ATPase is the multiplicity of alpha and beta isoforms. Three isoforms exist for the alpha subunit, alpha 1, alpha 2, and alpha 3, as well for the beta subunit, beta 1, beta 2, and beta 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the alpha subunit are present in the brain, while only alpha 1 is present in kidney and lung, and alpha 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na+ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell. Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the alpha subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1999

Characterization of the fourth alpha isoform of the Na,K-ATPase.

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue-specific isoforms exist for each of these, alpha1, alpha2 and alpha3 and beta1, beta2 and beta3. We have proposed that an additional alpha isoform, alpha4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative alpha4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in alpha4 isoform cDNA transfected 3T3 cells. Using an alpha4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.

3T3 Cells↗

PubMed2002

Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that B1 and B2 have more (or more reactive) sialic acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

Alkaline Phosphatase↗

PubMed2003

Protein kinase C isoforms in the enteric nervous system.

C kinases (PKCs) are a family of enzymes essential for the transduction of signals in a diverse range of cell types, including neurons. The different isoforms vary in their activation requirements. Therefore, cell-specific expression of different isoforms has implications for PKC-mediated control of organ function. This study has investigated the types and distributions of PKC isoforms in the small intestine of the guinea-pig, with particular emphasis on their localisation in myenteric neurons, using immunohistochemistry and western blotting techniques. Three PKC isoforms, gamma, eta and theta, were detected in the calbindin-immunoreactive subset of intrinsic primary afferent neurons, but not in other myenteric neurons. Both gamma and theta immunoreactivities were also located in interstitial cells of Cajal. In contrast to these isoforms, immunoreactivity for PKCs lambda and epsilon was present in all myenteric neurons of the ileum. PKCalpha immunoreactivity was detected primarily in the glial network, as shown through double labelling with antibodies to the glial filament protein, S100b. Myenteric neurons were also weakly immunoreactive for this isoform. PKCdelta immunoreactivity was very highly expressed in smooth muscle, but was largely absent from neurons. Immunoreactivity for RACK1, a binding protein for PKCbeta, was detected in both calbindin-immunoreactive neurons and in smooth muscle cells. This study indicates a selective distribution of PKC isoforms to specific cell types. Isoform-specific activity of these enzymes could provide a means through which targeted modulation of intestinal function is achieved.

Animals↗

PubMed2006

In silico assessment of gene function involved in cysteine biosynthesis in Arabidopsis: expression analysis of multiple isoforms of serine acetyltransferase.

In plants, the inorganic sulfur is first fixed into cysteine by the cysteine biosynthetic pathway. This biosynthetic pathway of cysteine involves several enzymatic reactions. In Arabidopsis thaliana, multiple isoforms seem to participate in each enzymatic step for cysteine biosynthesis. To obtain more insights on the specific role of each isoform involved in the cysteine biosynthesis, in silico analysis of these isoforms using Arabidopsis expressed sequence tags (EST) database was carried out. This EST database analysis revealed distinct population distribution of ESTs among multiple isoforms, suggesting that each isoform has its particular expression pattern, presumably associated with its specific role in cysteine biosynthesis. As another in silico analysis, co-expression analysis of genes involved in sulfur metabolism in Arabidopsis was performed using a public transcriptome database of DNA microarrays. This co-expression analysis also suggested specific function and co-regulation of some isoform genes for cysteine biosynthesis by consideration on the clustering of co-expressed genes. From the results of sensitivity to feedback regulation, subcellular localization and expression of mRNA analyses, each serine acetyltransferase (SATase) isoform seems to have its specific role for cysteine biosynthesis. Similar expression patterns were observed between the experimental results of expression data for SATase isoforms and the in silico results of "digital northern" analysis using EST database.

Arabidopsis↗

PubMed1991

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle.

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.

Actins↗

PubMed1996

Selective pharmacological inhibition of distinct nitric oxide synthase isoforms.

Nitric oxide (NO) is produced in physiological and pathophysiological conditions by three distinct isoforms of NO synthase (NOS): endothelial NOS (ecNOS), inducible NOS (iNOS), and brain NOS (bNOS). Selective inhibition of iNOS may be beneficial in various forms of shock and inflammation, whereas inhibition of bNOS may protect against neuroinjury. This article surveys the enzymatic mechanism of NO production, lists the strategies and pharmacological tools for selective inhibition of distinct NOS isoforms, and considers the side-effects of the various approaches. Selective inhibition of NOS isoforms is achieved by: (a) targeting the differential co-factor (calmodulin or tetrahydrobiopterin) requirement of various NOS isoforms, and NOS; (b) targeting the differential substrate requirements of cells expressing various isoforms of NOS (L-arginine uptake blockers or arginase); (c) the use of pharmacological agents that are selectively taken up by cells expressing various isoforms of NOS (7-nitroindazole); or (d) developing pharmacological NOS inhibitors with isoform specificity. The amino acid-based NOS inhibitor, NG-nitro-L-arginine, shows a preference for ecNOS and bNOS over iNOS, whereas L-N6-(1-iminoethyl)lysine is selective for iNOS over bNOS. Certain non-amino acid-based small molecules, such as aminoguanidine and certain S-alkylated isothioureas, also express selectivity towards iNOS and have anti-inflammatory and anti-shock properties. 7-nitroindazole, a bNOS-selective inhibitor, protects in central nervous system injury. Clearly, there are a number of distinct approaches that are worthy of further research efforts in order to achieve even more selective targeting of various NOS isoforms

Animals↗

PubMed1992

Creatine kinase MB isoforms for early diagnosis and monitoring of acute myocardial infarction.

MB isoforms of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) in 848 sera obtained from 80 patients with acute myocardial infarction (AMI) were studied by agarose gel isoelectric focusing. In 173 sera (20%) from 25 patients (31%), a new isoform designated as MB3 (pI 5.4) was detected at the cathodal side of MB2 (pI 5.2) in addition to the previously known MB2 and MB1 (pI 5.1). The new isoform MB3 was found in the extract of the cardiac muscle. MB3 was dominant in the sera at an earlier stage and a shorter period of time after AMI: 1-14 h (range), 1-29.5 h and 4-154 h for MB3, MB2 and MB1 dominant, respectively. MB3 was therefore found to be an earlier and a shorter phase indicator for AMI than MB2 or MB1. However, MB2 greater than 1 was the most prevalent pattern at the time of admission to the hospital. In AMI, specificity was 96.2%, 92.4% and 90.6%, and sensitivity was 20.4%, 88.9% and 97.6%, for MB3, MB2 and MB1 isoforms, respectively. CK-MB isoform patterns were biased to MB1 dominant in the deceased group, and to MB2 dominant in the surviving group. Therefore determination of CK-MB isoforms is also useful in the course of observation of AMI. The fourth isoform, MB0 (pI 5.0), was detected at the anodal side of MB1. MB0 was a minor band of the CK-MB isoform which appeared when serum CK-MB activity increased.

Acute Disease↗