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PubMed1990

Myosin isoforms and cell heterogeneity in vascular smooth muscle. I. Developing and adult bovine aorta.

Monoclonal anti-smooth muscle (SM-E7, SM-F11, and BF-48) and anti-nonmuscle (NM-A9 and NM-G2) myosin antibodies, Western blotting, and immunocytochemical procedures were used to study myosin isoform composition and distribution in the smooth muscle (SM) cells of bovine aorta differentiating in vivo and in vitro. Two myosin heavy chain (MHC) isoforms were identified by SM-E7 in adult aorta: SM-MHC-1 (Mr = 205 kDa) and SM-MHC-2 (Mr = 200 kDa), respectively. When tested with the SM-F11 antibody, SM-MHC-2 isoform showed distinct antigenic properties compared to SM-MHC-1. Two bands of 205 and 200 kDa were also present in the aortic SM tissue from 3-month-old fetus and were equally recognized by the BF-48 antibody. The 200-kDa SM myosin isoform was labeled by SM-F11 but not by SM-E7, thus indicating the existence of a fetal-specific SM-MHC-2 isoform. At the cellular level, both developing and adult bovine aortic tissues showed the existence of distinct patterns of myosin isoform expression. Three or even more aortic cell populations are differently distributed in areas which appear as (1) a network of interconnecting sheet-like or compact tissue (early fetus) and (2) enriched of collagenous-elastic or muscular tissue (adult animal). In addition, the SM-MHC-2 isoform of the fetal type appears to be uniquely distributed in cultured SM cells grown in vitro from adult bovine aortic explants. Our data indicate that in bovine aorta (1) MHC isoform expression is developmentally regulated and (2) the distribution of myosin isoforms is heterogenous both among and within aortic cells. These findings may be related to the distinct physiological properties displayed by SM during vascular myogenesis.

Animals↗
PubMed1992

Contractile protein isoforms in muscle development.

The contractile proteins of skeletal muscle are often represented by families of very similar isoforms. Protein isoforms can result from the differential expression of multigene families or from multiple transcripts from a single gene via alternative splicing. In many cases the regulatory mechanisms that determine the accumulation of specific isoforms via alternative splicing or differential gene expression are being unraveled. However, the functional significance of expressing different proteins during muscle development remains a key issue that has not been resolved. It is widely believed that distinct isoforms within a family are uniquely adapted to muscles with different physiological properties, since separate isoform families are often coordinately regulated within functionally distinct muscle fiber types. It is also possible that different isoforms are functionally indistinguishable and represent an inherent genetic redundancy among critically important muscle proteins. The goal of this review is to assess the evidence that muscle proteins which exist as different isoforms in developing and mature skeletal and cardiac muscles are functionally unique. Since regulation of both transcription and alternative splicing within multigene families may also be an important factor determining the accumulation of specific protein isoforms, evidence that genetic regulation rather than protein coding information provides the functional basis of isoform diversity is also examined.

Actins↗
PubMed1994

Expression of the beta-subunit isoforms of the Na,K-ATPase in rat embryo tissues, inner ear and choroid plexus.

We report evidence of the apical localization of the two Na,K-ATPase beta-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na,K-ATPase beta 1 and beta 2 subunits. These polyclonal rabbit antisera were raised against truncated beta-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the beta 1 isoform and five bands in the beta 2 isoform immunoblots. In E15 rat embryos, the beta 1 isoform was detected in brain, heart and kidney and the beta 2 isoform only in brain. While beta-subunit mRNA expression (Watts AG, Sanchez-Watts G, Emanuel JR, Levenson R (1991) Proc Natl Acad Sci USA 88, 7425-7429), immunoblotting and enzymatic activity have been determined (Zlokovic BV< mackic JB, Wang L, McComb JG, McDonough A (1993) J Biol Chem 268, 8019-8025), very little is known about the specific localization of each beta-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the beta 1 and beta 2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus.

Animals↗
PubMed1993

Four isoforms of tyrosine hydroxylase are expressed in human brain.

In contrast to nonprimate species, the RNA for human tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human tyrosine hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human tyrosine hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human tyrosine hydroxylase protein forms to determine the presence and distribution of each human tyrosine hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human tyrosine hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human tyrosine hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human tyrosine hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human tyrosine hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human tyrosine hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.

Adrenal Medulla↗
PubMed1994

Identification of novel alternatively spliced isoforms of the tropomyosin-encoding gene, TMnm, in the rat cochlea.

Analysis of a rat cochlear cDNA library for the expression of the non-muscle (nm) tropomyosin (TM)-encoding gene (TMnm), demonstrated that four nm isoforms were present. These four TMnm variants are NM-1, NM-2, NM-3 and NM-4. Nucleotide (nt) sequencing revealed that all these isoforms expressed the nm exon 1b sequence, but varied in their usage of the alternatively spliced exons 6a and b and 9a/b and d, representing skeletal muscle (sk) (exons 6b and 9a/b) and nm (6a and 9d) sequences. A novel exon 9 (designated as exon 9c) was associated with the NM-4 isoform and was also found to be expressed in the cochlea. Comparisons of the nt and amino acid (aa) sequences demonstrated a high homology between rat, mouse and human sequences encoding the 'classical' nm isoform, TM30nm, which is designated herein as NM-1. The rat NM-1 nt homology with mouse and human sequences also included the 3' untranslated region. Homologies were found between aa sequences of the C termini of NM-1 and the TM alpha smooth muscle and TM beta nm isoforms, between the sk sequence of NM-3 and the TM alpha and beta sk isoforms, and between the novel 9c sequence of NM-4 and the TM alpha brain-1 isoform. These data predict that the nm isoforms share biochemical properties described for the other TM isoforms.

Alternative Splicing↗
PubMed2004

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme.

Two isoforms of ferredoxin-NADP(+) reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure-function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP(+) with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k(cat)/K(m)), as an NADPH-dependent diaphorase, higher than those of both the root ( approximately 2-fold) and the leaf enzyme ( approximately 5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.

Catalysis↗
PubMed1997

Overexpression of alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells -- H-7 promotes neurite outgrowth.

Since the alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain, we compared the effect of overexpression of the beta isoform of CaM kinase II with that of the alpha isoform. The subcellular distribution of the alpha isoform was different from that of the beta isoform, although the catalytic properties of the alpha and beta isoforms expressed in transfected cells were similar to those of brain CaM kinase II. The alpha isoform was found in the soluble fraction more than in the particulate fraction, whereas most of the beta isoform bound to subcellular structures. In the cell overexpressing alpha and beta isoforms of CaM kinase II, neurite extension was promoted when compared with the morphology of neo transfectants. Neurite outgrowth of cells overexpressing CaM kinase II was further stimulated by the treatment of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a selective but not absolutely specific inhibitor of protein kinase C. The morphological change was rapid and observed within 1 h followed by H-7 treatment. Morphological changes, such as the number of cells with neurites and length of neurites were greater in the beta cells than in the alpha cells. Chelerythrine, a specific inhibitor of protein kinase C, also stimulated the neurite outgrowth of these cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing CaM kinase II stimulated with H-7. These results suggest that CaM kinase H and protein kinase C play an important role in the control of cell change, and that the subcellular distribution of CaM kinase II is important for regulating cellular functions efficiently.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Regional differences between the immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II alpha and beta isoforms in the brainstem of the rat.

The distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) alpha and beta isoforms in the brainstem of adult rats was investigated using an immunohistochemical method with two monoclonal antibodies which specifically recognize the alpha and beta isoform, respectively. We found that these isoforms were differentially expressed by neurons in the substantia nigra, red nucleus, dorsal cochlear nucleus, pontine nuclei and inferior olivary nucleus. Neurons in the inferior olivary nucleus express the alpha isoform, but not the beta isoform. In contrast, neurons in the substantia nigra, red nucleus and pontine nuclei were immunostained with the beta antibody, but not with the alpha antibody. In the dorsal cochlear nucleus, neurons in layers I and II were alpha-immunopositive, whereas neurons in layers III and IV were beta-immunopositive. Therefore, the distribution of the CaM kinase II alpha-immunopositive neurons is completely different from that of CaM kinase II beta-immunopositive neurons. Next we examined the possible coexistence of CaM kinase II alpha isoform and glutamate or that of CaM kinase II beta isoform and glutamic acid decarboxylase (GAD) in the single neuron by double immunofluorescence labelling using a pair of anti-alpha and anti-glutamate antibodies, or a pair of anti-beta and anti-GAD antibodies. The results indicated that neurons expressing anti-alpha immunoreactivity were also immunopositive against anti-glutamate antibody, and neurons expressing beta isoform were also immunopositive against anti-GAD antibody, suggesting that alpha-immunopositive neurons are classified as excitatory-type neurons, and on the contrary, beta-immunopositive neurons are classified as inhibitory-type neurons. In conclusion, the present study confirmed that alpha- and beta-isoforms of CaM kinase II are differentially expressed in the nuclei of the brainstem and have different roles.

Animals↗
PubMed1998

mRNA encoding four isoforms of the plasma membrane calcium pump and their variants in rat kidney and nephron segments.

To survey the presence of the four different isoforms of the plasma membrane calcium pump (PMCA) and their alternative splicing variants in the rat kidney, three major zones (cortex, outer medulla, and inner medulla) were macrodissected and probed for the presence of mRNA encoding these isoforms and their variants at the splicing site C by using reverse transcription-polymerase chain reaction (RT-PCR). Both the cortex and the outer medulla showed PMCA 1b, 2b, 3(a and c), and 4b. Semiquantitative comparisons indicated that isoform 2b is more abundant in the cortex than in the outer medulla and conversely, that isoform 3 (a and c) is more abundant in the outer medulla than in the cortex. The inner medulla showed only mRNA for isoforms 1b and 4b. The nephron segments present in the cortex and outer medulla were microdissected and analyzed by RT-PCR. Isoforms 1b, 2b, and 4b were found in all nephron segments but were found more frequently in tubular segments with high rates of Ca2+ reabsorption, suggesting that these isoforms may be involved in transepithelial transport. On the other hand, mRNA encoding isoform 3 (a and c) was most abundant in descending thin limb of Henle but was detected also in glomeruli and cortical thin ascending limb. Its distinct localization suggests that this isoform might have another function, such as in intracellular signalling.

Alternative Splicing↗
PubMed1998

Leptin receptor isoforms expressed in human adipose tissue.

Leptin and its structural gene, Ob, are exclusively expressed in adipose tissue. Leptin is secreted into the blood and is responsible for fat mass regulation via leptin receptors in the hypothalamus. This has been considered the major role of leptin, but leptin receptor isoforms are expressed not only in the brain but also in most other tissues in humans and rodents: heart, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, and colon. This implicates leptin regulation in other systems apart from fat mass regulation, and leptin action has been demonstrated in human fetal development and reproductive development, liver metabolism, hematopoiesis, and insulin secretion. Four splice variants of the leptin receptor have been identified in humans: the long isoform huOb-R and the shorter isoforms B219.1 to B219.3. It is known that the long isoform has full intracellular signaling capacity, and is responsible for anorectic action in the hypothalamus. The roles of the other isoforms are yet to be elucidated. Here, we report the identification by reverse transcriptase-polymerase chain reaction (RT-PCR) of three leptin receptor isoforms coexpressed in human visceral adipose tissue: the long isoform huOb-R and the short isoforms huB219.1 and huB219.3. The possible roles of these isoforms are discussed.

Adipose Tissue↗
PubMed1997

The multiple murine 3 beta-hydroxysteroid dehydrogenase isoforms: structure, function, and tissue- and developmentally specific expression.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. To date five distinct isoforms have been identified in the mouse. The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated. The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups. 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases. These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones. In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads. 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver. 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty. In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform. Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally. Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development. The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3. Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert. Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod Helix pomatia.

A quantitative assay based on real-time detection polymerase chain reaction (rtdPCR) was applied to analyze basal and metal-induced mRNA levels of two metallothionein (MT) isoforms (Cd-MT and Cu-MT) in organs of the terrestrial gastropod Helix pomatia. The results show that specific Cd-MT mRNA levels increase with Cd tissue burden, identifying hepatopancreas and gut as the main organs of Cd accumulation and, accordingly, the predominant organs of Cd-MT mRNA expression. In situ hybridization localized this isoform in epithelial cells of hepatopancreas, gut, and kidney. In contrast to the observed Cd-dependent inducibility of the Cd-binding MT isoform, gene expression of the Cu-binding MT could not be induced by either Cd or Cu exposure. Only very low mRNA amounts of the Cu-MT isoform were found in snail hepatopancreas and kidney, whereas the mantle exhibited high basal mRNA levels of this isoform. In situ localization revealed that the Cu-MT gene expression was restricted to one cell type, the so-called rhogocytes, which are present to various extents in the different organs examined. These results suggest a metal-specific sharing of functions between the two MT isoforms. The Cd-MT isoform apparently plays a crucial role in Cd detoxification, as demonstrated by the inducibility of this isoform, as well as its specific localization in the main metabolic and Cd storing organs. The predominant presence of Cu-MT in rhogocytes of snail mantle strengthens the hypothesis that this isoform may regulate Cu availability in hemocyanin synthesis.

Animals↗
PubMed1998

AH receptor-controlled transcriptional regulation and function of rat and human UDP-glucuronosyltransferase isoforms.

Transcriptional regulation and function of rat and human PAH-inducible UDP-glucuronosyltransferase (UGT) isoforms have been studied. 1. At least two PAH-inducible UGT isoforms are expressed in a variety of tissues, the rat isoforms UGT1A6 and UGT1A7, and the human isoforms UGT1A6 and UGT1A9. 2. For the rat and human UGT1A6 isoforms two modes of tissue- and cell-specific regulation were found, PAH-inducible and constitutive expression. 3. Transient transfection studies, using human UGT1A6/CAT fusion constructs and colon carcinoma Caco-2 cells, revealed that PAH induction of human UGT1A6 is mediated by the Ah receptor. 4. Cell-expressed UGT isoforms were used to study their function in PAH metabolism. Rat UGT1A7 and human UGT1A9 appear to be more efficient than the corresponding UGT1A6 isoforms in catalyzing glucuronide formation of PAH phenols and diphenols. Several isoforms may act together in the formation of benzo(a)pyrene-3.6-diol diglucuronide, the major glucuronide found in rat bile. The results suggest complex modes of transcriptional regulation of PAH-inducible UGTs. They also suggest a major role of these UGT isoforms in detoxication of PAHs.

Animals↗
PubMed1997

Developmental changes in regulation of the Na+, K(+)-ATPase alpha 3 isoform by thyroid hormone in ferret heart.

Ferret heart expresses the alpha 1- as well as the alpha 3-isoform of the Na+, K(+)-ATPase. We have shown previously that the alpha 3 isoform is differentially upregulated during postnatal cardiac development and that in adult ferrets expression of alpha 3 is not responsive to regulation by thyroid hormone (TH). Since developmental-stage dependent effects of TH have been reported previously, the present study examined whether effects of TH on expression of the Na+, K(+)-ATPase isoforms in ferret heart is modulated during development and possible mechanisms were examined. Ferrets of different age groups were treated with TH and the relative abundance of Na+, K(+)-ATPase isoforms in ferret myocardium was determined by immunoblotting. Thyroid hormone (T3; 50 micrograms/100 g body weight on 3 alternating days, s.c.) increased protein levels of the alpha 3 isoform, but not that of alpha 1 or beta 1, in myocardium of 5-day-old and 3-week-old ferrets. By contrast, in myocardium of 6- and 8-week-old ferrets T3 failed to increase protein levels of alpha 1 and alpha 3. To determine whether elevated plasma levels of TH during development plays a role in the transition, mature ferrets were first made hypothyroid before TH treatment. In these hypothyroid ferrets expression of the alpha 3 isoform remained unresponsive to TH (T4, 0.5 mg/kg for 7 days, s.c.). The transition from TH-responsive to TH-unresponsive appears to be isoform-specific because in skeletal muscle of 8-week-old ferrets and in hypothyroid ferrets the alpha 2 isoform is upregulated by TH. Finally, there appears to be functional thyroid hormone receptors throughout development because in each age group TH effectively induced expression of alpha-MHC in the myocardium. In conclusion, these findings demonstrate that expression of alpha 3 isoform in the myocardium of newborn ferret is responsive to TH; however, the responsiveness terminates between 3- and 6-weeks of age. Neither elevated endogenous TH level nor a lack of functional thyroid hormone receptor appears to be responsible for the transition from TH-responsive to TH-unresponsive.

Animals↗
PubMed2000

Induction and alternative splicing of delta isoform of Ca(2+)/calmodulin-dependent protein kinase II during neural differentiation of P19 embryonal carcinoma cells and during brain development.

Since the expression of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) is regulated during brain development, the developmental change of the enzyme was investigated during the neural differentiation of murine P19 embryonal carcinoma cells. CaM kinase II activity was induced during the differentiation of P19 cells treated with retinoic acid. Expression of the enzyme was induced 2 days after the treatment and maximized at 5 days. The enzyme activity increased about approximately 8-fold. The enzyme protein was shown to differ between differentiated and undifferentiated cells. The delta isoform of CaM kinase II was found as the major isoform in P19 cells by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). A total of four and three alternatively spliced variants of delta isoform were detected in P19 cells by RT-PCR analysis and by immunoblotting, respectively. Although multiple alternatively spliced forms have been reported, the major splice variants of delta isoform in differentiated cells were delta l and delta 9 isoforms, which were specifically detected in differentiated cells. In undifferentiated cells, the major splice variant corresponded to delta 2 isoform. These results indicated that the expression of delta isoform of CaM kinase II was induced, and the splicing pattern of the isoform changed, during neural differentiation. Cell type distinctive changes of splicing pattern of delta isoform were also observed not only during differentiation of cultured neuronal cells, but also during development of rat forebrain and cerebellum.

Alternative Splicing↗
PubMed1998

Over-expression of the 18 kD and 21/23 kD fibroblast growth factor-2 isoforms in PC12 cells and Schwann cells results in altered cell morphology and growth.

Basic fibroblast growth factor (FGF-2) occurs in different isoforms which represent alternative translation products from a single mRNA. The question of whether the presence of multiple FGF-2 isoforms has physiological implications is compelling but unresolved so far. However, it has been shown recently that the FGF-2 isoforms are differentially regulated in sensory ganglia and peripheral nerve following nerve injury and, moreover, in the adrenal medulla during postnatal development and after hormonal stimuli suggesting that the isoforms may serve different physiological functions. To investigate isoform-specific effects we have established immortalized Schwann cells and PC12 cells stably over-expressing the 18 kD and the HMW isoforms. We found that the over-expression of the different isoforms alters morphology and growth of the Schwann cells. PC12 cells over-expressing the 18 kD FGF-2 were found to differentiate towards the neuronal phenotype whereas over-expression of the HMW isoforms resulted in a stabilization of the endocrine phenotype. Taken together, these data corroborate the idea of FGF-2 isoform-specific functions.

Animals↗
PubMed2000

Clinical utility of CKMB isoform determinations in patients who present to the emergency department with continuous or resolved chest pain.

The object of this investigation was to demonstrate that the enhanced sensitivity of the diagnosis of acute myocardial infarction (AMI) using a more-inclusive criterion of CKMB Isoforms may detect earlier stages of AMI (designated Isoform Type 2) than the currently accepted marker for AMI by CKMB Isoform (designated Isoform Type 1) in a busy, urban Emergency Department (ED). Two features characterized the study of CKMB Isoforms in a prospective cohort of 223 ED patients: first, nontraumatic chest pain within 12 h before presentation, thought to be of ischemic etiology; and second, normal or nondiagnostic electrocardiogram (EKG). Patients were further divided into two groups characterized as either recent but resolved chest pain at ED visit, or ongoing or staccato chest pain. Sensitivity (S), specificity (SP), positive (PPV) and negative (NPV) predictive values, and 95% confidence intervals (CI) for AMI diagnosis were determined. Two criteria for AMI diagnosis by CKMB Isoforms were tested. The first and currently recommended criterion was identified as Isoform Type 1. An AMI diagnosis by Type 1 criterion requires both CKMB2> or =2.6 IU/L and CKMB2/CKMB1> or =1.7. The second criterion for AMI diagnosis was identified as Isoform Type 2, which is defined as either CKMB2> or =2.6 IU/L or CKMB2/CKMB1> or =1.7. Both Isoform types are predictive of AMI by the gold standard, and addition of EKG changes results in a small improvement. Type 1 demonstrates SP 0.94 (CI 0.90, 0.97) and NPV 0.90 (CI 0.86, 0.94), and Type 2 demonstrates S 0.90 (CI 0.80, 0.97) and NPV 0.97 (CI 0.93, 0.99) for AMI diagnosis. Type 2 criteria can confidently exclude the immediate risk of AMI in patients with resolved chest pain whereas in patients with continuous chest pain, Type 1 criteria may identify those at high risk for AMI.

Clinical Enzyme Tests↗
PubMed1997

Limited specificity and large overlap of the functions of the mouse RAR gamma 1 and RAR gamma 2 isoforms.

The RAR gamma gene generates two major isoforms, RAR gamma 1 and RAR gamma 2, which originate from two distinct promoters. We report here the engineering of mice lacking RAR gamma 1, but in which RAR gamma 2 is normally expressed. The effect of this null mutation has been compared with those previously described for RAR gamma 2 and all RAR gamma isoforms (total RAR gamma gene inactivation), both in single mutants and in double mutants bearing additional null mutations in their RAR alpha, RAR beta or RXR alpha genes. RAR gamma 1 mutants, but not RAR gamma 2 mutants, displayed a subset of the abnormalities exhibited by total RAR gamma null mutants (growth deficiency, abnormal cricoid cartilage and occasional cervical vertebra defects), suggesting that RAR gamma 1 is the main isoform mediating the corresponding RAR gamma functions. Interestingly, cricoid cartilage defects were also found in a fraction of heterozygote animals for the RAR gamma 1, RAR gamma or RAR alpha mutations, indicating that wild type levels of RARs are required for the normal morphogenesis of this structure. Compound RAR alpha/RAR gamma 1 and RAR alpha/RAR gamma 2 double null mutants exhibited only a small fraction of the defects found in RAR alpha/RAR gamma double null mutants. Moreover, these defects were often partially penetrant, or corresponded to a less severe form. However, they occurred preferentially in certain compound mutants, demonstrating that given isoforms mediate specific functions of RAR gamma in the context of a RAR alpha null background. In a RXR alpha null background, both RAR gamma 1 and gamma 2 isoform mutations resulted in increased severity of the RXR alpha null ocular phenotype. Together, the present observations indicate that the functions of the two RAR gamma isoforms overlap to a large extent, but also that each of these isoforms exhibits a limited functional specificity. Furthermore, the occurrence of morphological defects in heterozygote mutants for a single RAR isoform provides a basis for explaining the strong conservation of these isoforms during vertebrate evolution.

Animals↗