Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “isoforms”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 829 records · Page 46Linked to original sources
PubMed2004

Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene.

PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active. However, one formed (Hyb1) by swapping the unique C-terminal portion of PDE4A7 with that common to all active PDE4 isoforms was catalytically active. Compared with the active PDE4A4B isoform, Hyb1 exhibited a similar K(m) value for cAMP and IC50 value for rolipram inhibition, but was less sensitive to inhibition by Ro-20-1724 and denbufylline, and considerably more sensitive to thermal denaturation. The unique C-terminal region of PDE4A7 was unable to support an active catalytic unit, whereas its unique N-terminal region can. The N-terminal portion of the PDE4 catalytic unit is essential for catalytic activity and can be supplied by either highly conserved sequence found in active PDE4 isoforms from all four PDE4 subfamilies or the unique N-terminal portion of PDE4A7. A discrete portion of the conserved C-terminal region in active PDE4A isoforms underpins their aberrant migration on SDS/PAGE. Unlike active PDE4A isoforms, PDE4A7 is exclusively localized to the P1 particulate fraction in cells. A region located within the C-terminal portion of active PDE4 isoforms prevents such exclusive targeting. Three functional regions in PDE4A isoforms are identified, which influence catalytic activity, subcellular targeting and conformational status.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Immobilized pH gradient focusing of alkaline proteins: analysis of the isoform composition of purified human non-secretory ribonucleases from kidney, liver and spleen.

Previous studies on the isoform composition of human ribonucleases (RNAases) have resulted in confusing and inconsistent results, presumably due to methodological problems in electrofocusing of alkaline proteins. In the present study, immobilized pH gradient (IPG) carrier ampholyte (CA) isoelectric focusing (IEF) and conventional CA-IEF have been evaluated for the analysis of the isoforms of human non-secretory RNAases purified from kidney, liver and spleen. CA-IEF proved unsuitable since the alkaline RNAase isoforms migrated into the cathode. IPG-CA-IEF, however, resolved the RNAase isoforms and marker proteins in the basic region of the gel matrix. The three RNAases had comparable isoform profiles, each with two protein bands with approximate pI values of 10.3 and 10.4. Western blotting showed that the two protein bands of each RNAase were immunoreactive (with polyclonal antibodies that recognize RNAase), indicating that the protein bands are RNAase isoforms. The present results provide reliable pI data on human RNAase isoforms and suggest that IPG-CA-IEF should be a suitable technique for analysing the isoforms of other alkaline proteins.

Blotting, Western↗
PubMed1997

Mitochondrial creatine kinase isoform expression does not correlate with its mode of action.

In adult mammalian ventricular tissue, mitochondrial creatine kinase (mi-CK), which is bound to the outer surface of the mitochondrial inner membrane, is functionally coupled to oxidative phosphorylation. This is shown, in saponin-permeabilized rat ventricular fibres, by a shift in the apparent K(m) of mitochondrial respiration for ADP from 300 +/- 56 microM to 111 +/- 40 microM (P < 0.001) on the addition of 25 mM creatine, due to a local accumulation of ADP close to the ATP/ADP translocator. We have found that in atrial fibres, the apparent K(m) for ADP is high, but is not decreased by creatine, suggesting an absence of coupling in this tissue, as has previously been observed in smooth muscle. mi-CK is encoded by two different genes, which are expressed in a tissue-specific manner: the sarcomeric isoform is expressed in ventricular and skeletal muscles, while the ubiquitous isoform is expressed in smooth muscle, brain and other tissues. In order to determine whether a specific function can be attributed to the expression of a specific isoform, we investigated mi-CK mRNA expression by Northern blot analysis. Hybridization with synthetic oligonucleotides specific for each mi-CK isoform showed the expression of only the sarcomeric isoform in rat atria. This result was confirmed by PCR using primers specific for each isoform. In addition, electrophoretic analysis of CK isoforms showed no difference in the octamer/dimer ratio of mi-CK in the atria and ventricles. In atria, unlike the soleus or ventricles, the maximum potential rate of mitochondrial phosphocreatine synthesis was lower than the maximal rate of ATP production by the mitochondria. The total CK/adenylate kinase ratio was also lower in atria than in the other tissues, suggesting a greater contribution of adenylate kinase to adenine nucleotide compartmentation in this tissue. The functional differences between mi CK in the two cardiac tissues seem to imply a specific arrangement of the proteins in the intermembrane space rather than the expression of specific isoforms.

Adenosine Diphosphate↗
PubMed1997

Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells.

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-alpha, -gamma,-delta, -epsilon and -zeta. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-delta and the partial depletion of PKC-alpha but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-delta and perhaps PKC-alpha are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-delta anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-delta anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-delta and -epsilon anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-delta and -epsilon isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-delta.

3T3 Cells↗
PubMed1997

Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type.

AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5' ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5' end. Extreme 5' regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3' untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins.

AMP Deaminase↗
PubMed1997

Gender-related differences in the regulatory influence of thyroid hormone on the expression of myosin isoforms in young and old rats.

The effects of 4 weeks of thyroid hormone (3.5.3'-triiodothyronine. T3) treatment on the expression of myosin heavy chain (MyHC) isoforms were examined in young (3-6 months; and old (20-24 months) female rats, and compared with those in age-matched male rats (Larsson et al. 1995). In control rats ageing was associated with a type IIA to I MyHC isoform switching in the slow-twitch soleus and a type IIB to IIX MyHC isoform switching in the fast-twitch extensor digitorum longus muscle (EDL). Gender- and muscle-specific differences were observed in the regulation of MyHC isoforms by T3. In the soleus, dramatic downregulation of the type I and upregulation of the type IIA MyHC isoform were observed in both females and males, but upregulation of the IIX MyHC isoform was observed only in male rats. In EDL, T3 treatment had no significant influence on the MyHC isoform composition in the males irrespective of the age of the animal. In the females, on the other hand, T3 treatment resulted in a significant MyHC transformation from IIA to IIB, probably via IIX myosin, in spite of the fact that type IIA mRNA has been reported to be downregulated in both females and males. It is concluded that the regulation of MyHC isoforms by thyroid hormone differs between females and males, presumably as a result of a gender-related difference in the translational or post-translational regulation of MyHC synthesis.

Age Factors↗
PubMed2002

Isoform specificity for oestrogen receptor and thyroid hormone receptor genes and their interactions on the NR2D gene promoter.

Oestrogens are critical for the display of lordosis behaviour and, in recent years, have also been shown to be involved in synaptic plasticity. In the brain, the regulation of ionotropic glutamate receptors has consequences for excitatory neurotransmission. Oestrogen regulation of the N-methyl-d-aspartate receptor subunit 2D (NR2D) has generated considerable interest as a possible molecular mechanism by which synaptic plasticity can be modulated. Since more than one isoform of the oestrogen receptor (ER) exists in mammals, it is possible that oestrogen regulation via the ERalpha and ERbeta isoforms on the NR2D oestrogen response element (ERE) is not equivalent. In the kidney fibroblast (CV1) cell line, we show that in response to 17beta-oestradiol, only ERalpha, not ERbeta, could upregulate transcription from the ERE which is in the 3' untranslated region of the NR2D gene. When this ERE is in the 5' position, neither ERalpha nor ERbeta showed transactivation capacity. Thyroid hormone receptor (TR) modulation of ER mediated induction has been shown for other ER target genes, such as the preproenkephalin and oxytocin receptor genes. Since the various TR isoforms exhibit distinct roles, we hypothesized that TR modulation of ER induction may also be isoform specific. This is indeed the case. The TRalpha1 isoform stimulated ERalpha mediated induction from the 3'-ERE whereas the TRbeta1 isoform inhibited this induction. This study shows that isoforms of both the ER and TR have different transactivation properties. Such flexible regulation and crosstalk by nuclear receptor isoforms leads to different transcriptional outcomes and the combinatorial logic may aid neuroendocrine integration.

3' Untranslated Regions↗
PubMed2002

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.

Animals↗
PubMed1999

Immunological evidence that the beta isoform of Ca2+/calmodulin-dependent protein kinase IV is a cerebellar granule cell-specific product of the CaM kinase IV gene.

Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) exists as two monomeric isoforms, alpha and beta. In this study, we raised an antibody against the beta isoform and provided immunohistochemical evidence for specific expression of the beta isoform in cerebellar granule cells as a single gene-derived translational product distinct from the alpha isoform. Immunohistochemical examination showed that the beta-immunoreactivity was confined to the nuclei of the cerebellar granule cells, in contrast to the more widespread immunoreactivity for the alpha isoform in both nuclei and cytoplasm of the cerebellar granule cells and many other neurons with dominant nuclear localization. In developing cerebella, the beta-immunoreactivity gradually appeared in the internal granule cells during the postnatal 2nd and 3rd weeks, while the alpha-immunoreactivity had already appeared in the internal granule cells in the 1st postnatal week. Unlike the alpha isoform, beta-immunoreactivity was not detected in the Purkinje cells at any developmental stages. The differential expression of the alpha and beta isoforms suggests that each isoform may be involved in different cerebellar functions.

Aging↗
PubMed2003

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

OBJECTIVE: To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. MATERIALS AND METHODS: Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. RESULTS: There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. CONCLUSION: Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

Animals↗
PubMed1999

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis.

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.

Animals↗
PubMed1999

Regulation of Na+, K+-ATPase isoforms in rat neostriatum by dopamine and protein kinase C.

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.

Animals↗
PubMed2002

Transient CD44 variant isoform expression and reduction in CD4(+)/CD25(+) regulatory T cells in C3H/HeJ mice with alopecia areata.

Alopecia areata, an autoimmune disease affecting anagen stage hair follicles, can be induced by grafting spontaneous alopecia areata affected skin to normal-haired C3H/HeJ mice. As the onset of alopecia areata can be significantly retarded by anti-CD44 variant isoform 10 treatment, it was interesting to explore the underlying disease mechanism. Two weeks after transplanting alopecia areata affected skin, expression of CD44 variant isoforms 3, 6, 7, and 10 was strikingly upregulated as compared with sham-grafted mice. By 6 wk after grafting, CD44 variant isoform levels had returned to normal, whereas in draining lymph nodes, CD44 variant isoform expression was slightly decreased. Leukocytes in the skin of mice with chronic alopecia areata expressed a hematopoietic isoform of CD44 and CD44 variant isoform 6 at an elevated level, but CD44 variant isoform 3 expression was reduced. Cytokine expression in leukocytes of chronic alopecia areata affected skin was higher than in normal-haired controls. Cytokine expression also increased postsurgery in sham and alopecia areata grafted mice, but remained elevated only in mice receiving alopecia areata affected skin. Finally, from the skin of mice with chronic alopecia areata and of mice transplanted with alopecia areata affected skin, an increased number of CD4(+) and CD8(+) cells, but a strongly decreased number of CD4(+)/CD25(+) regulatory T cells was recovered. Thus, expression of CD44 variant isoforms is important for the migration of leukocytes during the initial period of alopecia areata. CD44, however, is apparently not involved in the maintenance of the disease state, which is characterized by high cytokine expression levels, an increased number of CD4(+) and CD8+ cells, but a low level of CD4(+)/CD25(+) suppressor cells.

Alopecia Areata↗
PubMedDate not supplied

Spatial and temporal expression of transforming growth factor-beta isoforms during ovine excisional and incisional wound repair.

To elucidate the role for transforming growth factor-beta isoforms (beta(1), -beta(2), and -beta(3)) in wound repair, we used isoform-specific antibodies to detect the spatial and temporal expression of the latent and mature/active transforming growth factor-beta isoforms by immunohistochemical localization through 21 days after excisional and incisional wounding of ovine skin. Although incisional and excisional wounds showed similar patterns of transforming growth factor-beta immunoreactivity, we found a differential temporal and spatial expression of the latent and mature transforming growth factor-beta isoforms throughout wound repair. Specifically, 1 day after wounding, there was a marked increase in transforming growth factor-beta isoforms in the epithelium adjacent to the wound, epidermal appendages, and the cells and matrix of the granulation tissue. At this time, transforming growth factor-beta(3) isoform was the most abundant. Most notably, the epidermis adjacent to the wound was intensely immunoreactive for all transforming growth factor-beta isoforms 1 day after injury. However, the migrating epithelium, derived from both the hair follicles and the wound margins, was completely devoid of immunoreactive transforming growth factor-beta until reepithelialization was complete. Within the inflammatory exudate, there was a distinct band of leukocytes that was immunoreactive for transforming growth factor-beta(2) and -beta(3) 1 day after injury and 1 day later for transforming growth factor-beta(1). Although transforming growth factor-beta(1) and -beta(2), latent transforming growth factor-beta(2), transforming growth factor-beta(3), and latent transforming growth factor-beta(3) immunostaining was present in the numerous fibroblasts and other dermal cells, latent transforming growth factor-beta(1) was only associated with the extracellular matrix. In general, immunoreactivity remained high until day 7 after wounding and slowly subsided over time. However, by day 21, immunostaining had not returned to normal and the original wound was replete with immunoreactive fibroblasts and a dense, immunostained extracellular matrix. Thus, although the dynamic presence of transforming growth factor-beta isoforms exemplifies its positive role in the wound repair process, its persistence together with its known potent effects on matrix accumulation, supports its role in scar formation.

Journal Article↗
PubMed2003

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.

Adult↗
PubMed2002

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes.

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the alpha1, alpha3, beta2, and beta3 subunit isoforms and the gamma modulator were present. Northern analysis of the erythroid progenitor cells again showed that beta2 but not beta1 or alpha2 isoforms were present. The erythroid cells display a unique beta subunit expression profile (called beta-profiling) in that they contain the message for the beta2 isoform but not beta1, whereas leukocytes and platelets are known to have the message for the beta1 but not for the beta2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of alpha1, alpha3, (no alpha2), beta2, beta3, and gamma together now with clear evidence that beta1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that alpha1, alpha3, beta1, beta2, beta3, and gamma are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the alpha1 and alpha3 isoforms of the alpha subunit, the beta1, beta2, and beta3 isoforms of the beta subunit, and the gamma modulator.

Cells, Cultured↗
PubMed1992

Selectively amplified expression of an isoform of the vacuolar H(+)-ATPase 56-kilodalton subunit in renal intercalated cells.

The intercalated cells of the kidney collecting duct are specialized for physiologically regulated proton transport. In these cells, a vacuolar H(+)-ATPase is expressed at enormous levels in a polarized distribution on the plasma membrane, enabling it to serve in transepithelial H+ transport. In contrast, in most eukaryotic cells, vacuolar H(+)-ATPases reside principally in intracellular compartments to effect vacuolar acidification. To investigate the basis for the selective amplification of the proton pump in intercalated cells, we isolated and sequenced cDNA clones for two isoforms of the approximately 56-kDa subunit of the H(+)-ATPase and examined their expression in various tissues. The predicted amino acid sequence of the isoforms was highly conserved in the internal region but diverged in the amino and carboxyl termini. mRNA hybridization to a cDNA probe for one isoform (the "kidney" isoform) was detected only in kidney cortex and medulla, whereas mRNA hybridization to the other isoform of the approximately 56-kDa subunit and to the H(+)-ATPase 31-kDa subunit was found in the kidney and other tissues. Immunocytochemistry of rat kidney with an antibody specific to the kidney isoform revealed intense staining only in the intercalated cells. Staining was absent from proximal tubule and thick ascending limb, where H(+)-ATPase was detected with a monoclonal antibody to the 31-kDa subunit of the H(+)-ATPase. This example of specific amplification of an isoform of one subunit of the vacuolar H(+)-ATPase being limited to a specific cell type suggests that the selective expression of the kidney isoform of the approximately 56-kDa subunit may confer the capacity for amplification and other specialized functions of the vacuolar H(+)-ATPase in the renal intercalated cell.

Adult↗
PubMed1995

Neuronal cell expression of inserted isoforms of vertebrate nonmuscle myosin heavy chain II-B.

Previous work has demonstrated that unique isoforms of nonmuscle myosin heavy chain II-B (MHC-B) are expressed in chicken and human neuronal cells (Takahashi, M., Kawamoto, S., and Adelstein, R. S. (1992) J. Biol. Chem. 267, 17864-17871). These isoforms, which appear to be generated by alternative splicing of pre-mRNA, differ from the MHC-B isoform present in a large number of nonmuscle cells in that they contain inserted cassettes of amino acids near the ATP binding region and/or near the actin binding region. The insert near the ATP binding region begins after amino acid 211 and consists of either 10 or 16 amino acids. The insert near the actin binding region begins after amino acid 621 and consists of 21 amino acids. Using a variety of techniques, we have studied the distribution and expression of the inserted MHC-B isoforms. In the developing chicken brain, mRNA encoding the 10-amino acid insert gradually increases after embryonic day 4, peaks in the 10-14-day embryo, and then declines. In contrast, the mRNA encoding the 21-amino acid insert appears just before birth and is abundantly expressed in the adult chicken cerebellum. There is a marked species difference between the distribution of the inserted isoforms in adult tissues. The mRNA encoding MHC-B containing the 10-amino acid insert near the ATP binding region is expressed at low levels in the adult chicken brain, but makes up most of the MHC-B mRNA expressed in the human cerebrum and approximately 90% of MHC-B in the human retina. It is also expressed in neuronal cell lines. The mRNA encoding MHC-B containing the 21-amino acid insert is abundantly expressed in the chicken cerebellum and human cerebrum, but is absent from the retina and cell lines. Employing human retinoblastoma (Y-79) and neuroblastoma (SK-N-SH) cell lines, an increase in expression of mRNA encoding the 10-amino acid inserted isoform was seen following treatment by a number of agonists or by serum deprivation. In each case, expression of the inserted MHC-B isoform correlated with cell differentiation (neuronal phenotype) and inhibition of cell division. Using a rat pheochromocytoma cell line (PC12), we found that prior to treatment with nerve growth factor (NGF), there was no evidence for either inserted isoform, although noninserted MHC-B was present. NGF treatment resulted in the appearance of mRNA encoding MHC-B containing the 10-amino acid insert, concomitant with neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗