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PubMed1996

Altering culture conditions reveals strain-related differences in activity and immunoreactive isoforms of 3 beta-hydroxysteroid dehydrogenase-isomerase in mouse Leydig cells.

We previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity is higher in Leydig cells from C57BL/6J mice than those from C3H/HeJ mice. This study examines whether the differences in 3 beta HSD activity in Leydig cells from the two strains of mice are due to the expression of different 3 beta HSD isoforms and if a specific isoform corresponds with the amount of 3 beta HSD activity under various culture conditions. Leydig cells were plated in Waymouth's +15% horse serum (HS) medium. Some cultures were terminated 24 h later after plating (day 1) and assayed for 3 beta HSD activity or immunoreactivity. The remaining cultures were maintained in HS or changed to serum-free medium. We demonstrate for the first time that two 3 beta HSD immunoreactive isoforms are expressed in freshly isolated Leydig cells and those cultured for 1 day. The same two 3 beta HSD isoforms are detectable in both strains. Thus, the previously reported strain-related differences in 3 beta HSD activity are not due to the expression of different isoforms. When cultured for 8 days, the higher molecular weight 3 beta HSD immunoreactive band is no longer detectable in Leydig cells from either strain. When maintained in HS, 3 beta HSD activity in C57BL/6J Leydig cells decreases significantly by day 8, while 3 beta HSD activity in C3H/HeJ Leydig cells does not change through day 8. When Leydig cells were cultured in serum-free medium, 3 beta HSD activity is maintained in cultured Leydig cells from C57BL/6J and significantly increases in C3H/HeJ 3 beta HSD by day 8. These data suggest that HS has a strain-specific inhibitory effect on 3 beta HSD activity, causing a significant decrease in C57BL/6J 3 beta HSD activity and preventing an increase in C3H/HeJ. Densitometric analysis reveals a correspondence between changes in 3 beta HSD activity and the lower molecular weight isoform but not the higher molecular weight isoform. Treatment with cAMP induces the immunoreactive mass of the lower molecular isoform but not the higher molecular isoform of 3 beta HSD. Currently, it is unclear what the function of the higher molecular weight 3 beta HSD isoform is in mouse Leydig cells.

Animals↗
PubMed1992

Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol.

Rats were treated with 3-methylcholanthrene (MC) and DT-diaphorase from liver was partially purified on an azodicoumarol-Sepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and VIb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-1, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms are glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(beta-1,3)N-acetylgalactosamine, while isoform VIb reacts only with biotinylated lectins specific for mannose and N-acetylgalactosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-chromatofocusing revealed that the induction of the isoforms is not uniform, since isform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of only two peaks.

Amino Acids↗
PubMed1994

Selective expression of the high-affinity isoform of the folate receptor (FR-alpha) in the human placental syncytiotrophoblast and choriocarcinoma cells.

The folate receptor (FR), an essential component in the process of folate uptake in various cells, is known to exist in three isoforms, FR-alpha, FR-beta and FR-gamma, with differential tissue expression. Transfer of folate across the human placenta from mother to fetus involves participation of a folate receptor expressed in the syncytiotrophoblast, but the isoform identity of this receptor has not been established. Based on the tissue/cell type from which these isoforms have been cloned, it is currently believed that FR-alpha is the isoform expressed in adult tissues whereas FR-beta is the isoform expressed in fetal tissues including placenta. The present study, undertaken primarily to establish the isoform identity of the FR expressed in the placental syncytiotrophoblast, does not support this currently prevailing nomenclature. Reverse transcription coupled with polymerase chain reaction (RT-PCR) of total/poly(A)+ RNA from placenta, cultured trophoblast cells and JAR choriocarcinoma cells with primer pairs specific for either FR-alpha or FR-beta reveals that while both isoforms are detectable in the whole placental tissue, only FR-alpha is present in the normal trophoblast cells and in the choriocarcinoma cells. Northern analysis with probes designed to distinguish between the mRNA transcripts coding for these two isoforms corroborate the RT-PCR findings. Furthermore, the nucleotide sequences of the PCR products obtained from the trophoblast cells and JAR cells are identical to the nucleotide sequence of the FR-alpha cDNA. These studies establish that it is the FR-alpha isoform, and not the FR-beta isoform, which is selectively expressed in the placental trophoblast cells. FR-beta, which is known to be present in the placenta, most likely arises from the maternal decidua normally associated with this tissue.

Amino Acid Sequence↗
PubMed2004

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes.

OBJECTIVE: The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. METHODS: Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. RESULTS: The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Gö6983, rottlerin, Gö6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Gö6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Gö6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. CONCLUSION: Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

Acetophenones↗
PubMed2006

Differential effects of apolipoprotein E isoforms on lipolysis of very low-density lipoprotein triglycerides.

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor-deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.

Adenoviridae↗
PubMed2004

Modification of the tropomyosin isoform composition of actin filaments in the brain by deletion of an alternatively spliced exon.

Tropomyosin (Tm) in non-muscle cells is involved in stabilisation of the actin cytoskeleton. Some of the 40 isoforms described are found in the brain and exhibit spatial and developmental regulation. Non-muscle isoforms from the gamma Tm gene can be subdivided into three subsets of isoforms differing at the C-terminus, all of which are found throughout the brain and some of which are implicated in different aspects of neuronal function. We have approached the role of different gamma isoforms in neuronal function by knocking out a subset of isoforms. We show here that we can successfully knock out all isoforms containing the brain-specific 9c C-terminus. Brains from these mice did not show any gross abnormalities. Western analysis of adult brains showed that 9c isoforms are reduced in +/- and absent in -/- mice but that a compensation by 9a-containing isoforms resulted in total levels of gamma products remaining the same. No other Tm isoforms were altered. We have therefore specifically altered the Tm composition in these neurons which allows us to study the effects of these changes on the cytoskeleton of neurons during growth, differentiation and maturation and give us insights into the normal roles of these isoforms.

Actins↗
PubMed1998

Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue.

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate-phenol-chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde-1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase-polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase-polymerase chain reaction studies have confirmed expression of the phospholipase C-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle.

Animals↗
PubMed2002

Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts.

BACKGROUND: In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease. METHODS: In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD). RESULTS: A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment. CONCLUSIONS: The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.

Adult↗
PubMed2002

Balloon catheterization induces arterial expression of new Tenascin-C isoform.

Migration of smooth muscle cells (SMCs) across the internal elastic lamina is a key step in the development of atherosclerotic or restenotic plaques. Cell movement is a complex and highly dynamic phenomenon, involving the continuous formation and breakage of attachments with the underlying substratum. Tenascin-C (Tn-C), a counter-adhesive extracellular matrix protein, is comprised of several isoforms with distinct biological activities. Neither the structure nor function of these isoforms in SMCs has been defined. We have used primers and RT-PCR to fully identify Tn-C isoforms expressed by SMCs. Cloning and sequence analysis of the PCR product indicated that SMCs express a Tn-C isoform with only repeats A1 and A2 of fibronectin type III repeats. Using A1A2-specific antibodies, cDNA probes and RNase mapping, we observed that the A1A2 isoform is predominantly expressed by cultured SMCs derived from aorta of newborn rats, and its expression is up-regulated by PDGF-BB. In contrast, the expression of this isoform is markedly down-regulated in the SMCs derived from adult rat aorta. Western and Northern blots of injured rat carotid arteries revealed that the A1A2-isoform is expressed in response to injury. Using cultured SMCs, we found that the recombinant A1A2 protein that was found in the newly discovered Tn-C isoform promotes SMC chemotaxis. We conclude that Tn-C isoforms are expressed in a regulated fashion in vascular system. Our findings suggest a new role of Tn-C isoforms in the remodeling of vascular wall.

Animals↗
PubMed2002

Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia.

Protein kinase C (PKC) is reported to play a role in maturation of the myeloid cell and functions of the mature neutrophil. The neutrophils in chronic myeloid leukemia (CML) exhibit defects in several functions. As a step towards understanding the role of PKC in the defects in function of the leukemic cells, this study investigates the expression of PKC isoforms, their subcellular distribution, levels and kinase activity in the normal and leukemic neutrophils. It also investigates changes in representative PKC isoforms during myeloid maturation. This study confirms the presence of PKC alpha, beta and delta and shows, for the first time, the presence of non conventional PKC isoform theta, atypical PKC isoform lambda/iota and PKC isoform mu in normal human neutrophils. In unstimulated cells all the detected PKC isoforms showed a predominantly cytosolic localisation in normal and CML neutrophils. Cytosol-membrane distribution of PKC alpha and delta were significantly altered in leukemic neutrophils as compared to normal cells. Cytosolic levels of all PKC isoforms were reduced in CML neutrophils with PKC alpha, beta, iota, theta, and mu showing a significant decrease. Cytosolic levels of PKC delta contrary to the trend observed for other PKC isoforms showed a slight increase in CML cells, while its membrane levels were significantly reduced in CML neutrophils. Total PKC kinase activity in CML neutrophil cytosol was significantly reduced, while specific kinase activity of two representative isoforms, PKC alpha and delta, from normal and CML neutrophils were similar, thereby increasing the significance of the altered levels of PKC isoforms in CML, and highlighting their role in the defects in function exhibited by the leukemic neutrophils. The levels of PKC delta and iota increased and decreased respectively as the leukemic myeloid cell matured from the blast to the neutrophil, while the levels of PKC alpha and beta were not altered. This suggests a role for PKC delta and iota in the maturation of the leukemic myeloid cell.

Cell Fractionation↗
PubMed1997

Isolation and nucleotide sequence of human liver glycogen debranching enzyme mRNA: identification of multiple tissue-specific isoforms.

Glycogen storage disease type III (GSD-III) is caused by a deficiency of glycogen debranching enzyme (AGL) activity. Patients are found to have deficient AGL activity in both muscle and liver, and also enzyme deficiency in the liver, but not in muscle. To determine the molecular basis of enzymatic variability in GSD-III and to elucidate the mechanism for control of tissue-specific expression of AGL, we previously cloned and sequenced the human muscle AGL cDNA. Here we report the isolation and nucleotide sequence of liver AGL cDNA and the tissue distribution of the isoform mRNAs. The predominant form of human liver AGL cDNA (isoform 1) contained 400 bp of 5' untranslated region, 4596 bp of coding region, and 2371 bp of 3' untranslated region. The liver AGL mRNA sequence was identical to the previously published muscle sequence (isoform 5) for most of the length, except for the 5' end, in which the liver sequence diverged completely from the muscle sequence. The divergence began with the transcription start point and extended 82 nucleotides downstream from the translation initiation codon. Six isoforms of AGL mRNA were identified and sequenced from liver and muscle. These isoforms differed only at the 5' end. Tissue distribution studies showed that liver, kidney and lymphoblastoid cells expressed predominantly isoform 1; whereas muscle and heart expressed not only isoform 1, but also muscle-specific isoform mRNAs (isoforms 2, 3 and 4). Defining tissue-specific AGL isoform mRNAs is an important step toward understanding the molecular basis of enzymatic variability in GSD-III.

Amino Acid Sequence↗
PubMed1998

Human progesterone receptor A and B isoforms in CHO cells. I. Stable transfection of receptor and receptor-responsive reporter genes: transcription modulation by (anti)progestagens.

A hormone-dependent transcription modulation system was established on the basis of a two-step transfection procedure of the human progesterone receptor isoforms (hPR-A and hPR-B, respectively) and a progesterone receptor-responsive reporter (MMTV-Luc). In the first step, stable transfection of the hPR-A and hPR-B isoform-encoding cDNAs was performed in the steroid receptor-negative CHO K1 cell line. Individual clones were characterized for hPR-isoform expression with respect to Western immuno-blotting, transcriptional activation and hormone binding. With respect to the latter characteristic, individual hPR-isoforms demonstrated similar dissociation constants (Kd for hPR-A: 0.5 +/- 0.3 and hPR-B: 0.8 +/- 0.3 nM, respectively) irrespective of the amount of receptor isoform expressed (Bmax varying from 4.1 to 33.2 nM). The Kd values observed for individual hPR-isoforms were comparable to those found for human breast tumor MCF-7 cells (Kd for hPR-A + hPR-B: 0.6 +/- 0.3 nM). In the second step, hPR-isoform expressing CHO clones were supertransfected with a MMTV-Luc reporter construct resulting in permanent cell lines useful for testing the activity of natural and synthetic steroids in their ability to modulate gene transcription. Both isoform-specific reporter cell lines responded in a similar ranking order towards different progesterone reference compounds such as Org 2058, progesterone (Prog), R5020, norethisterone (NE), and medroxy progesterone acetate (MPA). Moreover, a good correlation was observed between the relative binding affinity (RBA) and the transcriptional activation potency of these compounds towards the individual hPR-isoforms. The latter correlation could not only be demonstrated for the progestagenic agonist reference compounds but was also observed for the progestagenic antagonist reference compounds like Org 33628, Org 31710, RU 38486 and ZK 98299. The major difference observed between the individual PR-isoforms was related to the degree of stimulation of the reporter gene (MMTV-based) within the cellular CHO context. Therefore, these cell lines can be used for the determination and quantitation of the activity of (anti)progestagenic compounds in vitro but may also be useful to predict the activity of compounds in vivo (see also II Comparison of binding, transactivation and ED50 values of several synthetic (anti) progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats).

Animals↗
PubMed1991

Multiplicity of acidic subunit isoforms of crotoxin, the phospholipase A2 neurotoxin from Crotalus durissus terrificus venom, results from posttranslational modifications.

Crotoxin, the major toxin of the venom of the South American rattlesnake, Crotalus durissus terrificus, is made of two subunits: component B, a basic and weakly toxic phospholipase A2, and component A, an acidic and nontoxic protein that enhances the lethal potency of component B. Crotoxin is a mixture of isoforms that results from the association of several isoforms of its two subunits. In the present investigation, we have purified four component A isoforms that, when associated with the same purified component B isoform, produced different crotoxin isoforms, all having the same specific enzymatic activity and the same lethal potency. We further determined by Edman degradation the polypeptide sequences of these four component A isoforms. They are made of three disulfide-linked polypeptide chains (alpha, beta, and gamma) that correspond to three different regions of a phospholipase A2 precursor. We observed that the polypeptide sequences of the various component A isoforms all agree with the sequence of an unique precursor. The differences between the isoforms result first by differences in the length of the various chains alpha and beta, indicating that component A isoforms are generated from the proteolytic cleavage of the component A precursor at very close sites, possibly by the combined actions of endopeptidases and exopeptidases, and second by the possible cyclization of the alpha-NH2 of the N-terminal glutamine residue of chains beta and gamma. These observations indicate that the component A isoforms are the consequence of different posttranslational events occurring on an unique precursor, rather than the expression of different genes.

Amino Acid Sequence↗
PubMed1997

Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres.

Recent studies on single mammalian skeletal muscle fibres revealed a correlation between the kinetics of stretch-induced delayed force increase (stretch activation) and the isoforms of the myosin heavy chain. This observation suggests a causal relation between stretch activation and myosin heavy chain. However, the assumption is weakened by the fact that isoforms of other myofibrillar proteins tend to be coexpressed with myosin heavy chain isoforms. The relation between the isoforms of the tropomyosin-binding troponin subunit and myosin heavy chain is unknown. For a variety of reasons, tropomyosin-binding troponin subunit is a possible candidate for being involved in stretch activation. Therefore, we measured stretch activation of single, maximally Ca(2+)-activated skinned rat skeletal muscle fibres and characterized them by their myosin heavy chain composition, as well as by the isoform species of tropomyosin-binding troponin subunit. Four myosin heavy chain isoforms (I, IIa, IId or IIx and IIb) and six tropomyosin-binding troponin subunit isoforms (TnT1s, TnT2s, TnT1f, TnT2f, TnT3f, TnT4f) were distinguished. The following preferential coexpression patterns of the myosin heavy chain and tropomyosin-binding troponin subunit isoforms were observed: MHCI-TnT1s, MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f. Stretch activation kinetics was found to be correlated with the myosin heavy chain isoform complement also in fibres not displaying one of the preferential MHC-TnTf isoform coexpression patterns. This corroborates the assumption of a causal relation between myosin heavy chain and stretch activation.

Animals↗
PubMed1994

Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha- and beta-subunit isoforms in rat kidney using RT-PCR.

To characterize the expression of genes encoding the alpha- and beta-subunit isoforms of the Na(+)-K(+)-ATPase in rat kidney, we used reverse transcription (RT)-PCR of microdissected renal structures combined with quantitation of subunit isoform mRNAs in the major renal parenchymal zones. Transcripts for alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms were detected by RT-PCR in microdissected glomeruli, proximal convoluted tubules, medullary thick ascending limbs of Henle, cortical and inner medullary collecting ducts. The truncated alpha 1 (alpha 1-T) isoform was also amplified from cortex, outer and inner medulla and isolated glomeruli, but it was not detected in these nephron segments. The DNA sequence of the renal alpha 1-T PCR product was identical to that of the cDNA previously cloned from aortic smooth muscle cells. RNA dot-blot analysis indicated that the alpha 1, alpha 2, and alpha 3 isoforms contributed approximately 70%, approximately 20%, and approximately 10%, respectively, of the total alpha isoform mRNA in each parenchymal zone. RNase protection assays determined that the beta 1 and beta 2 isoforms accounted for approximately 95% and approximately 5%, respectively, of the beta isoform mRNA in each zone. These data provide definitive evidence for the differential expression of mRNAs encoding all the alpha and beta isoforms in the renal parenchyma, and for the coexpression of these isoforms in the nephron segments examined. The results suggest the potential expression of up to eight different Na(+)-K(+)-ATPase isoenzymes in the kidney, and for multiple molecular levels of regulation of renal Na(+)-K(+)-ATPase expression.

Animals↗
PubMed2004

Isoforms of the APC tumor suppressor and their ability to inhibit cell growth and tumorigenicity.

Mutation of the APC tumor suppressor gene is one of the earliest events in the development of most colorectal tumors. The APC gene encodes multiple protein isoforms through a complicated pattern of expression and alternative splicing. The role that each isoform plays in cellular physiology is unknown, although the presence of some of these isoforms in postmitotic cells suggests a role in controlling cell growth or promoting differentiation. Three APC isoforms that differ in their amino-terminal domains were evaluated by gene transfer experiments using a colon cancer cell line that lacks functional APC. All three isoforms alter cellular morphology and affect cell growth by elongating the G1 phase of the cell cycle. The conventional APC and brain-specific APC isoforms suppress the tumorigenic phenotype of cultured cells, while the 0.3 APC isoform does not. In support of these experiments, BrdU incorporation as a marker for S-phase entry occurs at a higher level in transiently transfected cells with 0.3 APC when compared to cells transfected with the other isoforms. All three APC isoforms colocalize with microtubules and dramatically reduce beta-catenin activity to the same extent in transiently transfected cancer cells, suggesting that the different effects of each isoform on tumorigenesis may be nontranscriptional in origin.

Cell Division↗
PubMed1996

Protein kinase C isoforms epsilon, eta, delta and zeta in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states.

The Ca(2+)-insensitive protein kinase C (PKC) isoforms epsilon, eta, delta and zeta are possible direct downstream targets of phosphatidylinositol 3-kinase (P13-K), and might therefore be involved in insulin signalling. Although isoform-specific changes in PKC expression have been reported for skeletal muscle and liver in insulin-resistant states, little is known about these isoforms in adipocytes. Therefore we studied (1) expression and subcellular localization of these isoforms in murine adipocytes, (2) translocation of specific isoforms to membranes in response to treatment with insulin and phorbol 12-myristate 13-acetate (PMA) and (3) regulation of expression in insulin-resistant states. The PKC isoforms epsilon, eta, delta and zeta are expressed in adipocytes. Immunoreactivity for all isoforms is higher in the membranes than in the cytosol, but subcellular fractionation by differential centrifugation shows an isoform-specific distribution within the membrane fractions. PMA treatment of adipocytes induces translocation of PKC-epsilon and -delta from the cytosol to the membrane fractions. Insulin treatment does not alter the subcellular distribution of any of the isoforms. 3T3-L1 adipocytes express PKC-epsilon and -zeta, and PKC-epsilon expression increases with differentiation from preadipocytes to adipocytes. PKC-epsilon expression decreases in an adipose-specific and age/obesity-dependent manner in two insulin-resistant models, the brown-adipose-tissue-deficient mouse and db/db mouse compared with control mice. We conclude that, although none of the isoforms investigated seems to be activated by insulin, the decrease in PKC-epsilon expression might contribute to metabolic alterations in adipocytes associated with insulin resistance and obesity.

3T3 Cells↗
PubMed2002

Influence of endothelin receptor antagonists on myocardial protein kinase C isoforms in uraemic cardiomyopathy.

Increased endothelin-1 (ET-1) levels were found in patients with chronic renal failure and these correlate with the severity of renal failure. Increased mortality due to cardiovascular problems is observed in patients with elevated ET-1 concentrations. The aim of this study was to find out the influence of ET-1 and ET receptor antagonists on myocardial protein kinase C (PKC) regulation in uraemic cardiomyopathy. Male rats were subtotally nephrectomized and treated with an ET(A)-receptor antagonist (30 mg x kg(-1) x day(-1), LU302146) or an ET(AB)-receptor antagonist (30 mg x kg(-1) x day(-1), LU302872) for 12 weeks. One group was left untreated (SNX) and one group was sham-operated (sham). Systolic blood pressure, myocardial weight and the changes of the protein kinase C isoforms in the heart were determined. PKC isoforms alpha and delta were investigated by Western blot analysis using specific antibodies. In the SNX group, systolic blood pressure rose to 154+/-5 mmHg after 12 weeks. The ET(A) receptor antagonist prevented this increase in blood pressure, but ET(AB) antagonism did not. Left ventricular weight increased in SNX; this increase was inhibited by the ET(A) receptor antagonist. In comparison with the sham group, PKC isoform alpha increased by 19% in SNX animals. When the SNX animals were treated with ET(A) or ET(AB) antagonists, PKC isoform alpha levels decreased by 31%. PKC isoform delta levels decreased by 35% in SNX animals. Treatment with both ET(A) or ET(AB) antagonists increased PKC isoform delta levels to normal. In the myocardium of uraemic rats PKC isoforms are differentially regulated with an increase in alpha isoform but a decrease in delta isoform. ET receptor blockers normalize these PKC isoforms.

Animals↗