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PubMed2005

Expression and tissue distribution of p63 isoforms in human ocular surface epithelia.

The functional significance of p63 in regulating cell proliferation in various stratified epithelial cells has previously been proposed. More than six isoforms have been reported for this protein; however, it is not yet clearly understood how functionally different these isoforms are. To investigate how these isoforms are used in ocular surface epithelia, we studied the spatial distribution of p63 isoforms within human ocular surface epithelia. Individual layers (basal, intermediate, and superficial) of the human ocular surface epithelia (cornea, limbus, and conjunctiva) were selectively obtained using a laser micro-dissection device. These samples were equally amplified and subjected to RT-PCR analysis with primer pairs, which specifically amplify each of five isoform-determining regions or each of six p63 isoforms. Regarding the N-terminal region, the TA domain was not detected in all samples, while a DeltaNp63 specific region was detected in the basal-intermediate region of all types of epithelia and in the superficial layer of the limbus. Regarding the C-terminal region, an alpha-isoform specific region was detected in all layers of the conjunctiva and limbus, as well as in the basal to intermediate layers of the cornea. A beta-isoform specific region was detected in the basal to intermediate layers of the limbus. A gamma-isoform specific region was detected in almost all layers of all epithelia. Among the six p63 isoforms, only DeltaNp63alpha was detected in the basal to intermediate layers of the limbus and conjunctiva. These results suggest that DeltaNp63alpha is the most dominant isoform within human ocular surface epithelia. This isoform may contribute, at least in part, to the maintenance of cell proliferative capacity within the ocular surface epithelia.

Aged↗
PubMed2001

Characterisation of agonist binding on human 5-HT2C receptor isoforms.

The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.

Animals↗
PubMed2001

Differential crosstalk between estrogen receptor (ER)alpha and ERbeta and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE.

Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERalpha and ERbeta are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERalpha and beta with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE) derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERalpha and beta isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERalpha transactivates to a greater extent than ERbeta. Although neither of the TRbeta isoforms have an effect on ERalpha transactivation from the consensus ERE, the liganded TRalpha1 inhibits the ERalpha transactivation from the consensus ERE. In contrast, the liganded TRalpha1 facilitates ERbeta-mediated transactivation. The crosstalk between the TRbeta isoforms with the ERalpha isoform, on the consensus ERE, is different from that with the ERbeta isoform. The use of a TRalpha1 mutant, which is unable to bind DNA, abolishes the ability of the TRalpha1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Animals↗
PubMed2002

A study on CD45 isoform expression during T-cell development and selection events in the human thymus.

CD45 molecules are known to appear as various isoforms generated by alternative splicing of variable exons 4, 5, and 6, but the detailed profile of CD45 isoform expression during thymocyte development has not been revealed. We examined the CD45 isoforms expressed in the various human thymocytes' subsets defined by CD3, CD4, and CD8 expressions using RT-PCR and 4-color flow cytometry. RT-PCR study revealed that RABC, RAB, RBC, RB, and R0 isoforms were expressed in thymocytes while any of RAC, RA, or RC isoforms were not detected. RABC, RAB and RBC isoforms were expressed at CD3(-)CD4(-)CD8(-) and CD3(+)CD4(+)CD8(-) stages, but were barely detectable at CD3(-)CD4(+)CD8(+) stage. RB isoform was consistently expressed at a relatively high level at all stages. R0 isoform was expressed at a low level at CD3(-)CD4(-)CD8(-) and CD3(-)CD4(+)CD8(-) stages but upregulated at CD3(+)CD4(+)CD8(+) and CD3(+)CD4(+)CD8(-) stages. In combination with the results obtained by 4-color flow cytometric study, CD45 isoform expression on human thymocytes were determined to be RABC(+)RAB(+/-)RBC(+)RB(+)R0(+/-) at CD3(-)CD4(-)CD8(-) stage, RABC(-)RAB(-)RBC(-)RB(+)R0(+) at CD3(-)CD4(+)CD8(-) and CD3(-)CD4(+)CD8(+) stages, RABC(+/-)RAB(+)RBC(+)RB(++)R0(++) at CD3(+)CD4(+)CD8(+) stage, and RABC(+)RAB(+)RBC(+)RB(++)R0(+) at CD3(+)CD4(+)CD8(-) stage. Bcl-2 expression was upregulated between CD3(-)CD4(+)CD8(+)CD45R0(+) and CD3(+)CD4(+)CD8(+)CD45R0(+) stages. Expression of CD45R0 epitope was upregulated between CD3(-)CD4(+)CD8(+)CD69(-) and CD3(+)CD4(+)CD8(+)CD69(+) stages while CD45RA epitope expression was unchanged. Thus, when thymocytes are positively selected, CD45R0 isoform expression seems to be upregulated while CD45RABC isoform expression stays at a very low level. In summary, various isoforms of CD45 were shown to be tightly regulated during thymocyte development and through the selection process.

Antigens, CD↗
PubMed2002

Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG.

We have previously demonstrated that a hyperglycosylated isoform of chorionic gonadotropin (hCG) (B152 hCG) is detected in the blood and urine in early pregnancy and is subsequently rapidly replaced by the hCG isoform (B109 hCG) characteristic of later pregnancy. In the current study we have extended our work on the origin of these isoforms. We have used a combination of in situ and in vitro approaches. Localization studies in placental tissues showed that monoclonal antibody B109 stained very specifically syncytiotrophoblast (STBs) from first and second trimester tissues. At term, STBs exhibited no B109 staining at all. Immunostaining with B152 antibody, that recognize the hyperglycosylated isoform of hCG, revealed only punctate staining of STBs in most villi of first trimester tissue. Both antibodies B109 and B152 failed to stain cytotrophoblasts (CTBs). To assess the functional relevance of these observations we analyzed conditioned media from purified CTBs using two immunometric assays, one of which (B152-B207*) has primary specificity for the hyperglycosylated, choriocarcinoma-like hCG and the other (B109-B108*) having primary specificity for the later pregnancy hCG isoform. Regardless of gestational age, isolated CTBs secreted predominantly B152 hCG isoform in contrast to placental villi (predominantly STBs), which released primarily the B109 hCG isoform. Isolated CTBs, however, failed to immunostain with both B109 and B152 antibodies. To resolve this contradiction, we cultured CTBs in the presence of brefeldin A, a drug known to block secretion by inhibiting protein translocation from the endoplasmic reticulum to the Golgi vesicles. Brefeldin A treated CTBs stained strongly with B109 and did not stain or stained weakly with B152 antibody. We assume that treatment with brefeldin A impaired glycosylation of beta subunit and consequently inhibited the production of hyperglycosylated form of hCG recognized by B152. In summary, our in vitro experiments indicate that both isoforms of hCG are produced by villus CTBs and that the dominant isoform is the one recognized by antibody B152. STBs produce primarily the less glycosylated B109 hCG isoform. This data suggests that at the beginning of pregnancy villus CTBs are the major source of the B152 hCG isoform. This finding is supported by our clinical data that show that the dominant hCG isoform in the blood and urine of pregnant women in the first 6 weeks of pregnancy is recognized by B152 (). The inversion of the B152/B109 ratio observed after 6-7 weeks of pregnancy can be explained by the reduction of number of villus CTBs and/or by maturation of STBs.

Brefeldin A↗
PubMed2002

Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain.

The appearance of 14-3-3 proteins in the cerebrospinal fluid is characteristic of some neurodegenerative conditions which include sporadic Creutzfeldt-Jakob disease. Although 14-3-3 proteins are physiochemically well characterised and are known to be present in neuronal cells little is known of the neuroanatomical localisation of the individual isoforms. Using 14-3-3 isoform specific antibodies we have examined the distribution of the isoforms in normal murine brain and the changes observed during neurodegeneration as a result of ME7 scrapie infection. In normal brain there are two major patterns of immunolabelling. The beta, gamma, eta and zeta isoforms which exhibit a similar distribution pattern showing labelling of neuronal cell bodies often in particular anatomical nuclei. However the individual isoforms exhibit variation revealing subtle differences in location. The tau isoform was found only in the hippocampus and medulla, and the epsilon isoform was found throughout grey matter of the CNS. In the scrapie-infected murine brain, where severe pathological changes occur during the course of the disease, significant differences in the 14-3-3 isoform distribution were observed in the hippocampus and in the thalamus. Importantly, both the 14-3-3 eta isoform and prion protein were seen in the same neurones in both the cerebellar roof nuclei and in the lateral hypothalamic nuclei. Our study of 14-3-3 isoform distribution in adult murine brain clearly demonstrates a heterogeneous pattern of neurolocation for specific 14-3-3 isoforms. The fact that isoform labelling in terminal scrapie CNS is lost in some brain areas, but increases in others, suggests that the processing of these proteins during neurodegeneration may be much more complex than previously recognised.

14-3-3 Proteins↗
PubMed2000

The isoform-specific stretch of hSos1 defines a new Grb2-binding domain.

hSos1 isoform II, defined by the presence of a 15 amino acid stretch in its carboxy-terminal region, exhibits higher Grb2 affinity than hSos1 isoform I. In this study, we investigated the cause for this difference and observed that, in addition to the four currently accepted Grb2-binding motifs, a number of additional, putative SH3-minimal binding sites (SH3-MBS) could be identified. The isoform II-specific 15 amino acid stretch contained one of them. Indeed, we demonstrated by site-directed mutagenesis that these SH3-MBS were responsible for the Grb2 interaction, and we found that C-terminal fragments of the two hSos1 isoforms (lacking the four cannonical Grb2-binding motifs, but containing the SH3-minimal binding sites) were able to bind Grb2, with the isoform II fragment showing higher Grb2 affinity than the corresponding isoform I fragment. Furthermore, we provide evidence that C-terminal truncated mutants of either hSos1 isoform, containing only the SH3-minimal binding sites, were able to originate in vivo stable complexes with Grb2. Although, Grb2-binding remains higher in both full-length isoforms, compared to the C-terminal truncated mutants, these mutants were also able to activate Ras, supporting a potential role of this C-terminal region as negative modulator of Sos1 activity. These findings document the existence of a new, functional, SH3-minimal binding site located in the specific stretch of hSos1 isoform II which may be responsible for the increased Grb2 affinity of this isoform in comparison to isoform I, and for the physiological properties differences between both isoforms. Moreover, these SH3-minimal binding sites may be sufficient to attain stable and functional hSosl-Grb2 complexes.

3T3 Cells↗
PubMed2004

A single-fibre study of the relationship between MHC and TnC isoform composition in rat skeletal muscle.

In the present study, we investigated the possibility that MHC (myosin heavy chain) and TnC (troponin C) isoforms exist in specific combinations in rat-skeletal-muscle fibres. Single fibres (numbering 245) from soleus (predominantly slow-twitch) and sternomastoid (predominantly fast-twitch) muscles of adult rats were analysed for MHC and TnC isoform composition, using alanine-SDS/PAGE for separating MHC isoforms, and a novel method (based on the previously reported influence of Ca2+ on the mobility of Ca2+-binding proteins in SDS gels) for unequivocal identification of TnC isoforms in single-fibre segments. In this study, all fibres that contained only one MHC isoform (slow or fast) contained only the matching TnC isoform and all fibres that contained multiple fast MHC isoforms contained only the fast TnC isoform. Fibres expressing both slow and fast MHC isoforms displayed either both TnC isoforms or only one TnC isoform of a type depending on the relative proportion of fast/slow MHC present. Our results suggest a close relationship between MHC and TnC isoform composition in non-transforming skeletal muscles of adult rat.

Animals↗
PubMed2000

Soluble isoforms of CEACAM1 containing the A2 domain: increased serum levels in patients with obstructive jaundice and differences in 3-fucosyl-N-acetyl-lactosamine moiety.

CEACAM1 (biliary glycoprotein or CD66a) is a member of the carcinoembryonic antigen (CEA) subgroup of the CEA family. Eleven RNA isoforms derived from the splicing of a single CEACAM1 gene have been described. Some of the CEACAM1 isoforms have been recognized by the CD66 antibodies in T and B lymphocytes, natural killer cells, granulocytes and epithelial cells in several human tissues. Although it is also present in soluble form in bile and serum, and elevated levels have been found in the serum of patients with liver diseases, it is not known which isoforms are primarily involved. In order to learn more about the distribution and properties of particular CEACAM1 isoforms, we have prepared a monoclonal antibody specific for the A2 domain of CEACAM1, designated TEC-11. This antibody does not cross-react with other members of the CEA family. Immunoblotting analysis revealed that the TEC-11 epitope was present in all cell types expressing CEACAM1 containing the A2 domain [CEACAM1(A2)], including granulocytes (160 000 MW isoform) and sperm cells (140 000 MW isoform). A 115 000 MW isoform of CEACAM1(A2) was present in human serum, bile, saliva and seminal fluid. Human bile, saliva and seminal fluid also contained the 160 000 MW CEACAM1(A2) isoform. Significantly higher serum levels of the 115 000 MW CEACAM1(A2) isoform were detected in patients with obstructive jaundice. The 160 000 MW isoform of CEACAM1(A2) in bile, but not a 115 000 MW isoform in serum and bile, carried the 3-fucosyl-N-acetyl-lactosamine moiety. The combined data indicate that various isoforms of CEACAM1(A2) are present in different body fluids where they could take part in different CEACAM1-mediated functions.

Animals↗
PubMed2002

Expression of multiple troponin T isoforms in chicken breast muscle regeneration induced by sub-serous implantation.

Chicken fast-muscle type (F-type) troponin T (TnT) isoforms are classified into two types, leg-muscle type (L-type) and breast-muscle type (B-type), which are generated by exclusion and inclusion of exon x series-derived sequences in mRNAs, respectively. The B-type isoforms are further classified into neonatal breast-muscle (BN), young chicken breast-muscle (BC), and adult chicken breast-muscle (BA) subtypes. It is known that the multiple F-type TnT isoforms are transiently expressed in the breast muscle tissue during normal development. To examine whether the transition of the isoforms was fixed in muscle cell lineage, breast muscle pieces (pectoralis major) of 1-day old chicks were cultured under gizzard serous membrane of the same chicks for 60 days at the longest. TnT isoform expression of the implants was monitored by immunoblotting and immunostaining using anti-F-type TnT against both L-type and B-type isoforms, anti-exon x3 against only B-type isoforms, and anti-S-type TnT against slow-muscle-type (S-type) isoforms. Muscle fibers in the implant degenerated first, and then new myotubes expressing L-type isoforms were formed by the fusion of myoblasts from surviving satellite cells. When the maturation of the myotubes into myofibers proceeded, BN-, BC-, and BA-subtype isoforms were expressed in the order of developmental stage specific-manner, indicating that the order of appearance of these isoforms was fixed in muscle cell lineage. In immunostaining of the implants recovered on the 60th day after implantation, at least three kinds of the regenerated myofibers were observed, expressing mainly B-type, both B-type and L-type, and only L-type isoforms. The immunohistochemical results suggested that the regulation of alternative splicing of F-type TnT pre-mRNAs was different among individual myofibers, and that the regulation was programmed in myogenic cells, probably satellite cells, which were the primary source of the fibers.

Animals↗
PubMed2004

Clinico-biological features and prognostic significance of PML/RARalpha isoforms in adult patients with acute promyelocytic leukemia treated with all trans retinoic acid (ATRA) and chemotherapy.

Debate exists over the clinical relevance of molecular heterogeneity of acute promyelocytic leukemia (APL). Based on the genomic breakpoint in PML gene, three different PML/RARalpha isoforms are recognized: intron 3 [short (S)], intron 6 [long (L)] and exon 6 [variable (V)]. Studies on the prognostic significance of PML/RARalpha isoforms have reported contradictory results. This discrepancy may be related to differences in the treatment protocols, as some studies used ATRA alone during induction therapy. We analyzed the clinical course of 61 consecutive newly diagnosed patients with a genetically confirmed diagnosis of APL, treated with ATRA and chemotherapy at Princess Margaret Hospital from January 1994 to January 2002. The results of RT PCR at diagnosis were available on 48 patients. In this study, we report on clinico-biological features and prognostic significance of PML/RARalpha isoforms in these 48 patients. Of 48 patients, 19(40%) had the S isoform and 29 (60%) had the L/V isoform. Median white blood cell (WBC) count for patients with S isoform was 8.6 [interquartile range Q1-Q3 i.e. IQR 3.2-29] compared to 1.8 [IQR 1.0-4.9] for the L/V isoform group (P 0.001). No difference was seen in number of patients achieving of molecular remission after induction and consolidation treatment in the two-isoform groups. The patients with S isoform had significantly inferior relapse-free survival (RFS) at 3 years compared to L/V isoform patients [48% (95% C.I. 19 77) vs. 92% (95% C.I. 82-100), P0.006]. In a univariate analysis, S isoform status (P 0.006) and high WBC count ( > or = 5 x 10(9)+/l) (P 0.017) were significant prognostic factors for RFS. No difference in overall survival was seen between the two isoform groups (P 0.35). Our results suggest that based on molecular characterization, it may be possible to identify a subgroup of APL patients at higher-risk of relapse.

Adolescent↗
PubMed2001

Leucocyte intracellular pH and Na+/H+ exchanger isoform-1 activity in postpartum women with pre-eclampsia.

OBJECTIVE: To investigate leucocyte Na+/H+ exchanger isoform 1 activity in postpartum pre-eclamptics. DESIGN: Exchanger isoform-1 activity and intracellular resting pH were established in leucocytes isolated from two study groups. SAMPLE: Leucocytes isolated from 10 women who had had pre-eclamptic pregnancies more than five months postpartum, and from 10 age-matched normotensive women who were more than five months postpartum. SETTING: Hypertension Clinic, Antenatal Assessment Area, Leicester Royal Infirmary. METHODS: A well validated technique involving flurometry using a pH sensitive dye (BCECF-AM) was performed to determine exchanger isoform-1 activity and intracellular pH. Determination of exchanger isoform-1 protein abundance was performed by western blotting. Exchanger isoform-3 protein abundance was examined to rule out the possibility of activity due to this particular isoform. RESULTS: Intracellular pH was significantly lower in the postpartum pre-eclamptic group (7.11 +/- 0.02), compared with the postpartum normotensive controls (7.33 +/- 0.04; P < 0.001). Exchanger isoform-1 efflux rate (in mmol/L/minute) was significantly higher in the postpartum pre-eclamptic group (35.91 +/- 3.1), compared with the postpartum normotensives (23.94 +/- 2.0; P = 0.005). Exchanger isoform-1 protein density was established to be similar among the two subject groups. No exchanger isoform-3 protein was identified by western blotting. CONCLUSION: Our results suggest that elevated exchanger isoform-1 activity is an important finding in women who have suffered from pre-eclampsia. This increased activity is not due to an increase in exchanger isoform-1 protein abundance or the presence of exchanger isoform-3.

Adult↗
PubMed2005

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner.

We found that Notched-fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP- and QAE-sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 degrees C for the QAE1 isoform, 0.11 degrees C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes 'active' when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co-operative manner.

Amino Acid Sequence↗
PubMed2004

Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers.

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr(2+) of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr(2+) concentration ([Sr(2+)]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr(2+) dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr(2+) than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (-log(10) [Sr(2+)]) curves is tightly (r(2) = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr(2+) activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr(2+), TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers.

Animals↗
PubMed2000

Progesterone receptor isoforms, PR-B and PR-A, in breast cancer: correlations with clinicopathologic tumor parameters and expression of AP-1 factors.

In the present study, we used Western blot analysis to determine the expression of the progesterone receptor (PR) isoforms, PR-B and PR-A, in breast tumors (n = 53), and correlated the expression patterns of the two isoforms with the clinicopathological parameters of these tumors and with expression of the AP-1 family of transcription factors. Expression of the two PR isoforms correlated significantly with each other, indicating that the expression of the two isoforms is probably regulated in a correlated fashion. Expression of both isoforms correlated significantly with expression of the estrogen receptor (ER). Furthermore, expression of PR-B was found to correlate significantly with the absence of ErbB2/neu. For the AP-1 factors, Fra-1 expression showed an inverse correlation with PR-B expression. In contrast, expression of FosB correlated significantly with expression of both isoforms, and the association was stronger with PR-B expression. An analysis of the ratio of expression of the two isoforms showed that most of the tumors expressed PR-A levels which were equal or higher than the corresponding PR-B expression levels (together 94% of the analyzed tumors) indicating that, in mammary carcinomas, a predominance of the PR-A isoform over the PR-B isoform seems to be the case. While there was no statistically significant correlation with age, staging and histological type, expression of both isoforms correlated with a more differentiated phenotype (G1/G2 grading). However, this association was stronger for PR-B. Also, a PR-A < or = PR-B expression level was associated with G1/G2 grading, while a PR-A > PR-B expression level showed an association with a more undifferentiated phenotype (G3 grading). The expression level of the two PR isoforms might prove to be of prognostic and/or predictive value, especially since the two isoforms have been shown to be functionally different and to modulate the response of tumor cells to progestins and antiprogestins differently.

Blotting, Western↗
PubMed1999

Functional analysis of dynamin isoforms in Drosophila melanogaster.

Dynamin and dynamin-like proteins are required for endocytosis, synaptic vesicle recycling and membrane trafficking. From the shibire locus in Drosophila melanogaster, six different isoforms of dynamin are generated by alternative splicing. However, the roles of the individual isoforms in cellular processes are unknown. To investigate functional differences among the dynamin isoforms, transgenic lines were generated that individually expressed each of 3 different isoforms under UASGAL4 control. The expression of the isoforms was controlled by neural promoter (elav)-driven GAL4, or by a shibire-promoter driven GAL4 transgene. Reporter gene expression indicated that the shi promoter is active during embryogenesis, and in larvae, pupae, and adults in a pattern consistent with normal dynamin expression. To assay for the ability of dynamin isoforms to function in vivo, the isoforms expressed via these GAL4 drivers were tested for the ability to rescue shibire phenotypes. When expressed at very high levels all individual isoforms tested rescued the temperature-sensitive paralytic phenotype of shi(ts2) flies; however, this rescue was partial, suggesting that no single tested isoform is sufficient for synaptic vesicle recycling in vivo. When tested for ability to rescue lethality induced by heat-pulsing larvae during development, shi- promoter driven expression of individual isoforms conferred significant resistance to heat treatment during larval development. However, all 3 isoforms were unable to rescue the lethality of shi12-12B mutants which are severely hypomorphic (or null) for shibire function. Taken together, these observations suggest that individual shibire isoforms have specific molecular activities in vivo.

Alternative Splicing↗
PubMed1998

The mechanisms of action of alpha- and beta-isoforms of antithrombin.

Antithrombin (AT) is the most important physiological inhibitor of thrombin. This effect can be increased more than a 1000-fold by heparin and heparin-like glycosaminoglycans, which induce a conformational change in the molecule. Two isoforms of AT exist in plasma: alpha and beta. The beta-isoform lacks one of four carbohydrate side-chains that are present on the alpha-isoform. The beta-isoform, which constitutes approximately 10% of plasma AT, has a higher affinity for heparin and heparin-like glycosaminoglycans than the alpha-isoform. In contrast to their distribution in plasma, the two isoforms of AT appear to be present in the same proportions in the vessel wall. After balloon injury of rabbit aorta, thrombin can be detected in the vessel wall, an effect that is inhibited by treatment with AT. The inhibitory effect of AT on thrombin coagulant activity in the injured vessel wall is attributable to the beta-isoform. The appearance of thrombin in the injured vessel wall can also be inhibited by heparin treatment, but this requires heparin to be circulating in plasma at the time of excision of the injured vessel wall. Thrombin has been suggested as a mitogen for smooth muscle cells. This effect of thrombin can be inhibited by AT, an inhibition that is increased by heparin in a concentration-dependent manner. The alpha-isoform of AT has a lower inhibitory capacity for the thrombin-induced proliferation of smooth muscle cells in the absence of heparin, compared with the beta-isoform, which is an effective inhibitor alone. This indicates that the beta-isoform of AT may use glycosaminoglycans produced by smooth muscle cells as a cofactor. In conclusion, the beta-isoform of AT appears to be an effective inhibitor of the thrombin coagulant activity induced by vessel wall injury. It is also a more effective inhibitor of the thrombin-induced proliferation of smooth muscle cells than the alpha-isoform.

Animals↗
PubMed2000

Androgen receptor isoforms in human and rat prostate.

AIM: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. METHODS: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4 degrees C, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. RESULTS: From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41 (17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. CONCLUSION: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.

Androgens↗