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PubMed2000

Expression of vascular endothelial growth factor isoforms and platelet-derived endothelial cell growth factor in bladder cancer.

We analyzed the expression of vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) isoforms and platelet-derived endothelial cell growth factor (PDECGF) mRNA in bladder cancer. We also attempted to determine if correlation exists between their expression level and conventional clinical variables in patients with bladder cancer. Tissues obtained from 60 patients with bladder carcinoma were used for analysis. Expression levels of VEGF isoforms and PDECGF were examined using reverse transcription-polymerase chain reaction (RT-PCR). Correlations between the expression levels of each VEGF isoform and PDECGF and histopathologic findings were evaluated. Four VEGF isoforms corresponding to VEGF121, 165, 189, and 206 were detected in bladder cancer tissue by RT-PCR. Gene expression of all VEGF isoforms as a ratio of the target to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed no correlation with pathologic stage of bladder cancer. However, with regard to relative expression levels of VEGF isoform, which is the ratio to the sum of total VEGF isoforms, the levels of VEGF206 and VEGF189 in tumor samples of grade pT2 or higher were significantly lower than those in tumors of grade pT1 or lower (P<.05). In contrast, the levels of VEGF121 in >/=pT2 tumors tended to be higher than those in </=pT1 tumors (P=.056). The expression level of PDECGF as a ratio to GAPDH in pT2</= tumors was significantly higher than that in either pTa or pT1 tumors (P<.05). Moreover, a higher expression level of PDECGF was observed in G3 tumors than in G1 tumors (P<.05). The results indicated that gene expression of VEGF isoforms do not play a significant role in tumor progression or invasion; however, the distribution of VEGF isoforms may play a role in tumor progression of bladder cancer. A high expression level of PDECGF correlated significantly with the tumor progression of bladder cancer.

Journal Article↗
PubMed1999

Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.

BACKGROUND: Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform. OBJECTIVES: We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific. STUDY DESIGN: Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas. RESULTS: Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE. CONCLUSION: Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.

Amino Acid Sequence↗
PubMed1994

Isoforms of the EP3 subtype of human prostaglandin E2 receptor transduce both intracellular calcium and cAMP signals.

The EP3 subtype of prostaglandin E2 receptor transduces diverse physiological responses in mammalian tissues through signaling pathways coupled to heterotrimeric G proteins. Distinct cDNA clones encoding five isoforms of the EP3 receptor were isolated from a human uterus cDNA library. The human EP3 receptor isoforms designated hEP3-I, I', II, III, and IV are derived from alternative RNA splicing and differ only in the distal sequences of their carboxyl-terminal cytoplasmic tails. The unique cytoplasmic tails consist of 31 amino acids for isoforms I and I', 29 for II, 6 for III, and 15 for IV. When stably expressed in CHO cell transfectants, all isoforms exhibited similar EP3-specific binding of [3H]-PGE2 and PGE2 analogs. The EP3-selective agonist M&B 28767 both decreased the intracellular cAMP concentration ([cAMP]i) and increased the intracellular concentration of calcium ([Ca2+]i) with quantitative differences among different isoforms, but none mediated an increase in [cAMP]i. Pertussis toxin treatment completely blocked the decrease in [cAMP]i, but not the increase in [Ca2+]i evoked by M&B 28767. PGE2-induced desensitization of [3H]PGE2 binding by isoforms III and IV was rapid and transient, whereas that by isoform II was slow and persistent. Reverse transcription-PCR amplification of EP3 receptor messages in human kidney and uterine tissue RNA detected expression of all isoforms with different abundancies. The dual signal transduction pathways and distinctive tissue distribution of isoforms of the EP3 receptor are consistent with its mediation of diverse functions of PGE2.

Alprostadil↗
PubMed2004

Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells.

Monocytes and macrophages provide key targets for the action of novel anti-inflammatory therapeutics targeted at inhibition of PDE4 cAMP-specific phosphodiesterases. PDE4 enzymes provide the dominant cAMP phosphodiesterase activity in U937 human monocytic cells. Differentiation of U937 monocytic cells to a macrophage-like phenotype causes a marked reduction in total cellular PDE4 activity. Monocytic U937 cells express the long PDE4A4, PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 isoform. Differentiation of U937 cells to a macrophage-like phenotype causes a marked downregulation of PDE4D3 and PDE4D5, elicits a marked upregulation of PDE4B2 and induces the novel PDE4A10 long isoform. Comparable patterns are found in human peripheral blood monocytes and macrophages differentiated from them. Immunopurification of PDE4 subfamilies identifies long PDE4D isoforms as providing the major PDE4 activity in U937 monocytic cells. In U937 macrophage-like cells, the activity of the short PDE4B2 isoform predominates. No indication of either the expression or induction of PDE4C was evident. Activation of ERK exerts an inhibitory effect on total PDE4 activity in monocytic U937 cells, where the activity of long PDE4 isoforms predominates. The effect of ERK activation is switched to one of overall stimulation of total PDE4 activity in macrophage U937 cells, where the activity of the short PDE4B2 isoform predominates.10 The profound differentiation-induced changes in PDE4 isoform profile identified here suggests that the development of inhibitors specific for particular PDE4 isoforms may allow for selective effects on monocytes and macrophages to be achieved.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Novel protein isoforms of the APC tumor suppressor in neural tissue.

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 - 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of approximately 300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS - APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS-APC protein isoforms also interacts with beta-catenin. BS-APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind beta-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

Adenomatous Polyposis Coli Protein↗
PubMed2002

Expression of the two mRNA isoforms of the iron transporter Nramp2/DMTI in mice and function of the iron responsive element.

Nramp2/DMT1 is a transmembrane proton-coupled Fe(2+) transporter. Two different mRNAs are generated by alternative splicing; isoform I contains an iron responsive element (IRE), whereas isoform II does not. They encode two proteins differing at their C-terminal end and by their subcellular localization. IRE-mediated stabilization of isoform I mRNA is thought to stimulate DMT1 expression in response to iron deficiency. We have measured the two mRNAs by real-time quantitative PCR in several mouse tissues, in normal conditions or following injection of phenylhydrazine, a potent haemolytic agent. Isoform I mRNA is expressed in the duodenum and is induced by stimulation of erythropoiesis, whereas the non-IRE isoform is mostly induced in erythropoietic spleen. Surprisingly, both isoforms are highly expressed in the kidney and are not regulated by erythropoiesis. To evaluate the role of the IRE in regulating isoform I mRNA stability, in response to variations in cell iron status, several constructs were made in pCDNA3 with either a normal or a mutated IRE placed at the 3' end of a stable mRNA. These constructs were transfected into HT29 cells and mRNAs were analysed after growing cells in the presence or absence of exogenous iron. There was no difference in the level of expression of the different messages, suggesting that the IRE does not regulate stability of isoform I mRNA. The half-life of the endogenous IRE-mRNA was also measured following actinomycin D addition in iron- or desferrioxamine-treated cells. Decay of the mRNA was very similar in both conditions. These results suggest that additional transcriptional regulations at the promoter level, or iron-dependent regulation of alternative splicing are likely to participate in the induction of isoform I mRNA by iron deficiency.

Alternative Splicing↗
PubMed1997

Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations.

Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.

Amino Acid Sequence↗
PubMed2000

Identification of the human cytochrome P450 isoforms mediating in vitro N-dealkylation of perphenazine.

AIMS: To identify the human cytochrome P450 (CYP) isoforms mediating the N-dealkylation of the antipsychotic drug perphenazine in vitro and estimate the relative contributions of the CYP isoforms involved. METHODS: cDNA-expressed CYP isoforms were used to identify the isoforms that are able to mediate the N-dealkylation of perphenazine, which is considered a major metabolic pathway for the drug. Using human liver microsomal preparations (HLM), inhibition studies were carried out to establish the relative contributions of the CYP isoforms involved in the N-dealkylation reaction. RESULTS: CYP isoforms 1A2, 3A4, 2C8, 2C9, 2C18, 2C19 and 2D6 were able to mediate the N-dealkylation of perphenazine. Reaction velocities and their relative abundance in HLM suggested that CYP1A2, 3A4, 2C19 and 2D6 were the most important contributors to N-dealkylation. Apparent Km values of CYP1A2 and CYP2D6 were in the range 1-2 microM, and Km values of CYP2C19 and CYP3A4 were 14 microM and 7.9 microM, respectively. Ketoconazole inhibition of N-dealkylation mediated by a mixed HLM indicated that CYP3A4 accounted for about 40% of perphenazine N-dealkylation at therapeutically relevant concentrations. The contribution of the CYP isoforms 1A2, 2C19 and 2D6 amounted to 20-25% each as measured by the percentage inhibition obtained by addition of furafylline, fluvoxamine or quinidine, respectively. HLM-mediated N-dealkylation of perphenazine accounted for 57% of the total amount of substrate consumed during incubation. CONCLUSIONS: The present in vitro study suggests that CYP isoforms 1A2, 3A4, 2C19 and 2CD6 are primarily involved in the N-dealkylation of perphenazine. The relatively modest role of CYP2D6 is at variance with in vivo studies, which indicate a greater contribution of this isoform. Alternative metabolic pathways, corresponding to 43% of the HLM-mediated metabolism of the drug, may depend more strongly on CYP2D6.

Alkylation↗
PubMed1996

Time-related effects of a progestogen on the isoforms of serum gonadotrophins in 17 beta-oestradiol treated post-menopausal women.

OBJECTIVE: It has previously been shown that 17 beta-oestradiol (E2) implants counteract the formation of more acidic isoforms of the gonadotrophins in post-menopausal women. A much lesser effect was observed on the charge of the gonadotrophin isoforms in women with chronic oral daily therapy with 2 mg E2 combined with a progestogen, 1 mg norethisterone acetate (NETA), in spite of similar serum levels of E2 and SHBG. The presence of the progestogen in the latter study may explain the difference observed. The present study investigated the effect of the progestogen NETA on the charge and concentration of serum FSH and LH in E2 implant treated women. DESIGN: A group of 8 post-menopausal women, mean age 65 years (range 50-80 years) treated with 20 mg E2 implants every 6 months, participated in the study. The women were given a daily oral medication of 5 mg NETA for a 4-week period starting at 4 weeks after the insertion of an E2 implant (mean serum E2 420 pmol/l). This treatment with NETA was repeated in 6 of the women starting at 18 weeks after the insertion of the E2 implant (mean serum E2 317 pmol/l). Blood samples were obtained at the start of the NETA therapy, after 2 and 4 weeks of treatment and at 4 weeks after the last NETA treatment. The effects of NETA therapy on the charge of the serum gonadotrophin isoforms was determined by electrophoresis in 0.1% agarose suspension and FSH, LH, E2, and SHBG were determined with fluoroimmunoassays. RESULTS: The NETA treatment decreased the serum FSH and LH levels after 2 weeks to 24 and 23% of the levels before NETA and after 4 weeks to 14.6 and 8.8%, which were 1.3 and 2.2% of the mean levels for non-treated post-menopausal women. Both FSH and LH isoforms became more acidic during the first 2 weeks of treatment. During the following 2 weeks of NETA treatment the isoforms of both FSH and LH became more basic again. Ten weeks later both the concentration and the charge of the gonadotrophins were similar to those before the NETA treatment. The changes in concentration and charge of the gonadotrophins during the second treatment period were similar to those during the first. All the changes were statistically significant (P < 0.05 - < 0.001). The mean SHBG level decreased (P < 0.01) from 84.5 to 70.6 nmol/l after 2 weeks and to 59.9 nmol/l after 4 weeks of NETA treatment and increased (P < 0.01) 10 weeks later to 77 nmol/l. CONCLUSION: In the oestradiol treated women, the effect of the progestogen norethisterone acetate on the charge of the gonadotrophin isoforms was time-related. The oestradiol effect on the charge was counteracted during the first 2 weeks of progestogen treatment and more acidic isoforms appeared in the circulation. During the following 2 weeks the isoforms became more basic again. The levels of the gonadotrophins were efficiently decreased after 2 weeks of progestogen treatment and further decreased after 4 weeks. The time-related effect of the progestogen on the gonadotrophin isoforms may be mediated via changes in the pattern of GnRH release from the hypothalamus. The observed gradual decrease in the SHBG level during the progestogen therapy may cause an increased oestradiol effect on the hypothalamus and pituitary.

Administration, Oral↗
PubMed1999

Acidic isoforms of chorionic gonadotrophin in European and Samoan women are associated with hyperemesis gravidarum and may be thyrotrophic.

OBJECTIVE: There is conflicting evidence concerning the role of human chorionic gonadotrophin (hCG) in the aetiology of hyperemesis gravidarum (HG); particular isoforms of hCG may be the critical factor. Ethnic differences in HG prevalence and putative thyrotrophic effects of hCG may also relate to differences in hCG isoform profiles. To address these issues we examined the relationship of hCG isoforms to HG and thyroid function tests in two groups of women from ethnic backgrounds with significantly different HG prevalence rates. PATIENTS AND DESIGN: We enrolled 10 European and 10 Samoan women with HG and an equally sized non-hyperemetic, gestational stage matched control group. MEASUREMENTS: We administered a questionnaire, generated serum hCG charge-isoform profiles by chromatofocusing and measured the serum concentrations of total hCG, oestradiol (E2), thyrotrophin (TSH) and free thyroxine (FT4). RESULTS: The mean serum total hCG levels were highest in the Samoan hyperemetics (176,268 IU/l), and overall higher in hyperemetics compared with controls (159,770 IU/l vs. 86,420 IU/l, P < 0.001). When compared with controls, hyperemetics displayed increased hCG concentrations in the more acidic half (pH < 4) of the chromatofocusing pH range (89,843 IU/l vs. 41,146 IU/l, P < 0.003). Serum E2 levels did not differ between the four groups, but correlated with the hCG concentration between pH 5.2 and 4.01. Mean serum TSH levels were significantly lower in hyperemetics than in controls (0.33 mIU/l vs. 1.19 mIU/l, P < 0.001) and correlated with the hCG concentration between pH 4.6 and 2.8, while serum FT4 correlated with the hCG concentration below pH 4.0. CONCLUSIONS: Acidic isoforms of hCG may play a role in the aetiology of HG and gestational thyrotoxicosis. Minor ethnic differences in hCG isoform profiles were observed, but the relationship of acidic hCG isoforms to HG and serum thyroid hormone levels was largely independent of the patients' ethnicity. The mechanisms by which acidic isoforms might provoke nausea remain uncertain, but do not seem to involve E2, while the longer half-life of acidic hCG isoforms may result in increased in vivo TSH receptor cross-talk with resultant thyrotrophic effects.

Adult↗
PubMed1997

Differential distribution of thyroid hormone receptor isoform in rat dorsal root ganglia and sciatic nerve in vivo and in vitro.

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific [125I]-labeled T3 binding sites. Thyroid hormone receptors (TR) include several isoforms (TR alpha(1), TR alpha(2), TR beta(1), TR beta(2...)) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms. Using a panel of antisera to specific for the TR alpha-common (alpha(1) and alpha(2)), TR alpha-1 or TR beta-1 isoforms, we detected TRs isoform localization at the cellular level during DRG and sciatic nerve development and regeneration. Immunohistochemical analysis revealed that during embryonic life, sensory neurons express TR alpha-common and TR beta-1 rather than TR alpha-1. The number of TR alpha-common and TR beta-1 positive neurons as well as the intensity of labeling increased during the first two postnatal weeks and remained more or less stable in adult life. TR alpha-1 immunoreactivity, which was undetectable in embryonic sensory neurons, became discreetly visible in neurons after birth. In developing DRG and sciatic nerves, Schwann cells exhibited TR alpha-common and TR alpha-1 rather than TR beta-1 immunolabeling. The appearance of TR alpha-common and alpha-1 isoform immunoreactivity in the sciatic nerve was restricted to a short period ranging from E17 up to two postnatal weeks. By comparing TR alpha-common and TR alpha-1 immunostaining we can deduce that Schwann cells primarily express TR alpha-1. Afterwards, in adult rat sciatic nerve TR alpha isoforms was no more detected. However transection of sciatic nerve caused a reexpression of TR alpha isoforms in degenerating nerve. The prevalence of TR alpha in Schwann cells in vivo was correlated with in vitro results. The differential expression of TR alpha and beta by sensory neurons and Schwann cells indicates that the feedback regulation of circulating thyroid hormone could occur by binding to either the alpha or beta TR isoforms. Moreover, the presence of multiple receptor isoforms in developing sensory neurons suggests that thyroid hormone uses multiple signaling pathways to regulate DRG and sciatic nerve development.

Aging↗
PubMed1994

Role of innervation for development and maintenance of troponin subunit isoform patterns in fast- and slow-twitch muscles of the rabbit.

This study investigates the neural influence on the establishment and maintenance of muscle type-specific expression patterns of the three troponin (Tn) subunits, troponin T (TnT), troponin C (TnC), and troponin I (TnI) during postnatal development and in the adult rabbit. For this purpose, we followed changes in the expression of fast and slow TnT, TnC, and TnI isoforms at the protein and mRNA level in slow- and fast-twitch muscles. During postnatal development all fast Tn isoforms increased in fast-twitch muscle. Sequential transitions (TnTs-->TnT3f-->TnT1f) occurred in the TnT isoform pattern. These changes occurred in parallel with sequential transitions in the pattern of myosin heavy chain (HC) isoforms. Neonatal slow-twitch muscle displayed more mature (slow) isoform patterns for both TnT subunits and myosin HCs than fast-twitch muscle. Although the expression of slow TnC in slow-twitch muscle required innervation, denervation had little effect on slow TnT and TnI which seemed to be controlled by an intrinsic program. In fast-twitch muscle, denervation enhanced the expression of all slow Tn subunit isoforms. In addition, it led to a pronounced increase of the slow TnT2s isoform such that the amount of TnT2s exceeded that of TnT1s. The effects of denervation together with previous data on low-frequency stimulated muscle indicate that the expression of fast Tn isoforms in fast-twitch muscle is neurally controlled. The pattern of slow Tn isoforms in slow-twitch muscle seems to be regulated by an intrinsic program and, in addition, by neural influences.

Animals↗
PubMed1998

Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus).

Group V grass pollen allergens belong to the major grass pollen allergens causing reactions of type I allergy. cDNAs coding for two isoforms of the group V allergen of velvet grass pollen (Holcus lanatus), a widespread grass species, were isolated from a cDNA library by hybridization with a 5'-terminal reverse-transcribed PCR-derived cDNA probe. Amino acid sequences of the two isoforms, designated rHol l 5.01 and rHol l 5.02, revealed high similarity between them (78% identity) and to group V allergens and their isoforms found in other grass species. Recombinant Hol l 5 isoforms were expressed in Escherichia coli and purified as fusion proteins. To compare their immunological reactivities with group-V-specific monoclonal antibodies and patients' IgE, immunoblotting, ELISA and histamine release assay were performed. Interestingly, monoclonal antibody Bo9, specific for group Vb isoforms of timothy grass, bound only to isoform rHol l 5.01, not to rHol l 5.02. On the other side, IgE reactivities of patients' sera revealed no differences between the two isoforms when investigated by immunoblotting and only slight differences when investigated by ELISA. In histamine release assay both isoforms released comparable amounts of histamine from basophils of four individual patients. Thus, the two group V isoforms of velvet grass pollen exhibit differential binding when tested with monoclonal antibodies, i.e. different structure of single epitopes, but negligible differences concerning overall IgE-binding capacity and histamine-releasing capacity.

Allergens↗
PubMed2000

Differential assembly of human tau isoforms in the presence of arachidonic acid.

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.

Actin Cytoskeleton↗
PubMed2002

Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform.

The light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo requires the presence of Rubisco activase, a nuclear-encoded chloroplast protein that consists of two isoforms arising from alternative splicing in most plants. We examined the function of each isoform by characterizing Rubisco activation in transgenic Arabidopsis plants that express only one or both isoforms, as compared with the wild type. In plants expressing only the shorter isoform, Rubisco activity was as high as in the wild type under saturating light, but the activity was not down-regulated at intensities limiting for photosynthesis. In contrast, in plants expressing only the longer isoform, Rubisco activity was down-regulated at limiting light, but the activity was slightly lower and increased much more slowly at saturating light intensities as compared with the wild type. Light regulation of Rubisco similar to that in the wild-type plants was observed in the progeny of a genetic cross of these two transformants in which both isoforms were again present. When the capacity to redox regulate the activity of the larger activase isoform was eliminated by replacement of the critical cysteine residues in the carboxyl-terminal extension unique to this isoform, Rubisco activity in saturating light was similar to the wild type, but the ability of the larger isoform to down-regulate Rubisco activity at limiting light intensities in transgenic plants was almost abolished. These results indicate that the light modulation of Rubisco under limiting light is mainly due to the ability to regulate the activity of Rubisco activase by redox changes in the stroma.

Arabidopsis↗
PubMed1992

Diversity among tight junctions in rat kidney: glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1.

ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.

Aging↗
PubMed1993

Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain.

An isoform of the estrogen receptor messenger RNA (ER-mRNA) was identified in RNA from the brain of lizards and rats. Poly(A)+ RNA from brain and uteri was reverse transcribed using gene-specific primer for the ER. The resulting complementary DNA was amplified in a polymerase chain reaction followed by cloning and sequencing of the amplified products. This isoform lacks exon four and is designated delta 4 ER-mRNA. Although several isoforms of the ER have been described from cancerous cells, to our knowledge, none has been identified previously in the brain. Furthermore, the delta 4 isoform is the only isoform detected in normal tissue. The delta 4 isoform appeared most abundant in RNA from brain tissue, whereas uterine RNA contained only trace amounts of the isoform. Apparently, tissue-specific alternative splicing accounts for these differences in abundance. Because exon four encodes a part of the steroid-binding domain, we predict that the corresponding protein encoded by the isoform will not bind estradiol and may therefore belong to a growing subclass of the steroid/thyroid/vitamin superfamily known as orphan receptors. We predict that the putative delta 4 protein may function as a ligand-independent transcription factor that acts on the same DNA response elements as the conventional ER. The abundance of this isoform in the brain relative to the uterus raises fundamental questions regarding the regulation of estrogen-responsive genes in different tissues.

Animals↗
PubMed1993

Functional expression of the alpha 2 and alpha 3 isoforms of the Na,K-ATPase in baculovirus-infected insect cells.

Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha 2, alpha 3, and beta 1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha 2 and alpha 3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha 2 or alpha 3 with beta 1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-32P]ATP or 32Pi to identify the phosphorylated intermediates of the alpha 2 and alpha 3 isoforms. [3H]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha 3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.

Animals↗