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PubMed1995

Distinct functional properties of three human paired-box-protein, PAX8, isoforms generated by alternative splicing in thyroid, kidney and Wilms' tumors.

The mammalian paired box (Pax) genes encode a family of transcription factors involved in embryogenesis. The murine and human Pax8 genes are expressed in developing and adult thyroid as well as in the developing secretory system and at the lower level in adult kidney. In the secretory system expression is localized to the induced, extensively differentiating parts that undergo a transition from mesenchyme to epithelium. The human PAX8 gene generates at least five different alternatively spliced transcripts encoding different PAX8 isoforms. These isoforms differ in their carboxy-terminal regions downstream of the paired domain that has been shown previously to be responsible for the DNA binding. The PAX8a isoform contains a 63 amino-acid serine-rich region that is absent in the isoform PAX8b whereas PAX8c reveals a novel 99-amino-acid proline-rich region. This proline-rich region arises due to an unusual reading-frame shift in the PAX8 transcript. RNAse protection and RT(reverse transcription)-PCR analysis show the expression of all three PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. Band-shift assay indicates a greatly reduced binding affinity of the isoform PAX8c to a DNA sequence from the promoter of the thyroperoxidase gene compared to the binding of PAX8a and PAX8b to this sequence. Deletion analysis of murine PAX8a indicates that its activating domain residues at the carboxy terminus of the protein which is shared by isoforms PAX8a and PAX8b. In accordance with these data PAX8a and PAX8b activate transcription from a thyroglobulin promoter as well as from a cotransfected synthetic PAX8-specific promoter/chlorampericol acetyltransferase (CAT) reporter containing a Pax8-binding oligonucleotide in front of the basal herpes simplex virus thymidine kinase (HSV-TK) promoter (P11/12-TK-CAT). However if the basal HSV-TK promoter of this reporter is substituted by a minimal adenovirus E1b TATA element, PAX8a and PAX8b fail to activate transcription. Of the three chimaeric forms containing the GAL4 DNA-binding domain at the amino-terminal end fused to the corresponding carboxy-terminal regions of the PAX8 isoforms beginning immediately downstream of the paired domain only a GAL4-PAX8b fusion significantly activates transcription from a cotransfected GAL4-specific upstream-activating-sequence (UAS)-TK-CAT reporter. Substitution of the basal HSV-TK promoter in this reporter by the minimal E1b TATA element does not affect this activation. These results indicate that the PAX8 isoforms display different functional properties and may also function differently in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternative Splicing↗
PubMed1996

Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles.

The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross-reinnervation by a fast nerve of the slow-twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast-twitch Semimembranous accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow-type (1S', 1S and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type 1 MyLC corresponding most probably to an embryonic MyLC. Five-month cross-reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co-expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type II fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast-type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98% type IIx/d and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow-type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast-twitch or slow-twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast-twitch SMa muscle, and is barely expressed in cross-reinnervated slow-twitch SMp muscle. In contrast, type IIa and type IIx/d are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I.

Animals↗
PubMed1997

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV).

Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alphabeta(II) and alphabeta(IV) isoforms than to the alphabeta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alphabeta(II), alphabeta(III) and alphabeta(IV) were 13358, 4558 and 10828 M(-1) s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33 x 10(5), 1.56 x 10(5) and 5.44 x 10(5) M(-1), respectively. The differences in kinetic parameters among different beta-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.

Colchicine↗
PubMed1997

Structural differences in the crossbridge head of temperature-associated myosin subfragment-1 isoforms from carp fast skeletal muscle.

We determined the primary structures of the three acclimation-temperature-associated isoforms of myosin subfragment-1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending 5'-regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10 degrees C- and 30 degrees C-acclimated carp, together with the region that encodes an intermediate structure [Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M. & Watabe, S. (1997) J. Exp. Biol. 200, 27-34]. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10 degrees C- and intermediate-type, between the 10 degrees C- and 30 degrees C-type, and between the intermediate- and 30 degrees C-type myosin heavy chains, respectively. However, isoform-specific differences were clearly observed between the 10 degrees C- and 30 degrees C-type heavy chains in the first 60 amino acid residues from the N-terminus, where the intermediate-type showed an intermediate feature in its sequence compared to the 10 degrees C- and 30 degrees C-type isoforms. Other striking differences were observed in two surface loops between the 10 degrees C- and 30 degrees C-type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP-binding pocket, and six out of twenty were different in loop 2 on the actin-binding site. The loops connecting beta-sheets that are known to surround the ATP-binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10 degrees C- and intermediate-type isoforms were significantly higher in carp acclimated to 10 degrees C and 20 degrees C than carp acclimated to 30 degrees C, whereas the level of the 30 degrees C-type isoform was significantly higher in carp acclimated to 30 degrees C than those acclimated to 10 degrees C and 20 degrees C.

Amino Acid Sequence↗
PubMed1999

Isoform-specific regulation of the sodium pump by alpha- and beta-adrenergic agonists in the guinea-pig ventricle.

1. Guinea-pig ventricle was used in the RNase protection assays to determine which alpha-isoforms of the Na+-K+ pumps are present, and ventricular myocytes were used in whole cell patch clamp studies to investigate the actions of alpha- and beta-adrenergic agonists on Na+-K+ pump current. 2. RNase protection assays showed that two isoforms of the alpha-subunit of the Na+-K+-ATPase are present in guinea-pig ventricle. The mRNA for the alpha1-isoform comprises 82 % of the total pump message, the rest being the alpha2-isoform. 3. We have previously shown that beta-adrenergic agonists affect Na+-K+ pump current (Ip) through a protein kinase A (PKA)-dependent pathway. We now show that these beta-effects are targeted to the alpha1-isoform of the Na+-K+ pumps. 4. We have also previously shown that alpha-adrenergic agonists increase Ip through a protein kinase C (PKC)-dependent pathway. We now show that these alpha-isoform effects are targeted to the alpha2-isoform of the Na+-K+ pumps. 5. These results suggest the effects of adrenergic activation on Na+-K+ pump activity in the heart can be regionally specific, depending on which alpha-isoform of the Na+-K+ pump is expressed.

Adrenergic alpha-Agonists↗
PubMed1993

Differential expression and subcellular localization of protein kinase C alpha, beta, gamma, delta, and epsilon isoforms in SH-SY5Y neuroblastoma cells: modifications during differentiation.

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis.

Cell Differentiation↗
PubMed2004

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms.

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.

Amino Acid Sequence↗
PubMed2005

Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen.

Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.

Amyloid beta-Peptides↗
PubMed1996

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U-purinoceptors. Both downregulation and inhibition studies show that PKC-alpha is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-epsilon (PKC-zeta is unresponsive to PMA), or an as yet uncharacterized PKC isoform.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

CD44 isoform expression in periodontal tissues: cell-type specific regulation of alternative splicing.

CD44 functions as a receptor for various extracellular matrices and plays crucial roles in homotypic and heterotypic cell-cell interactions. Recently, the molecular structure of CD44 has been extensively analyzed and multiple isoforms produced by alternative splicing of messenger RNA have been identified. In this study, we examined the expression of CD44 isoforms on different cell types isolated from periodontal tissue. In order to examine tissue differences in CD44 isoform expression, we established in vitro cell culture of human gingival fibroblasts (HGF), human periodontal ligament cells (HPDL) and human gingival epithelial cells (HGEC). These cells all expressed CD44 protein and messenger RNA. However, immunoprecipitation and Northern blot analysis revealed that HGEC expressed larger CD44 isoforms than HGF and HPDL. Reverse transcription-polymerase chain reaction with primers flanking the insertion site of alternatively spliced exons was used to study details of the heterogeneity. All cells examined expressed a major band in the absence of alternatively spliced exons and additional larger bands. In particular, HGEC contained more abundant high molecular mass species. In vitro stimulation by IL-1 beta, TNF alpha or phorbol 12-myristate 13-acetate induced an increase in total CD44 messenger RNA in HGF but not change in overall patterns of CD44 isoform expression. However, the isoform expression of HGEC was sensitive to cell density. The amount of larger isoform was decreased by culturing cells beyond confluence. These findings suggest that CD44 isoform expression is cell type-specifically regulated in periodontium and altered according to growth phase of HGEC.

Alternative Splicing↗
PubMed1994

Myosin heavy chain isoforms in single fibres from m. vastus lateralis of sprinters: influence of training.

The myosin heavy chain (MHC) composition of single fibres from m. vastus lateralis of a group of male sprint athletes (n = 6) was analysed, before and after a three months period of intensive strength- and interval-training, using a sensitive gel electrophoretic technique. Significant improvements were observed after training in almost all of a series of performance tests. After training the sprinters revealed a decrease in fibres containing only MHC isoform I (52.0 +/- 3.0% vs. 41.2 +/- 4.7% (mean +/- SE) (P < 0.05)) and an increase in the amount of fibres containing only MHC isoform IIA (34.7 +/- 6.1% vs. 52.3 +/- 3.6% (P < 0.05)). Fibres showing co-existence of MHC isoforms IIA and IIB decreased with training (12.9 +/- 5.0% vs. 5.1 +/- 3.1% (P < 0.05)). Only one out of 1000 fibres analysed contained only MHC isoform IIB. In contrast, a higher amount of type IIB fibres (18.8 +/- 3.6% vs. 10.5 +/- 3.9%, (P < 0.05)) was observed with myofibrillar ATPase histochemistry. The majority of histochemically determined type IIB fibres of sprinters seems therefore to contain both MHC isoforms IIA and IIB. Sprint-training appears to induce an increased expression of MHC isoform IIA in skeletal muscles. This seems related to a bi-directional transformation from both MHC isoforms I and IIB towards MHC isoform IIA.

Adult↗
PubMed2004

Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle.

Characterization of expression of, and consequently also the acute exercise effects on, Na(+),K(+)-ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at approximately 40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) mRNA and crude homogenate protein expression, using Real-Time RT-PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na(+),K(+)-ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 +/- 69 s; mean +/-s.e.m.) immediately increased alpha(3) and beta(2) mRNA by 2.4- and 1.7-fold, respectively (P < 0.05), whilst alpha(1) and alpha(2) mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for beta(1) and beta(3) mRNA, reflecting variable time-dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the alpha(1)-alpha(3) and beta(1)-beta(3) isoforms. Thus, human skeletal muscle expresses each of the Na(+),K(+)-ATPase alpha(1), alpha(2), alpha(3), beta(1), beta(2) and beta(3) isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na(+),K(+)-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na(+),K(+)-ATPase up-regulation.

Adult↗
PubMed2003

Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat.

The aim of this study was to determine the selectivities of chemical inhibitors for human cytochrome P450 (P450) isoforms toward the corresponding rat P450 isoforms by using cDNA-expressed rat P450s (CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, CYP3A1, and CYP3A2). Among the inhibitor probes for human P450s used in this study, only sulfaphenazole showed a selective inhibitory effect on the activity of the corresponding rat P450 isoform (CYP2C6). Furafylline also preferentially inhibited the activity of rat CYP1A2. However, methoxalen and ketoconazole more strongly inhibited the activities of other P450 isoforms than those of the corresponding rat P450 isoforms, CYP2A1 and CYP3A1/2, respectively. On the other hand, quinidine and aniline had little effect on the activities of the corresponding rat P450 isoforms, CYP2D2, and rat CYP2E1, respectively. These results suggest that chemical probes that have been used for human P450 isoforms do not always exhibit the same selectivity for the corresponding rat P450 isoforms. However, it appears that sulfaphenazole can be used as a selective inhibitor for rat CYP2C6. In addition, furafylline may also be a relatively selective inhibitor for rat CYP1A2.

Aniline Compounds↗
PubMed2004

Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid.

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

Acetone↗
PubMed2005

The role of protein kinase C isoforms in modulating injury and repair of the intestinal barrier.

Gastrointestinal cells express a diverse group of protein kinase C (PKC) isoforms that play critical roles in a number of cell functions, including intracellular signaling and barrier integrity. PKC isoforms expressed by gastrointestinal epithelial cells consist of three major PKC subfamilies: conventional isoforms (alpha, beta1, beta2, and gamma), novel isoforms (delta, epsilon, theta, eta, and mu), and atypical isoforms (lambda, tau, and zeta). This review highlights recent discoveries, including our own, that some PKC isoforms in gastrointestinal epithelia monolayer cell culture are involved in injury to, whereas others are involved in protection of, intestinal barrier integrity. For example, certain PKC isoforms aggravate oxidative damage, whereas others protect against it. These findings suggest that the development of agents that selectively activate or inhibit specific PKC isoforms may lead to new therapeutic modalities for important gastrointestinal disorders such as cancer and inflammatory bowel disease.

Animals↗
PubMed2002

Differential internalization of the prostaglandin f(2alpha) receptor isoforms: role of protein kinase C and clathrin.

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F(2alpha) (PGF(2alpha)). Alternative mRNA splicing gives rise to two isoforms, FP(A) and FP(B), which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP(A) and FP(B) receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF(2alpha) and were examined by immunofluorescence microscopy. In the absence of PGF(2alpha), FP(A)-expressing cells were labeled predominantly on the cell surface; however, FP(B)-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP(B) isoform. After treatment with PGF(2alpha), FP(A)-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP(A)-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF(2alpha)- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP(B) isoform, suggesting that the mechanisms of receptor internalization differ between the FP(A) and FP(B) isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF(2alpha)-induced internalization of the FP(A) isoform but did not block constitutive internalization of the FP(B) isoform. In conclusion, the FP(A) receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP(B) isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.

Cell Line↗
PubMed1995

Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Alternative Splicing↗
PubMedDate not supplied

Ontogenic changes in hepatic metallothionein isoforms in prenatal and newborn rats.

The relative quantities of the two metallothionein isoforms (MT-1 and MT-2) in prenatal and newborn rat livers have been quantified using a competitive enzyme-linked immunosorbent assay with two specific polyclonal antisera. Both MT isoforms were detected in fetal liver on gestation day 16 and their levels continued to increase into the neonatal period. A concomitant increase was observed in plasma MT levels in the pregnant rats. The level of MT-2 was higher (75%) than that of MT-1 in the liver of both prenatal and neonatal rats. Both hepatic MT isoforms showed a significant correlation (P < 0.05) with the hepatic Zn level. Immunohistochemical localization of MT isoforms showed no apparent difference in intracellular distribution. Both MT isoforms were mainly cytoplasmic in fetal rat liver. Intense intranuclear staining was observed in newborn rat liver when the total MT concentration reached a maximum of over 1 mg MT/g tissue. The nuclear MT staining decreased with age, and at day 7, approximately equal concentrations of MT were found in nucleus and cytoplasm. When the newborn rats (1 day old) were injected with Zn (10 mg/kg), both MT isoforms were induced on days 2 and 7. Injection of Cd (1 mg/kg) increased the hepatic MT levels only on day 7. The level of MT-2 remained higher than that of MT-1 in all cases. These results suggest that both MT isoforms play a similar role in the metabolism of Zn during early development and the relative abundance of the MT isoforms may be a species-specific phenomenon.

Aging↗