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PubMed1998

Expression of Na+,K(+)-ATPase alpha and beta subunit isoforms in the motor neurons of the rat spinal cord.

The data concerning the distribution of Na+,K(+)-ATPase alpha and beta subunit isoforms in the spinal cord and partly in the motor neurons of the ventral horns are limited. The lumbo-sacral portion of the spinal cord of adult rats was immunotested with polyclonal antibodies (UBI, NY) specific for alpha 1, alpha 2, alpha 3 and beta 1, beta 2 isoforms. After paraformaldehyde perfusion and postfixation, free-floating 50 microns thick vibratome sections were immunostained with Vectastatin Elite ABC. Sites of bound primary antibodies were visualized by incubation in DAB-H2O2 substrate medium. The histochemical technique revealed immunostaining for all five isoforms of Na+,K(+)-ATPase in the motor neurons. The findings show a principal similarity in the distribution pattern of the immuno-like reactivity for alpha 1 and alpha 2 isoforms, the staining of the pericarya being more or less continuous with that of the microenvironment. The immunostaining for beta 2 (in comparison with alpha 1 and alpha 2) outlines the pericarya of the motor neurons slightly better, whereas the staining for beta 1 outlines them extremely sharply. The immunostaining pattern for the alpha 3 isoform differs considerably from that for the other isoforms. The immuno-like reactivity for this isoform is concentrated at the surface of the pericarya and processes of the motor neurons. Accumulation of alpha 3 immunoreactivity on the surface of the motor neurons might reflect the intensive traffic of the alpha 3 isoform from the pericaryon to the plasma membrane and the processes of the neurons. The findings from the investigations performed here support the opinion, that, in addition to the conventional catalytic role in Na+,K(+)-ATPase activity, Na+,K(+)-ATPase isozymes play a part in different specific phenomena in the nervous system.

Animals↗
PubMed1999

Differential cellular and subcellular localization of protein phosphatase 1 isoforms in brain.

Protein phosphatase 1 (PP1) is a gene family with a number of important functions in brain. Association with a wide variety of regulatory/targeting subunits is thought to be instrumental in directing the phosphatase to specific subcellular locations and substrates. By using antibodies directed against specific PP1 isoforms, we asked whether PP1 isoforms are differentially distributed in brain. Immunoblotting detects in brain the PP1gamma2 isoform, which had previously been thought to be testis specific, in addition to alpha, beta, and gamma1 isoforms. PP1 isoform expression varies modestly in extracts from different subdissected brain regions and is relatively constant during postnatal development, except for an about twofold increase in PP1gamma2. By immunohistochemical analyses of rat brain, PP1beta and PP1gamma1 cellular expression is widespread but quite distinct from one another. Subcellular fractionation studies demonstrate that PP1beta and PP1gamma1 are selectively associated with different cytoskeletal elements: PP1beta with microtubules, PP1gamma1 with the actin cytoskeleton. Double-immunofluorescence labeling of cultured cortical neurons further reveals a strikingly different and nonoverlapping localization of PP1beta and PP1gamma1: whereas PP1beta localizes to a discrete area of the soma, PP1gamma1 is highly enriched in dendritic spines and presynaptic terminals of cultured neurons. These results show that PP1 isoforms are targeted to different neuronal cytoskeletal compartments with a high degree of specificity, presumably by isoform-specific association with regulatory/targeting proteins. Furthermore, the synaptic localization of PP1gamma1 indicates that it is this isoform that is involved in the regulation of synaptic phosphoproteins such as neurotransmitter receptors and ion channels implicated in synaptic plasticity.

Animals↗
PubMed2000

Expression and regulation of pyruvate dehydrogenase kinase isoforms in the developing rat heart and in adulthood: role of thyroid hormone status and lipid supply.

Activation of the pyruvate dehydrogenase (PDH) complex (PDHC) promotes glucose disposal, whereas inactivation conserves glucose. The PDH kinases (PDHKs) regulate glucose oxidation through inhibitory phosphorylation of PDHC. The adult rat heart contains three PDHK isoforms PDHK1, PDHK2 and PDHK4. Using Western-blot analysis, with specific antibodies raised against individual recombinant PDHK1, PDHK2 and PDHK4, the present study investigated PDHK isoform expression in the developing rat heart and adulthood. We identified clear differences in the patterns of protein expression of each of these PDHK isoforms during the first 3 weeks of post-natal development, with most marked up-regulation of isoforms PDHK1 and PDHK4. Distinctions between the three cardiac PDHK isoforms were also demonstrated with respect to post-neonatal maturational up-regulation; with greatest up-regulation of PDHK1 and least up-regulation of PDHK4 from the post-neonatal period until maturity. The study also examined the role of thyroid hormone status and lipid supply on PDHK isoform expression. We observed marked selective increases in the amount of PDHK4 protein present relative to total cardiac protein in both hyperthyroidism and high-fat feeding. Overall, our data identify PDHK isoform PDHK1 as being of more potential regulatory importance for glucose oxidation in the adult compared with the neonatal heart, and cardiac PDHK4 as a PDHK isoform whose expression is specifically responsive to changes in lipid supply, suggesting that its up-regulation during early post-natal life may be the perinatal switch to use fatty acids as the energy source. We also identify regulation of pyruvate sensitivity of cardiac PDHK as a physiological variable, a change in which requires factors in addition to a change in lipid supply.

Aging↗
PubMed2001

RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.

Endothelium, Vascular↗
PubMed2001

Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line.

AIM: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. METHODS: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. RESULTS: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3H-DHT) to these three isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. CONCLUSION: The expression of AR isoforms is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.

Dihydrotestosterone↗
PubMed1992

Expression of four protein kinase C isoforms in rat fibroblasts. Differential alterations in ras-, src-, and fos-transformed cells.

In the accompanying study (Borner, C.B., Guadagno, S. N., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892-12899) we found that R6 embryo fibroblasts express four isoforms of PKC, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta whose subcellular distribution, activation, and down-regulation are differentially regulated. Furthermore, we demonstrated that overproduction of an exogenous cPKC beta I isoform in these cells (R6-PKC3) altered the TPA-induced down-regulation of nPKC delta and nPKC epsilon. In this paper we show that transformation of R6 or R6-PKC3 cells with a variety of different oncogenes results in differential alterations in expression of individual PKC isoforms. R6 or R6-PKC3 cells transformed by an activated c-H-ras oncogene displayed a marked increase in the expression of both cPKC alpha and nPKC delta, decreased expression of nPKC epsilon, and no change in the expression of nPKC zeta. These alterations occurred at both the mRNA and protein levels but did not significantly affect the subcellular distribution of any of the four isoforms. Studies using actinomycin D and nuclear run-off assays indicated that the increased expression of cPKC alpha in ras-transformed cells was due to increased de novo transcription rather than increased mRNA stability. Qualitatively similar, but less extensive changes in the expression of the four PKC isoforms were seen in v-fos-transformed R6-PKC3 cells. Decreased expression of nPKC epsilon was also seen in the v-src-transformed R6- and R6-PKC3 lines; however, the cellular level of cPKC beta I appeared to be a limiting factor in mediating the effects of v-src on the increased expression of cPKC alpha and nPKC delta. Interestingly, no major changes in the levels of expression of any of the four PKC isoforms were found when R6 cells were transformed by myc, neu/erb-B2, or mos oncogenes. These results demonstrate that transformation of R6 cells by the oncogenes ras, src, and fos differentially alter the expression of three isoforms of PKC in the same host cell, and they suggest that individual isoforms may play distinct roles in mediating cellular transformation by specific oncogenes.

Alkaloids↗
PubMed2006

Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells.

High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

Breast Neoplasms↗
PubMed1990

Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane.

The sequence of more than 1,000 amino acid residues, derived from two different isoforms, has been determined from peptides generated from purified human erythrocyte membrane Ca2(+)-ATPase (hPMCA). Several of these peptide sequences correspond to the previously reported, cDNA deduced sequence of the "teratoma" Ca2+ pump isoform hPMCA1 (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159). The complete primary structure of a novel isoform (hPMCA3) has been determined by molecular cloning and nucleotide sequencing of its corresponding cDNA. This new member of the plasma membrane Ca2+ pump family consists of 1,205 amino acid residues with a calculated Mr of 133,930, and it shows 88% similarity (75% identity) with the previously sequenced pump isoform. Specific probes detect major mRNA species of 5.6 kilobases for hPMCA1, and of 7.5 kilobases for hPMCA3, on Northern blots of human K562 erythroleukemic cell RNA. A large number of peptide sequences match perfectly with only one or the other of these isoforms and all peptides (with 6 exceptions corresponding to a contaminant protein or to a third minor Ca2+ pump isoform) are found in either only one or in both of the isoforms. The two erythrocyte Ca2+ pumps display high sequence divergence in a few localized regions that may determine isoform-specific functional specializations; for example, the putative extracellular loop separating transmembrane domains 1 and 2, the highly negatively charged region previously suggested to be involved in Ca2+ binding, and the site of cAMP-dependent protein kinase phosphorylation.

Amino Acid Sequence↗
PubMed1990

Chick tropoelastin isoforms. From the gene to the extracellular matrix.

Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.

Amino Acid Sequence↗
PubMed1989

Hereditary and dietary effects on apolipoprotein[a] isoforms and Lp[a] in baboons.

Baboons possess Lp[a] that is similar to human Lp[a], including the presence of the unique protein, apo[a]. Baboon apo[a] occurred in at least nine isoforms distinguishable by size. Isoforms were resolved by 3-12% polyacrylamide gradient gel electrophoretic separation of serum proteins, and were detected with baboon apo[a]-specific antibodies. Thirty one different apo[a] isoform phenotypes were detected in a population of 165 unrelated baboons. Identical isoform phenotypes were observed in different samples from individual baboons, and isoform phenotypes were unaffected by changes in diet. In one experiment, 16 baboons were fed a series of five diets differing in amounts of cholesterol and saturated or unsaturated fats. There was no significant effect of diet on serum Lp[a] levels. In another group of baboons (n = 70) controlled for age and dietary history, enrichment of the diet with cholesterol and saturated fat caused a small, but significant (P less than 0.005), increase (means = 0.6 mg/dl) in serum Lp[a] concentration. Analysis of two large sire families suggested that apo[a] isoform patterns and serum Lp[a] concentrations were inherited. Putative parental alleles responsible for specific isoform bands appeared to segregate randomly. Heritability (h2) of serum Lp[a] concentration was estimated to be 0.95 +/- 0.04. We conclude that apo[a] isoform phenotypes and serum Lp[a] concentrations are inherited, and that Lp[a] concentrations are only slightly influenced by diet.

Animals↗
PubMed1989

Developmental and muscle-specific regulation of avian fast skeletal troponin T isoform expression by mRNA splicing.

We have investigated the developmental regulation of the avian fast skeletal muscle troponin T (TnTf) gene of the Japanese quail. Sequence analysis of troponin T mRNA, cDNA clones, and a genomic DNA segment demonstrate that the avian, fast skeletal TnTf protein isoforms are produced from a single gene. This TnTf gene is expressed in skeletal muscle, but not in adult cardiac muscles or in non-muscle tissues. In addition to known TnT isoforms, three new isoforms of TnT are described. These isoforms arise by regulated alternative RNA splicing of exons in the 5' and 3' regions of TnTf transcripts. Alternative splicing of the 5' TnTf exons involves splicing of multiple exons in different combinations (i.e. not mutually exclusive), whereas 3' alternative splicing involves mutually exclusive splice choices between two exons (alpha or beta exons). S1 nuclease protection and primer extension analyses show that alternative splicing of both 5' and 3' exons is precisely regulated and coordinated in physiologically different striated muscles, which express distinct, restricted combinations of 5' and 3' alternatively spliced exons in mRNA transcripts. In contrast, different embryonic muscles and clonal embryonic myoblast cultures coexpress the 3' alternative splice choices. This indicates that alternative splicing of TnTf mRNAs is controlled in different adult muscles by specific trans factors, and not by the restricted expression of different spliced forms in different embryonic myoblast lineages. Comparison of TnTf isoform expression in quail and chicken flight muscle (Wilkinson, J. M., Moir, A. J., and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47-56) to TnTf isoforms of the rat (Breitbart, R. E., and Nadal-Ginard, B. (1986) J. Mol. Biol. 188, 313-324), and rabbit (Pearlstone, J. R., Carpenter, M. R., and Smillie, M. B. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1902-1906) indicates that the avian gene contains an additional exon(s) not present in mammalian genes. The alternative exon sequences TnTf mRNAs expressed in anatomically distinct quail muscles can be correlated with sequences in TnTf protein isoforms in these chicken muscles. Thus, the regulated splicing of alternative exons in TnT transcripts, and not selective translation of stochastically spliced TnT mRNAs, regulates TnTf isoform expression in specific muscles.

Aging↗
PubMed1987

Isolation and functional comparison of bovine cardiac troponin T isoforms.

We report on the isolation of two bovine cardiac troponin isoforms which differ in sequence near the amino terminus of troponin T (Risnik, V. V., Verin, A. D., and Gusev, N. B. (1985) Biochem. J. 225, 549-552). The isoforms were isolated by direct separation on DEAE-cellulose and were also obtained by reconstitution of urea-dissociated subunits. The two isoforms were compared for their effects on processes involving interactions of troponin with tropomyosin and actin. The two isoforms had similar abilities to promote tropomyosin polymerization. They allowed equal potentiation, by high concentration of myosin subfragment 1, of the thin filament-activated MgATPase rate. In the presence of lower concentrations of myosin subfragment 1, the MgATPase rate was 96% sensitive to Ca2+, regardless of which troponin isoform was present. The Ca2+ concentration required to activate the MgATPase rate was similar but not identical for thin filaments containing one isoform or the other. In the presence of the smaller isoform, the Ca2+-activation curve is shifted 0.1 to 0.15 pCa units to the left. At 10(-6) M Ca2+ the MgATPase rate is 50% greater when the smaller troponin T isoform is present than when the larger is present. These results indicate that the variable region of troponin T influences the overall response of the thin filament to Ca2+, and raises the possibility that regulation of this region by mRNA splicing may modulate muscle function.

Actin Cytoskeleton↗
PubMed1986

Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties.

Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.

Actins↗
PubMed1987

Isoforms of creatine kinase isoenzymes in serum in acute myocardial infarction after intracoronary thrombolysis.

Kinetics of the catalytic activities of creatine kinase (CK;EC 2.7.3.2) for three CK-3 and two CK-2 isoforms in serum were studied in 20 patients with myocardial infarction randomly assigned to receive either intracoronary urokinase (group A) or conventional therapy (group B). The temporal characteristics of isoform changes described were (a) time at which the isoform activities are significantly greater than initial values, (b) maximal rate (Ka) at which isoforms are released into blood, (c) time lag from onset of pain until maximum activity value, (d) peak value of each serum isoform, and (e) rate (Kd) at which each isoform is cleared from serum. Thrombolytic treatment induced earlier peak times in group A: for CK-3(3), 7.4 vs 20.0 h; for CK-3(2), 11.6 vs 24.8; for CK-3(1), 18.6 vs 34.3; for CK-2(2), 9.1 vs 17.8; and for CK-2(1), 11.8 vs 26.8 (numbers given are medians; for all isoforms, P less than 0.05). Ka values were at least twofold greater and the first increase was significantly earlier in the urokinase group. Consequently, the ratio for CK-3(3) to CK-3(1) activities peaked significantly earlier in group A. Isoform peak activities and Kd were not significantly different between the two groups.

Adult↗
PubMed1985

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals.

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.

Amino Acid Sequence↗
PubMed1985

Plants have isoforms for acyl carrier protein that are expressed differently in different tissues.

Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.

Acyl Carrier Protein↗
PubMed1995

FGF-8 isoforms differ in NIH3T3 cell transforming potential.

We previously identified Fgf-8 as a frequently activated gene in tumors from mouse mammary tumor virus-infected Wnt-1 transgenic mice, suggesting that Fgf-8 is a proto-oncogene. We further determined that multiple, secreted protein isoforms that differ at their mature amino termini are encoded by alternatively spliced mRNAs transcribed from the gene. We now present evidence that there are differences in the potency of NIH3T3 cell transformation displayed by three of the FGF (fibroblast growth factor)-8 isoforms. We find that stable transfection of a cDNA for the FGF-8b isoform leads to marked morphological transformation of NIH3T3 cells and rapid tumorigenicity of the transfected cells in nude mice. In contrast, transfection of a cDNA for the FGF-8a or FGF-8c isoform results in moderate morphological changes in the NIH3T3 cells, and the transfected cells are weakly tumorigenic in nude mice. All three transfections result in cells that express comparable amounts of Fgf-8 mRNA and that produce the FGF-8 protein isoforms. The morphological changes observed in NIH3T3 cells can be reproduced by the addition of recombinant FGF-8 protein isoforms to the culture medium. Therefore, these results indicate that there are differences in the potency of transformation of NIH3T3 cells by FGF-8 protein isoforms and suggest that these FGF-8 isoforms may have different in vivo functions.

3T3 Cells↗
PubMed1995

CD44 isoforms correlate with cellular differentiation but not with prognosis in human breast cancer.

CD44 is a transmembrane glycoprotein occurring in several isoforms with different extracellular regions. The various transcripts are encoded by one gene locus containing 20 exons, of which at least 10 can be alternatively spliced in nascent RNA. Isoforms encoded by the variant exons (termed CD44v) are highly restricted in their distribution in nonmalignant tissue as opposed to the standard form of CD44 (CD44s) abundant in many tissues. Specific variant isoforms containing exon 6v have been shown to render nonmetastatic rat tumor cells metastatic. Based on the prominent role in rat metastasis formation, CD44v isoforms were suggested to be involved in human tumor progression. Correlations between prognosis and expression of CD44v have been reported for gastric and colon carcinoma, for non-Hodgkin's lymphoma, and recently for breast carcinoma. We evaluated the expression of CD44 isoforms in node-positive (n = 119) and node-negative (n = 108) cases of breast carcinoma by immunohistochemistry using CD44v exon-specific mAbs. In a subset of 43 cases of high-risk patients, reverse transcription-PCR was used to determine the exon composition of the transcripts. Protein and RNA expression data were probed statistically for their correlation to survival of the patients and clinical risk factors. In contrast to recently published data (M. Kaufmann et al., Lancet, 345: 615-619, 1995), in our cohort disease-free and overall survival data did not indicate significant correlations with the expression of the analyzed isoforms in univariate and multivariate analyses. Comparison of CD44 protein expression with established clinical risk factors for survival such as tumor size (pT1+pT2) and histological grading revealed correlations with the presence of CD44s (P = 0.02 and P = 0.03, respectively) and CD44-9v (P = 0.05 for histological grading). Carcinoma tissues with elevated estrogen and progesterone receptor levels showed positive correlation with CD44-6v (P = 0.001), while a trend for significant coexpression of CD44s and CD44-9v isoforms was observed in estrogen receptor-positive tissues (P = 0.08 and 0.06, respectively). In breast cancer, CD44s, CD44-9v, and CD44-6v are apparently markers for cellular differentiation but not for tumor progression. Our data suggest that steroid hormone receptors may be associated with the in vivo expression of CD44-6v-containing isoforms in human mammary carcinoma.

Adult↗