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PubMed2003

The thromboxane receptor antagonist PBT-3, a hepoxilin stable analog, selectively antagonizes the TPalpha isoform in transfected COS-7 cells.

The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1alpha,2alpha (Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [3H]SQ 29,548 [[1S-[1alpha,2alpha (Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TPalpha- and TPbeta-transfected COS-7 cells. These isoforms possess a different tail length, the alpha being shorter than the beta isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC50 value for PBT-3 = 2.0 +/- 0.3 x 10-7 M was observed for TPalpha, whereas this was one-sixth less active on the TPbeta isoform (IC50 = 1.2 +/- 0.2 x 10-6 M), suggesting selectivity for the TPalpha isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC50 was similar to that of the TPalpha isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TPalpha isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.

Adenoviruses, Human↗
PubMed1999

Acidic and basic troponin T isoforms in mature fast-twitch skeletal muscle and effect on contractility.

Developmentally regulated alternative RNA splicing generates distinct classes of acidic and basic troponin T (TnT) isoforms. In fast-twitch skeletal muscles, an acidic-to-basic TnT isoform switch ensures basic isoform expression in the adult. As an exception, an acidic segment in the NH2-terminal variable region of adult chicken breast muscle TnT isoforms is responsible for the unique exclusive expression of acidic TnTs in this muscle (O. Ogut and J.-P. Jin. J. Biol. Chem. 273: 27858-27866, 1998). To understand the relationship between acidic vs. basic TnT isoform expression and muscle contraction, the contractile properties of fibers from adult chicken breast muscle were compared with those of the levator coccygeus muscle, which expresses solely basic TnT isoforms. With use of Triton X-100-skinned muscle fibers, the force and stiffness responses to Ca2+ were measured. Relative to the levator coccygeus muscle, the breast muscle fibers showed significantly increased sensitivity to Ca2+ of force and stiffness with a shift of approximately 0.15 in the pCa at which force or stiffness was 50% of maximal. The expression of tropomyosin, troponin I, and troponin C isoforms was also determined to delineate their contribution to thin-filament regulation. The data indicate that TnT isoforms differing in their NH2-terminal charge are able to alter the sensitivity of the myofibrillar contractile apparatus to Ca2+. These results provide evidence linking the regulated expression of distinct acidic and basic TnT isoform classes to the contractility of striated muscle.

Aging↗
PubMed1989

Tissue-specific isoform regulation of Na+-K+-ATPase by thyroid hormone in ferrets.

We have shown previously that in the ferret heart there are two isoforms of Na+-K+-ATPase, alpha(+) and alpha, and that these isoforms undergo developmental changes. In the present study, we examine regulation of the isoenzymes by thyroid hormone, which is well known to increase activity of Na+-K+-ATPase in different tissue preparations. Ferrets were injected with L-thyroxine (T4) (0.5 mg/kg, sc) for 3 wk. The T4-treated ferrets gained 58 +/- 32 g body wt compared with 366 +/- 24 g for the control ferrets. Plasma 3,5,3'-triiodothyronine concentrations of the T4-treated animals increased about eightfold 16-18 h after the last injection. The number of alpha(+)- and alpha-subunits in heart homogenates estimated by [3H]ouabain binding was 3.5 +/- 0.1 and 2.5 +/- 0.1 pmol/mg protein, respectively [alpha(+)/alpha = 1.40]. In the T4-treated ferrets, there was no significant increase of the alpha(+)-isoform (3.2 +/- 0.2 pmol/mg protein), whereas the number of alpha-isoform increased significantly to 4.1 +/- 0.3 pmol/mg protein [alpha(+)/alpha = 0.78]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified cardiac plasma membrane preparations reveals similar pattern of changes of the isoenzymes. In the kidney, however, there was no significant change in the number of alpha-isoform, which is the predominant isoform in the kidney, and T4 does not appear to induce synthesis of alpha(+)-isoform in the kidney. It is concluded that thyroid hormone induces both isoform- and tissue-specific regulation of the Na+-K+-ATPase alpha-subunits in the ferret. These specific regulations of the isoforms seem to suggest important physiological roles of the isoenzymes.

Animals↗
PubMed1995

TGF-beta regulates expression of tenascin alternative-splicing isoforms in fetal rat lung.

Two distinct mRNA-splice isoforms of tenascin (TN) are expressed differentially during rat lung development. The unique temporal expression pattern of two TN isoforms suggests the expression of tenascin is strictly developmentally regulated in rat lung tissue. We investigated molecular mechanisms which modulate alternative-splicing expression of TN in lung development. The effect of transforming growth factor-beta 1 (TGF-beta 1) on regulation of expression of TN isoforms was examined by in vitro lung explant culture. Immunoblotting with anti-TN antibody detected two TN polypeptides in rat lung explant culture, the larger [relative molecular weight (M(r)) 230, TN230] polypeptide and the smaller (M(r) 180, TN180). TGF-beta 1 markedly induced the TN180 isoform and caused only a moderate increase of the TN230 isoform. The effects of TGF-beta 1 were shown to be dose dependent over a physiological range of TGF-beta 1 protein concentration. The induction of TN isoform biosynthesis by TGF-beta 1 was detected 12 h after addition of the growth factor, and the effects endured for up to 48 h at a dose of 5 ng/ml. By reverse transcriptase-polymerase chain reaction through amplification of the entire fibronectin type III (FN-III) splicing domain, two distinct TN isoforms were detected in total RNA isolated from gestational day 21 rat lung explant culture treated with TGF-beta 1 and from postnatal day 8 rat lung. The larger isoform contained five FN-III alternative splicing repeats [1,420 base pairs (bp)], but the shorter splicing isoform lacked four FN-III alternative splicing repeats (340 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗
PubMed1995

Unique localization of mRNA encoding plasma membrane Ca2+ pump isoform 3 in rat thin descending loop of Henle.

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.

Animals↗
PubMed1999

Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP.

The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of 22Na+ or 86Rb+ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl- cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl- cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl- cotransport in the mouse TAL by hormones that generate cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.

Animals↗
PubMed1999

Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing.

Three human glutaminase (hGA) isoforms were identified, two of which represent isoforms previously unidentified in any species. One isoform contains an open reading frame with high homology with the rat kidney-type glutaminase, suggesting that this isoform represents the human kidney-type glutaminase, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In addition, a third human glutaminase isoform was identified from a computer search and on further analysis was found to represent an additional unique isoform, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal muscle. hGAC is the predominant isoform expressed by a human breast cancer cell line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of five glutaminase genomic clones suggested that all three hGA isoforms originate from the same locus and therefore represent mRNA species that are produced by tissue-specific alternative splicing of a single pre-mRNA. Furthermore, an RT-PCR assay was developed that can be used to easily differentiate between hKGA and hGAC mRNA species.

Alternative Splicing↗
PubMed2006

Prostaglandin D synthase isoforms from cerebrospinal fluid vary with brain pathology.

Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS) has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls) with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson's disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

Amino Acid Sequence↗
PubMedDate not supplied

Isoforms of atopic allergens with reduced allergenicity but conserved T cell antigenicity: possible use for specific immunotherapy.

BACKGROUND: We analyzed the T cell activation potency and the IgE-binding properties (allergenicity) of nine isoforms of Bet v 1, the major allergen of birch pollen. METHODS: The capacity of recombinant Bet v 1 isoforms to bind serum IgE from allergic patients was evaluated by immunoblot experiments and skin prick tests. The potency of Bet v 1 isoallergens to activate T lymphocytes from birch-pollen-allergic patients was assayed using allergen-specific T cell clones. RESULTS: According to their ability to bind IgE from allergic patients in immunoblot experiments, Bet v 1 isoforms can be grouped into high-IgE-binding molecules and molecules with low/no IgE-binding activity. Representatively, isoform d was used in skin tests. Skin prick tests revealed no potency of this isoform to induce wheal and flare reactions in the skin of birch-pollen-allergic individuals. In contrast, isoform a and natural Bet v 1 displayed high allergenicity in vivo. On the other hand, Bet v 1 isoform d (low allergenicity) displayed significant higher T cell activation potency when compared to isoform a (high allergenicity). CONCLUSION: Based on these findings, we propose a new form of specific immunotherapy using hypoallergenic recombinant allergen isoforms.

Allergens↗
PubMed2006

Adenylyl cyclase isoform-selective regulation of vascular smooth muscle proliferation and cytoskeletal reorganization.

Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.

1-Methyl-3-isobutylxanthine↗
PubMed2004

The development of longitudinal variation of Myosin isoforms in the orbital fibers of extraocular muscles of rats.

PURPOSE: To examine the appearance of longitudinal variation of extraocular and embryonic myosin heavy chain (MyHC) isoforms during the development of orbital singly innervated fibers of rat extraocular muscles (EOMs). METHODS: EOMs were dissected from rat pups of various ages and stained with isoform-specific monoclonal antibodies to the embryonic and extraocular MyHC isoforms and to neurofilaments, as well as with labeled alpha-bungarotoxin. The orbital layers of whole muscles were examined by confocal microscopy. RNase protection assays for the embryonic (Myh3) and extraocular (Myh13) MyHC isoform mRNAs were also performed. RESULTS: At 10 days postpartum, the EOM MyHC RNA was first detected by RNase protection assay. At 11 days postpartum, the extraocular isoform was detected in the orbital fibers as two thin stripes just proximal and distal to the neuromuscular junction (NMJ). Over the next few weeks, the area occupied by the extraocular isoform increased to include the entire central region of the orbital fibers at and surrounding the NMJ. At the same time, the embryonic isoform became excluded from the region of the NMJ. CONCLUSIONS: The orbital layer fibers of rat EOMs contain a longitudinal variation in MyHC isoforms not seen in other skeletal muscles. Development of this longitudinal variation begins as a late event postpartum; and the first appearance of it may be closely linked to neural contact. This targeting of MyHC isoforms to distinct domains is unique to EOMs.

Animals↗
PubMed1994

The inverse association of plasma lipoprotein(a) concentrations with apolipoprotein(a) isoform size is not due to differences in Lp(a) catabolism but to differences in production rate.

Lipoprotein(a) (Lp[a]) is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL) but contains an additional protein called apolipoprotein(a) (apo[a]). Apo(a) is highly polymorphic in size, and there is a strong inverse association between the size of the apo(a) isoform and the plasma concentration of Lp(a). We directly compared the in vivo catabolism of Lp(a) particles containing different size apo(a) isoforms to establish whether there is an effect of apo(a) isoform size on the catabolic rate of Lp(a). In the first series of studies, four normal subjects were injected with radio-labeled S1-Lp(a) and S2-Lp(a) and another four subjects were injected with radiolabeled S2-Lp(a) and S4-Lp(a). No significant differences in fractional catabolic rate were found between Lp(a) particles containing different apo(a) isoforms. To confirm that apo(a) isoform size does not influence the rate of Lp(a) catabolism, three subjects heterozygous for apo(a) were selected for preparative isolation of both Lp(a) particles. The first was a B/S3-apo(a) subject, the second a S4/S6-apo(a) subject, and the third an F/S3-apo(a) subject. From each subject, both Lp(a) particles were preparatively isolated, radiolabeled, and injected into donor subjects and normal volunteers. In all cases, the catabolic rates of the two forms of Lp(a) were not significantly different. In contrast, the allele-specific apo(a) production rates were more than twice as great for the smaller apo(a) isoforms than for the larger apo(a) isoforms. In a total of 17 studies directly comparing Lp(a) particles of different apo(a) isoform size, the mean fractional catabolic rate of the Lp(a) with smaller size apo(a) was 0.329 +/- 0.090 day-1 and of the Lp(a) with the larger size apo(a) 0.306 +/- 0.079 day-1, not significantly different. In summary, the inverse association of plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the catabolic rates of Lp(a) but rather to differences in Lp(a) production rates.

Adult↗
PubMed1989

Expression of actin isoforms in developing rat intestinal epithelium.

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.

Actins↗
PubMed2006

Expression of full-length p53 and its isoform Deltap53 in breast carcinomas in relation to mutation status and clinical parameters.

BACKGROUND: The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity. RESULTS: In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Deltap53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Deltap53 have been predicted. We confirmed the expression of Deltap53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Deltap53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53. CONCLUSION: Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

Alternative Splicing↗
PubMed1993

Isoelectric charge of recombinant human follicle-stimulating hormone isoforms determines receptor affinity and in vitro bioactivity.

Recombinant human FSH (rhFSH) was obtained by expressing the human FSH alpha- and beta-subunit complementary DNAs in the chinese hamster ovary cell line. Isoforms of rhFSH were resolved into specific isoelectric (pI) fractions by chromatofocusing. rhFSH isoforms ranged from pI 3.0-5.5 with a modal value of pI 4.2. Analysis of the biological activity of specific pI isoforms of rhFSH was undertaken using both the rat granulosa cell aromatase (in vitro) bioassay and a RRA. More acidic isoforms (e.g. pI 3.5) showed significantly lower affinity (P < 0.05) for rat testicular FSH receptors than did the less acidic isoforms (e.g. pI 4.8). Consistent with the receptor binding affinity data, the more acidic fractions resulted in significantly less activation (P < 0.05) of rat granulosa cell aromatase activity, as measured by estrogen production, than did the less acidic isoforms. The observed bioactivities and their correlation with the pI values of the rhFSH isoforms are consistent with observations of differing bioactivities seen in both pituitary and urinary FSH isoforms. These results demonstrate that rhFSH, made in the chinese hamster ovary cell line, is both biologically active and has isoform profiles, and presumably carbohydrate structures, that closely resemble those seen in natural hFSH.

Animals↗
PubMed1993

Pulsatile administration of gonadotropin-releasing hormone does not alter the follicle-stimulating hormone (FSH) isoform distribution pattern of pituitary or circulating FSH in nutritionally growth-restricted ovariectomized lambs.

The experimental induction of puberty by GnRH administration to prepubertal lambs increases serum bioactive FSH (B-FSH) as measured in the rat Sertoli cell aromatase induction bioassay. Serum immunoreactive FSH (I-FSH) levels are unchanged. The increase in serum B-FSH is associated with an increase in the proportion of less acidic and more biopotent FSH serum isoforms. However, it is unknown if this effect of GnRH on serum FSH microheterogeneity is direct or mediated by gonadal factors. We have used the nutritionally growth-restricted ovariectomized lamb as a model of the neuroendocrine regulation of FSH isoform microheterogeneity. With this model, the hypothalamic-pituitary component of the neuroendocrine axis may be isolated from gonadal factors. In the present study, using the nutritionally growth-restricted ovariectomized lamb as a model, we investigated the role of GnRH on the regulation of FSH microheterogeneity. Specifically, we tested the hypothesis that GnRH increases the proportion of the less acidic (more biopotent) serum FSH isoforms. As an in vitro correlate, we investigated the effect of GnRH on gonadotropin secretion and FSH isoform distribution in ovine pituitary explant cultures. Seven ovariectomized nutritionally restricted lambs were administered GnRH (i.v., 2 ng/kg) for 36 h (at 2-h intervals for 24 h, then hourly for the final 12 h). Six others served as controls. Blood samples were withdrawn at 12-min intervals during the last 4 h for the measurement of serum immunoactive LH (I-LH) and I-FSH. Pituitary homogenates and serum from four animals from each group were individually chromatofocused, and the FSH isoform distribution patterns were determined. Pulsatile administration of GnRH to nutritionally growth-restricted lambs increased circulating I-LH concentrations from 0.6 +/- 1.0 to 5.9 +/- 3.1 ng/ml (P < 0.01), but did not significantly change circulating I-FSH (4.9 +/- 1.8 vs. 11.5 +/- 4.2 ng/ml) nor B-FSH concentrations (3.9 +/- 1.2 vs. 5.7 +/- 1.5 ng/ml). The pituitary content of I-FSH, B-FSH, and I-LH were unchanged. Neither serum nor pituitary FSH isoform distribution patterns were altered by pulsatile GnRH administration. However, compared to the pituitary FSH isoforms, a higher percentage of circulating FSH isoforms eluted in the salt peak of both groups of lambs. Similar to the in vivo studies, in vitro, GnRH increased the release of I-LH, as well as I-FSH, from pituitary explants, but did not significantly change the FSH isoform distribution in either the pituitary explant or media.(ABSTRACT TRUNCATED AT 400 WORDS)

Animal Nutritional Physiological Phenomena↗