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PubMed1994

Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line.

FSH has four asparagine-linked oligosaccharides with variable sialic acid contents, so that FSH is not a single molecule, but a heterogeneous group of isoforms. These isoforms differ in their biological properties and their distribution changes in various physiological states, allowing the modulation of FSH activity. Recombinant human (h) FSH has been produced in Chinese hamster ovary cells and has an isoform profile similar to those of both pituitary FSH standard and purified urinary FSH. These FSH preparations, however, do not contain the full spectrum of FSH isoforms found in the circulation. Production of recombinant hFSH in a cell line with a different pattern of glycosylation could broaden its isoform profile and potentially alter its biological activity. Thus, we transfected human embryonal kidney cells (293) with the human alpha and FSH beta genes to produce recombinant hFSH (hFSH-293) and determined its biological activity in a rat granulosa cell bioassay. Although hFSH-293 was immunologically indistinguishable from pituitary FSH standard, its biological potency was 3- to 6-fold higher than those of two different pituitary FSH standards. To investigate this increased potency, we separated the isoforms of hFSH-293 by chromatofocusing and determined their biological potencies in the rat granulosa cell bioassay. The isoform profile of hFSH-293 demonstrated a greater number of basic isoforms than that of pituitary FSH standard. Several of these basic isoforms exhibited enhanced in vitro biological potency, accounting for the increased biological potency of hFSH-293. This pattern of high in vitro biological activity and more basic isoforms is analogous to the FSH circulating during GnRH stimulation, pubertal induction, and ovulation.

Animals↗
PubMed1996

More basic isoforms of serum gonadotropins during gonadotropin-releasing hormone agonist therapy in pubertal children.

An acute challenge of exogenous GnRH elicits rapidly increased serum gonadotropin levels with qualitative changes to more basic isoforms of both FSH and LH. Chronic GnRH agonist therapy suppresses endogenous gonadotropins, and the serum levels of FSH and LH are low and fairly constant. A possible qualitative change in the gonadotropins during GnRH agonist therapy was investigated by determination of the median charge of the gonadotropin isoforms before and during therapy in 18 pubertal children. Two different GnRH agonists were studied: buserelin, given intranasally or as a sc implant for 1.5-34 months to five girls, aged 7-10 yr, and for 5-6 months to two boys, aged 11-13 yr; and triptorelin, administered as a depot preparation for 3-6 months to four girls, aged 9-12.5 yr, and for 1-24 months to seven boys, aged 10.5-12 yr. FSH and LH in serum and eluates after electrophoresis in 0.10% agarose suspension were measured with sandwich fluoroimmunoassays. The mean serum FSH and LH levels decreased significantly (P < 0.05) in girls during triptorelin therapy, whereas only the FSH level decreased (P < 0.05) in the boys. There were no significant (P > 0.05) changes in serum gonadotropin levels during buserelin therapy. All of the children had more basic serum isoforms of LH, and all but one had more basic forms of FSH during the GnRH agonist treatments. In a girl who had more basic gonadotropin isoforms after treatment with triptorelin for 2 and 6 months, a GnRH challenge elicited the release of still more basic isoforms. The changes in mean median charge to more basic gonadotropin isoforms were highly significant for both busereline (P < 0.01) and triptorelin (P < 0.001) treatment. An increased (P < 0.001) degree of charge heterogeneity was observed for FSH after triptorelin therapy. These findings show that there is a qualitative change in the isoforms of both FSH and LH in serum during GnRH agonist therapy in pubertal children. The changes in charge to more basic gonadotropin isoforms most likely reflect a direct effect at the pituitary level, leading to the synthesis and/or selective release of less sialylated and sulfated isoforms of the gonadotropins. The observed qualitative changes in the gonadotropin isoforms in these pubertal children may be part of the clinical effects of GnRH agonist therapy, leading to an arrest or regression of puberty.

Adolescent↗
PubMed2006

Differential response of progesterone receptor isoforms in hormone-dependent and -independent facilitation of female sexual receptivity.

Neurobehavioral effects of progesterone are mediated primarily by its interaction with neural progesterone receptors (PRs), expressed as PR-A and PR-B protein isoforms. Whereas the expression of two isoforms in the neural tissues is suggestive of their selective cellular responses and modulation of distinct subsets of PR-induced target genes, the role of individual isoforms in brain and behavior is unknown. We have previously demonstrated a critical role for PRs as transcriptional mediators of progesterone (ligand-dependent), and dopamine (ligand-independent)-facilitated female reproductive behavior in female mice lacking both the isoforms of PR. To further elucidate the selective contribution of the individual PR isoforms in female sexual receptive behavior, we used the recently generated PR-A and PR-B isoform-specific null mutant mice. We present evidence for differential responses of each isoform to progesterone and dopamine agonist, SKF 81297 (SKF), and demonstrate a key role for PR-A isoform in both hormone-dependent and -independent facilitation of sexual receptive behavior. Interestingly, whereas both the isoforms were essential for SKF-facilitated sexual behavior, PR-A appeared to play a more important role in the 8-bromo-cAMP-facilitated lordosis response, raising the possibility of distinct intracellular signaling pathways mediating the responses. Finally, we also demonstrate that antiprogestin, RU38486, was an effective inhibitor of PR-A-mediated, progesterone-dependent, but not SKF or 8-bromo-cAMP-dependent sexual receptivity. The data reveal the selective contributions of individual isoforms to the signaling pathways mediating female reproductive behavior.

Animals↗
PubMed1994

Structural and functional characterization of estrogen sulfotransferase isoforms: distinct catalytic and high affinity binding activities.

Estrogen sulfotransferase (EST) purified from the guinea pig (gp) adrenal gland consists of multiple charge isoforms with isoelectric points (pls) ranging from 6.5 to 5.2. Four individual isoforms were isolated for use in antibody production, as well as for tryptic fragmentation analysis. The multiple charge isoforms manifested a high degree of relatedness as evidence by complete immunocross-reactivity. This relatedness was further demonstrated by peptide mapping analysis, which revealed essentially identical elution profiles for three of the isoforms, whereas the fourth most acidic isoform, although similar to the other three isoforms, demonstrated some differences. To further explore the nature of the charge isoforms, native gpEST and EST expressed by CHO-K1 cells transfected with the gpEST cDNA were subjected to additional biochemical characterization. An interesting finding was that, in addition to EST activity, gpEST bound estradiol with a high affinity [dissociation constant (Kd) 10 nM]; furthermore, the binding activity was absolutely dependent on the cofactor, adenosine-3',5'-diphosphate (3',5'-ADP). An unexpected and novel finding was that only the most basic pl 6.5 isoform was catalytically active, while estradiol-binding activity was associated with a more acidic isoform (pl 5.5-5.2); however, it has not as yet been possible to definitively assign the binding activity to a specific isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1998

Cytoplasmic dynamics of myosin IIA and IIB: spatial 'sorting' of isoforms in locomoting cells.

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to 'sort' within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.

Actin Cytoskeleton↗
PubMed2002

Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish.

This paper describes the cloning and functional characterization of the heart muscle isoform of Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish Procambarus clarkii. The complete crayfish heart SERCA, identified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), consists of 4495 bp with a 3060 bp open reading frame, coding for 1020 amino acids. This isoform differs from the previously identified axial abdominal (tail) muscle SERCA solely in its C-terminal amino acids. The last nine amino acids of the tail muscle isoform are replaced by 27 hydrophobic amino acids in the heart isoform that have the potential to form an additional transmembrane domain. Consistent with other invertebrate studies, Southern blot analysis suggested that the heart and tail muscle isoforms are encoded from the same gene that is equally related to SERCA-1, -2 and -3 of vertebrates. The tissue distributions of these two isoforms have been assessed using isoform-specific probes and northern analysis. A cardiac-specific probe bound only to a 5.8 kb species in heart and had minimal cross-hybridization with 7.6 and 5.8 kb species in eggs and no hybridization with tail muscle. A tail-isoform-specific probe hybridized with a 4.5 kb species in tail muscle and cross-hybridized with a 4.5 kb species in eggs and 8.8 kb in heart muscle. Both isoforms are expressed in eggs suggesting that transcripts are formed early in development and are subsequently broadly expressed in all tissue types. Expression of the cardiac muscle SERCA isoform varied with the stage of moulting. Expression was high in intermoult and decreased in premoult. However, expression was restored rapidly in postmoult (within 2 days) unlike expression of tail muscle SERCA, which remained downregulated for weeks. Differences in contractility between the two muscle types in the postmoult period may explain these expression patterns.

Amino Acid Sequence↗
PubMed2001

Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.

Acid Phosphatase↗
PubMed1993

The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours.

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.

Blotting, Western↗
PubMed1999

Region-specific mRNA expression of phosphatidylinositol 3-kinase regulatory isoforms in the central nervous system of C57BL/6J mice.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) by receptor tyrosine kinases for growth factors is crucial for neuronal cell survival and proliferation. This class of kinases is comprised of heterodimers, each consisting of one regulatory and one catalytic subunit. Multiple isoforms of regulatory subunits exist, including p85alpha and its alternative splice products p50alpha and AS53/p55alpha, and p85beta and p55(PIK), which are derived from different genes. The regional distribution of these PI 3-kinase regulatory isoforms was mapped in the adult murine brain by in situ hybridization histochemistry. All isoforms were demonstrated in abundance in choroid plexus and anterior pituitary. In neuronal compartments, however, PI 3-kinase isoforms were distributed in a regionally specific manner. In general, the mRNAs for p85alpha, p50alpha, AS53, and p85beta were widespread, with the highest level in the olfactory system, in neuronal groups of the forebrain and hypothalamus, in the hippocampus, cortex, inferior and superior colliculus, pituitary, and cerebellum. However, each isoform had specific variations. Lower expression levels of these isoforms were found in the thalamus, diencephalon, mesencephalon, and brainstem. In contrast, abundant mRNA expression of p55(PIK) was limited to cerebellum and anterior pituitary, with moderate levels of p55(PIK) in the olfactory bulb and hippocampus and low levels elsewhere. The distribution pattern of PI 3-kinase isoforms in the brain indicates pluripotent signaling properties for PI 3-kinase isoforms p85alpha, p50alpha, AS53/p55alpha, and p85beta for a variety of receptor tyrosine kinases, whereas the restricted expression of p55(PIK) implies a regionally specific role for this isoform in neuronal signaling. The unique integrated expression profiles of PI 3-kinase isoforms in distinct neuronal compartments denote complex intracellular signaling pathways for each neuronal region to ensure specificity of receptor tyrosine kinase signal transduction.

Animals↗
PubMed1990

Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.

Animals↗
PubMed1991

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

[Comparative study of the action of O-phenanthroline metal complexes and a number of other compounds on the activity of brain arginase isoforms].

Two isoforms of arginase were isolated from rat brain tissue: isoform I-adsorbed on CM-Sephadex and isoform II-unadsorbed on the sorbent. In samples with arginine as a substrate Km values for isoforms I and II constituted 1.7 X 10(-3) M and 3.8 X 10(-3) M, respectively. EDTA and o-phenanthroline inhibited these both brain arginase isoforms. As shown in experiments with o-phenanthroline metal complex affecting the brain arginase activity some copper complexes were able to inhibit most effectively the activity of the isoform II. Activity of both these isoforms was inhibited also by dithiothreitol, proline and acetaldehyde. Complexes of o-phenanthroline with Co2+ and Fe2+ inhibited only slightly the activity of the isoform II and did not affect the isoform I. These data showed that brain arginase isoforms were markedly distinct in their properties from the ureotelic liver tissue arginase.

Animals↗
PubMed1995

Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the nonpigmented ciliary epithelium.

PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.

Aged↗
PubMed1995

CD44 isoform expression in primary and metastatic pancreatic adenocarcinoma.

CD44 is the transmembrane adhesion molecule which binds hyaluronate. The gene encoding CD44 is found on chromosome 11p and comprises 20 exons. Differential splicing of the 10 extracellular juxtamembranous exons (v1-10) generates the major isoforms of CD44. The major CD44 isoform found on hematopoetic cells (CD44s) contains none of the variably expressed exons, while the major isoform expressed on epithelial cells [CD44(v8-10)] contains exons v8-10. Metastasis-specific isoforms of CD44 were first documented in a model of rat pancreatic adenocarcinoma [CD44(v4-7), CD44(v6-7)] and subsequently in other cancers. This study is the first characterization of CD44 isoforms in primary and metastatic human pancreatic adenocarcinomas. CD44 isoforms were analyzed in specimens of 15 primary and 6 metastatic pancreatic adenocarcinomas as well as in 6 specimens of control pancreata by two different methods. Radiolabeled reverse transcriptase-PCR coupled with 8% PAGE allowed analysis of the major isoforms of CD44, while Southern blot hybridization with [alpha-32P]dCTP-labeled probes permitted analysis for metastasis-specific CD44 isoforms containing CD44(v6) or CD44(v8-10). No differences in the expression of CD44(v8-10) and CD44s were found among the primary and metastatic pancreatic adenocarcinomas, and control specimens of pancreata. However, a novel CD44(v6) isoform was found in metastatic lesions and may represent the human homologue of the rat pancreatic adenocarcinoma metastasis-associated CD44 isoform.

Adenocarcinoma↗
PubMed1995

Phosphor image analysis of human p53 protein isoforms.

Phosphor imaging was evaluated for detection, quantitation and resolution of multiphosphorylated protein isoforms separated by two-dimensional gel electrophoresis. A nuclear phosphoprotein, p53, was isolated by immunoprecipitation after biosynthetic labeling with 35S, 32P or 33P in cultured human cells. Of the three radionuclides, 35S was the most sensitive in detection after a 1-week exposure, although shorter exposure times were effective. In dividing cells, 11 35S-labeled isoforms were found, of which 10 were phosphorylated by 33P and 32P. Exposure of phosphonuclides for one half-life showed that 33P radiolabeling produced better resolution among isoforms than 32P but was less sensitive in detection. Volume integration showed phosphorylated isoforms comprised from 1% to 25% of total isoform signal. The relative phosphorylation of each p53 isoform was estimated by normalizing 33P or 32P isoform volumes with the corresponding 35S volume and showed progressive phosphorylation of acidic isoforms. Additionally, phosphor imaging capably detected quantitative changes among individual isoforms after experimental modulation of the isoform pattern by serum deprivation. The described electrophoretic isolation and quantitation procedures should find general application in discerning active and inactive phosphoisoforms for eventual identification.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man.

Crigler-Najjar syndrome type I (CN-I) is caused by an inherited absence of UDP-glucuronosyltransferase activity toward bilirubin (B-UGT), resulting in severe non-hemolytic unconjugated hyperbilirubinemia. Based on the expression of cDNAs in COS cells, two UGT isoforms in human liver, B-UGT1 and B-UGT2, have been reported to catalyze bilirubin glucuronidation. These isoforms, which are derived from a single gene, ugt1, have identical carboxyl-terminal domains that are encoded by four consecutive exons shared by both isoforms. A critical lesion in any of these common exons should inactivate both B-UGT isoforms, giving rise to CN-I. The amino-terminal domains of the B-UGT isoforms are unique, each being encoded by a different 5' exon. If both B-UGT isoforms contribute significantly to bilirubin glucuronidation, a mutation in one of these unique 5' exons should affect a single isoform, while the other isoforms should provide residual B-UGT activity. However, in two patients with CN-I, we found a mutation only in the unique exon of B-UGT1, the other exons being normal. To clarify this apparent paradox, we expressed the cDNA for each B-UGT isoform in COS cells and determined the specific B-UGT activity. These studies show that only B-UGT1 has quantitatively significant catalytic activity. Furthermore, we show that the mutation in B-UGT1 observed in each of the two CN-I patients inactivates B-UGT1. Together, the results indicate that B-UGT1 is the only physiologically relevant isoform in bilirubin glucuronidation.

Animals↗
PubMed1994

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms.

Minimal ectopic expression of the p58GTA protein kinase results in a provocative phenotype involving cell cycle delay, mitotic catastrophe, and decreased cell viability. In addition, this kinase is well conserved evolutionarily, ubiquitously expressed, and its genes map to a position on human chromosome 1 frequently deleted in the late stages of tumorigenesis. Here we report that the p58GTA protein kinase is a member of a larger subfamily of proteins. The mRNAs encoding these proteins are generated by alternative splicing from multiple duplicated genes. These isoforms range in size from 50 to 110 kDa. Divergence between the alternatively spliced isoforms is localized to the amino-terminal region of the molecule. The entire p58GTA open reading frame is conserved in most of these p58GTA isoforms. The predicted sequences of the larger isoforms encode bipartite nuclear localization signal sequences and extensive polyglutamic acid domains. Antibodies to the p58GTA isoform were used to confirm the presence of the alternatively spliced isoforms in different cell types as well as identify two additional isoforms that appear to arise from a separate gene(s). Cellular fractionation studies indicate that one of the isoforms is found only in the nucleus, and the remainder are found in both the cytoplasm and the nucleus. Expression and localization of some p58GTA isoforms suggest that they may have specialized cellular functions. Because of the large number of isoforms generated from multiple genes we propose naming these kinases PITSLRE alpha 1, alpha 2-1, alpha 2-2, alpha 2-3 alpha 2-4, beta 1, beta 2-1, and beta 2-2 based on the conserved sequence of the PSTAIRE box unique to p34cdc2 kinases and the gene from which they are transcribed.

Alternative Splicing↗
PubMed1997

Heterogeneous expression of DNA topoisomerase II alpha isoforms in tumor cell lines.

DNA topoisomerase II alpha is a nuclear enzyme essential for DNA metabolism and cell cycle progression. Previous studies have shown that human tumor cell lines can express more than one topoisomerase II alpha isoform through alternative splicing. A 160-kDa isoform of topoisomerase II alpha has been described in several cell lines selected for resistance to inhibitors of DNA topoisomerase, but its physiological function has not been defined. In the present study, we have identified two major (160 and 140 kDa) and two minor (150 and 145 kDa) isoforms of topoisomerase II alpha in drug-sensitive human leukemic CEM cells, all of which have lost C-terminal regions that produce epitopes recognized by specific antibodies. Reverse transcription-polymerase chain reaction and molecular cloning identified four alternatively spliced transcripts of topoisomerase II alpha from CEM cells. Furthermore, nucleotide sequencing indicated that the 160-kDa isoform is encoded by two transcripts derived from alternative splicing at a different C-terminal site and that the other two transcripts likely code for the 150-kDa isoform. Although the full-length topoisomerase II alpha resided in the cell nucleus, all altered isoforms, except the 160 kDa that was located in both cytoplasmic and nuclear extracts in about equal amount, were shown to be present predominantly in the cytosol. In contrast to the observations of other groups, we have not found an association of the topoisomerase II alpha isoforms with drug resistance. Rather, our results suggest that expression of topoisomerase II alpha isoforms is cell type specific or might be associated with the neoplastic phenotype of the cells. Thus, although T-lineage tumor cell lines examined (CEM, Jurkat, and H9) displayed altered topoisomerase II alpha isoforms, normal T cells expressed only a full-length copy of the gene. Together, these results suggest that expression of altered topoisomerase II alpha isoforms is not limited to drug resistance, but might be a feature of neoplastic cells.

Alternative Splicing↗