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PubMed2004

Expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature.

PURPOSE: Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion-signaling molecule with important roles in angiogenesis and inflammation. The alternative splicing of the PECAM-1 cytoplasmic domain modulates its adhesive properties during vascular development and angiogenesis. This study was designed to identify alternatively spliced PECAM-1 isoforms in human and murine retina and during postnatal vascularization of murine retina. METHODS: RT-PCR, DNA sequencing, and Western blot analysis were utilized to examine the expression pattern of alternatively spliced PECAM-1 isoforms in human and mouse retina, and during vascularization of murine retina. RESULTS: We demonstrate that the PECAM-1 cytoplasmic domain undergoes alternative splicing generating multiple isoforms in vascular beds of human and mouse retina. We detected full length PECAM-1 and an isoform that lacks exon 14 (Delta14) in human retina. Seven isoforms of PECAM-1 were detected in murine retina. These included full length, Delta12, Delta14, Delta15, Delta1215, Delta1415, and Delta12, 1415 PECAM-1 isoforms. The full length PECAM-1 was the predominant isoform detected in human retina, while the isoform lacking exon 1415 (Delta1415) was the predominant isoform detected in murine retina. In addition, the expression pattern of PECAM-1 isoforms changed during vascularization of murine retina. CONCLUSIONS: PECAM-1 mRNA undergoes alternative splicing generating multiple isoforms in human and murine retinal vasculature. The regulated expression pattern of these isoforms may influence endothelial cell adhesive properties impacting vasculogenesis and angiogenesis.

Alternative Splicing↗
PubMed1997

Vertebrates have conserved capping protein alpha isoforms with specific expression patterns.

Capping protein (CP), a ubiquitous actin binding protein composed of an alpha and a beta subunit, is important for actin assembly and cell motility. Lower organisms have one gene and one isoform of each subunit. Chickens have two very similar alpha-subunit isoforms. To determine if vertebrates in general contain multiple alpha isoforms and if those alpha isoforms have conserved sequences, we isolated and analyzed alpha subunit cDNA's in mice and humans. Both mice and humans also have two alpha isoforms. Phylogenetic analysis of the alpha isoform sequences reveals that vertebrates have two highly conserved subfamilies, alpha1 and alpha2. The alpha1 and alpha2 subfamilies are very similar to each other but can be defined and distinguished from each other by a small number of key amino acid residues. In addition, 3' untranslated cDNA sequences are conserved within the isoform subfamilies. To investigate the function of the alpha isoforms, we examined their expression in mouse cells and tissues. Endothelial cells contain only the alpha2 isoform, and erythrocytes contain almost exclusively the alpha1 isoform. Most tissues have both alpha1 and alpha2 isoforms but the ratio of alpha1:alpha2 varies widely. Together, these findings support the hypothesis that the CP alpha isoforms have conserved, unique and essential roles in vertebrates.

Actin Depolymerizing Factors↗
PubMed1994

Morphological, histochemical, and myosin isoform analysis of the diaphragm of adult horses, Equus caballus.

The horse provides an interesting model for study of the structure and function of the mammalian diaphragm. Multiple regions of diaphragm from seven adult horses were prepared for histochemistry, immunocytochemistry, myosin heavy chain electrophoresis, and native myosin electrophoresis. Two additional adults were dissected to demonstrate myofiber and central tendon morphology and stained for acetylcholinesterase to demonstrate motor endplates. All regions of the adult diaphragm were histochemically characterized by a preponderance of type I fibers with some type IIa fibers. Type IIb fibers were absent in all adult specimens. Myosin heavy chain electrophoresis supported the histochemical study: two isoform bands were present on SDS gels that comigrated at the same rate as rat type I and IIa myosin heavy chain isoforms. No isoform was determined to comigrate with rat type IIb heavy chain isoforms. Native myosin isoform analysis revealed two isoforms that comigrated with rat FM-4 and FM-3 (FM = fast myosin) and two isoforms that comigrated with rat SM-1 and SM-2 (SM = slow myosin) isoforms. In some samples, a third slow native myosin isoform was observed that comigrated at the same rate as the SM-3 of the equine biceps brachii muscle. This doublet (or "triplet") of slow isoforms is unique to some horse muscles compared with other adult animals studied. It is not known if these multiple slow native myosin isoforms confer some functional advantage to the equine muscles. The adult equine diaphragm also differs in its morphology by having a large central tendon compared to that in other mammals, and is predominantly slow in fiber type and myosin isoform composition.

Animals↗
PubMed1994

Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells.

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosyl-phosphatidyl-inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI-linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell-surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane-bound acetylcholinesterase varied between different subcellular fractions. The membrane-bound G2 isoform was greatly enriched in a high-speed "microsomal" fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell-surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane-bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface.

Acetylcholinesterase↗
PubMed1995

Regulated expression of heat shock factor 1 isoforms with distinct leucine zipper arrays via tissue-dependent alternative splicing.

HSF1 mediates the stress induced expression of heat shock proteins, referred to as the cellular stress response. Previous results indicated that mammalian cells express two distinct HSF1 protein isoforms, with molecular sizes of 69 kDa (HSF1-beta) and 71 kDa (HSF1-alpha). The purpose of this study was to determine the mechanism by which these two HSF1 protein isoforms are generated. Our results show that mammalian cells express two distinct HSF1 mRNA isoforms which arise via alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms differ by a single 66 bp exon of the HSF1 gene which is spliced into the HSF1-alpha mRNA isoform but skipped in the HSF1-beta mRNA isoform. This 66 bp exon encodes a 22 amino acid sequence, whose molecular weight (2.3 kDa) matches the difference in size between the HSF1-beta and HSF1-alpha protein isoforms (69 and 71 kDa). Further analysis reveals that this extra 22 amino acid sequence, whose insertion site in the HSF1-alpha isoform is located immediately adjacent to a C-terminal leucine zipper motif (leucine zipper 4) previously shown to be involved in maintenance of HSF1 in the non-DNA-binding control form, contains an additional, previously unidentified leucine zipper motif (leucine zipper 5). Our results also show that the levels of the two HSF1 isoforms are regulated in a tissue dependent manner, with testis expressing higher levels of the HSF1-beta isoform while heart and brain express higher levels of the HSF1-alpha isoform. These results demonstrate a new mechanism by which HSF1 expression is regulated in mammalian cells and suggest a potential role for the HSF1 isoforms in mediating tissue-dependent regulation of the cellular stress response.

Alternative Splicing↗
PubMed1991

Identification and pattern of transitions of some developmental and adult isoforms of fast troponin T in some human and rat skeletal muscles.

Using a monoclonal antibody (F24) in an immunoblotting procedure, the composition of fast troponin T in several adult and developing skeletal muscles of rat and human was studied. With the exception of diaphragm, four isoforms of fast troponin T (HF1-HF4) were detected in all the adult human skeletal muscles investigated. Another isoform of fast troponin T undetectable in the adult human skeletal muscles, designated the fetal isoform (HFF1), was found to be present in all fetal skeletal muscles at 20 weeks of gestation except the diaphragm. Unlike isoform HF4 that was undetectable in all the fetal skeletal muscles, isoforms HF1-HF3 were present in all the human fetal skeletal muscles including the diaphragm. At least five isoforms of fast troponin T (AF1-AF5) could be detected in adult rat skeletal muscles. An additional isoform designated (D) appeared to be present in the rat diaphragm. In some muscles one of the isoforms, AF1, could be further resolved into two to three variants. The proportions and the level of expression of AF1-AF5 isoforms varied not only in different muscles but in some cases also in different parts of the same muscle. In addition to the adult isoforms, four other developmental isoforms termed fetal (FF1 and FF2) and neonatal (NF1 and NF2), were detected during the early development in the rat skeletal muscles. Their presence was first detected during the late fetal to early neonatal period and these isoforms were generally undetectable in a majority of the muscles after 1-2 months of age although their low level of expression persisted in a small number of muscles.

Animals↗
PubMed2006

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms.

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca(2+)-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing alpha-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively alpha-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, alpha-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than beta-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.

Animals↗
PubMed1993

Purification and activation of phospholipase A2 isoforms from Naja mossambica mossambica (spitting cobra) venom.

Isoforms of phospholipase A2 (PLA2) from the venom of Naja mossambica mossambica (the spitting cobra) were purified by a combination of gel filtration on Bio-gel P-30 and ion exchange chromatography on DE-52 Cellulose and the purification followed by three types of polyacrylamide gel electrophoresis. SDS PAGE failed to resolve the active band into separate isoforms. Acid/urea PAGE, resolved the peptides and major protein components of the venom and was able to separate two PLA2 bands in the whole venom. Alkali/urea PAGE resolved four PLA2 bands in whole venom, but could resolve six distinct purified PLA2 species. Of the known isoforms, the acidic form (CM-1) was purified to homogeneity. The basic non-toxic isoform (CM-II) was shown to migrate as a close doublet of PLA2 isoforms. A novel minor purified isoform was identified with mobility intermediate between CM-I and the basic non-toxic isoform CM-II. CM-III was shown to contain a minor PLA2 contaminant. The analysis was facilitated by the fact that all of the isoforms could be eluted from the gels with > 60% recovery of activity. The venom therefore contains at least six isoforms of PLA2 which differ largely by their content of acidic acids. Oleoyl imidazolide treatment increased the haemolytic activity of all but the toxic PLA2 isoform in Naja mossambica mossambica, but partially inhibited the catalytic activity of acidic, toxic and newly purified isoforms whilst partially activating the non-toxic isoform.

Amino Acid Sequence↗
PubMed1996

Human larynx expresses isoforms of the oestrogen receptor.

Commercially available enzyme immunoassays (EIAs) were used for oestrogen (ER) and progesterone (PR) receptor determination in the cytosol fraction of 118 human larynx cancer specimens and in the corresponding histologically proven non-malignant tissues. Fifty-one ER positive cancerous samples had corresponding non-cancerous tissues also expressing the receptor. A high resolution isoelectric focusing (IEF) technique followed by immunoblotting with the H222 anti-ER monoclonal antibody was used to evaluate the presence of ER isoforms in the 51 ER positive human larynx cancer specimens and in their corresponding non-malignant tissues. In both tissues, four ER isoforms were detected, with isoelectric points (pI) similar to those obtained in breast and endometrium carcinomas (6.1, 6.3, 6.6 and 6.8). A significant difference in the expression of ER isoforms between cancerous and non-cancerous tissue was found; precisely, the 94.1% of the ER positive non-malignant specimens co-expressed the four isoforms while they were detected in only the 35.5% of the malignant specimens (P < 0.0001 by Fisher's exact test). In larynx cancer, the concentration values of ER and PR did not correlate, nevertheless tumours co-expressing the four ER isoforms had PR levels significantly higher than those which did not (P = 0.02 by Mann-Whitney Wilcoxon sum rank test). To investigate the possibility that the isoforms of the monomeric 4S form of the ER (those with pI 6.3, 6.6, and 6.8) could dimerise, a cold agarose gel electrophoresis technique was used on IEF-separated ER isoforms. In summary, the evidence shows that all the isoforms are able to form homodimers and that the isoforms at pI 6.3 and 6.8 are able to dimerise with that at pI 6.6 but, under the same experimental conditions, they do not form the 6.3/6.8 heterodimer. It was concluded that: (1) the four isoforms of the ER are co-expressed by the non-malignant human larynx and the cancer loses the capacity to express some of them; (2) the complete complement of ER isoforms (all four) is needed for PR expression; (3) the monomeric 4S isoform with pI 6.6 has the capacity to form homo- and heterodimers, while the remaining two are only able to homodimerise.

Breast Neoplasms↗
PubMed1992

Nature and time course of generation of isoforms of creatine kinase, MB fraction in vivo.

OBJECTIVES: This study was designed to characterize the nature and time course of carboxy-terminal lysine cleavages from the tissue isoform of MB creatine kinase (CK) in vivo. BACKGROUND: Rapid conversion of the tissue isoform of MM CK to two additional circulating isoforms with one or both carboxy-terminal lysines cleaved facilitates early detection of new tissue isoform release after acute myocardial infarction and coronary recanalization. Characterization of changes in plasma MB CK isoform profiles, potentially enhancing specificity even further, has been hindered by difficulties in separating the isoform products and elucidation of carboxy-terminal lysine cleavages underlying their formation. METHODS: Isoform species with carboxy-terminal lysine present on B-monomers were separated from those from which lysine had been cleaved by anion exchange chromatography. Carboxy-terminal lysine on M-monomers was assayed with the use of a monospecific antibody. RESULTS: MB CK in four pooled plasma samples from among 77 normal subjects exhibited carboxy-terminal lysine on 48 +/- 21% (mean +/- SEM) of B-monomers and 82 +/- 12% of M-monomers. Within the 1st 16 h after the onset of acute myocardial infarction, virtually all M- and B-monomers exhibited carboxy-terminal lysine, indicating release into plasma and the lack of rapid cleavage of lysine from the tissue isoform. After 20 to 30 h, 43 +/- 9% (three pools from 19 patients) of B-monomers and 95 +/- 10% of M-monomers exhibited lysine at the carboxyl terminus. After 40 to 50 h, 13 +/- 13% (four pools from 34 patients) of B-monomers and 46 +/- 19% of M-monomers still retained carboxy-terminal lysine. CONCLUSIONS: In contrast to MM CK, the tissue isoform of MB CK undergoes slow cleavage of lysine from both monomers in vivo. Sequential cleavage of lysine first from the carboxyl terminus of B-monomers and subsequently from M-monomers is consistent with generation of at least two additional isoforms. Development of assays capable of resolving all of the isoforms of MB CK that can occur in vivo might increase sensitivity for early detection of new tissue isoform release associated with acute myocardial infarction and coronary recanalization compared with currently available assays that resolve only two species.

Chromatography, Ion Exchange↗
PubMed1993

Three ryanodine receptor isoforms exist in avian striated muscles.

Two isoforms of the ryanodine receptor (termed alpha and beta) are coexpressed in avian fast twitch skeletal muscle, whereas a single isoform is expressed in avian cardiac muscle. We have investigated the relationship between these three proteins, comparing several different properties. First, the three receptor isoform subunits have different mobilities on SDS-polyacrylamide gels. Second, monoclonal antibodies against the chicken skeletal muscle receptor isoforms recognize shared and unique epitopes in each receptor protein, indicating there is not a simple antigenic relationship between the isoforms. Third, the three receptor isoforms exhibit different susceptibilities to proteolysis by trypsin, and limited tryptic digestion yields a different peptide map for each isoform. Fourth, in native sarcoplasmic reticulum membranes, the chicken muscle receptor isoforms are phosphorylated to different extents by the multifunctional calcium/calmodulin-dependent protein kinase II (beta > cardiac > alpha). Fifth, the sites phosphorylated by the calcium/calmodulin-dependent protein kinase in the chicken cardiac and skeletal receptor isoforms are not equivalent. A polyclonal serum, produced against a synthetic peptide containing the site phosphorylated by this kinase in the mammalian cardiac muscle receptor, by immunoprecipitation showed markedly different avidities for the receptor isoforms, and recognized only the cardiac receptor isoform on Western blots. Sixth, the chicken ryanodine receptor isoforms differ in the extent to which they bind azido[125I]calmodulin (alpha > beta > cardiac). These results indicate that three distinct ryanodine receptor proteins are expressed in chicken striated muscles.

Animals↗
PubMed1992

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.

Amino Acid Sequence↗
PubMed1988

Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities.

Crotoxin, the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus is a mixture of several isoforms that differ slightly in their molecular structure. The toxin consists of two nonidentical subunits: a basic and weakly toxic phospholipase A2, component B, and an acidic and nontoxic subunit, component A. In the present investigation, we have used fast-performance liquid chromatography (FPLC) on anionic and cationic exchange columns to purify isoforms of both crotoxin subunits. Two component A isoforms and four component B isoforms were obtained in a homogeneous state, and their purity was verified by isoelectric focusing in polyacrylamide gels. The amino acid composition of the purified component A and component B isoforms was in good agreement with the protein sequences determined previously with mixtures of isoforms. The amino acid compositions indicated that for both crotoxin components the isoforms differed only by the replacement of few amino acid residues. Eight crotoxin complexes have been prepared in a homogeneous state by reassociation of pure component A and component B isoforms. The quantitative comparison of enzymatic and pharmacological properties of the reconstituted crotoxins indicated that the two component A isoforms had identical properties, whereas the four component B isoforms fell in two classes: crotoxin complexes formed with component B isoforms of the first class were enzymatically less active and pharmacologically more potent than those obtained with component B isoforms of the second class.

Amino Acids↗
PubMed1992

Oestrogen receptor isoforms, their distribution and relation to progesterone receptor levels in breast cancer samples.

Oestrogen receptors (ER) in breast cancer tumours are highly heterogeneous. In this study, the variability in the profile of ER isoforms and its relation to progesterone receptor (PgR) levels in breast tumours has been studied. Using high resolution isoelectric focusing (IEF) 4 ER isoforms can be detected with pI values of 6.1 (corresponding to the 8S ER), and 6.3, 6.6 and 6.8 (all of which have a sedimentation pI values of 6.1 (corresponding to the 8S ER), and 6.3, 6.6 and 6.8 (all of which have a sedimentation coefficient of approximately 4S in sucrose density gradients). Data were obtained on the soluble receptors from supernatants of 66 ER-positive primary breast tumour homogenates using high resolution IEF. In 43 of these samples PgR levels were also measured. The isoform at pI 6.6 was present in 97.0% of tumours, the isoform at pI 6.1 in 83.3%, the pI 6.3 isoform 39.4% of tumours and the pI 6.8 isoform in only 33.3% of tumours. Only 12.1% of tumours studied contained the full complement of ER isoforms (pI 6.1, 6.3, 6.6 & 6.8). The ER isoforms at pI 6.1 & 6.8 were only found in PgR-positive (> 10 fmol PgR/mg protein) tumours. Some tumours contained only a single ER isoform at pI 6.6 or 6.1, but those at pI 6.3 and 6.8 were never found singly. Tumours containing 3 or 4 ER isoforms had significantly higher levels of PgR (> 90 fmol/mg protein) than those with only 1 or 2 (P < 0.001). The presence of ER isoforms at pI 6.3 and pI 6.8 also significantly correlated with high levels of PgR (P < 0.001). This variability in the ER isoform profile of breast tumours and their correlation with PgR levels may have a bearing on prognosis and tumour response to endocrine therapy.

Breast Neoplasms↗
PubMed1999

Expression of 3beta-hydroxysteroid dehydrogenase type I and type VI isoforms in the mouse testis during development.

Six isoforms of the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) have been identified in the mouse, each the product of a distinct gene. Two of these isoforms (type I and type VI) are detectable in the adult testis but changes in their expression during development are unknown. In this study we have examined changes in testicular expression and localization of mRNA encoding the type I and type VI isoforms of 3betaHSD. Total 3betaHSD (type I plus type VI) mRNA was measured by reverse transcription-polymerase chain reaction and showed a peak of expression at day 5 after birth followed by a decline and then a further rise after day 10 that continued up to adulthood. When each isoform was measured individually it was clear that the type I isoform was expressed at all ages from embryonic day 13 to adulthood. In contrast, the type VI isoform was only expressed at significant levels during fetal life on embryonic day 13 and then not again until after day 10 postnatally. Expression of the type VI isoform mRNA increased markedly after day 10 so that by adulthood it was the predominant 3betaHSD isoform present in the testis. Closer examination of the timing of type VI expression showed that the isoform mRNA was first detectable at a significant level on day 11. In-situ hybridization confirmed that the type I isoform is the only one expressed in the fetal/neonatal animal and showed that expression was limited to the interstitial tissue. In the adult, both type I and type VI expression was within the interstitial tissue. The timing of 3betaHSD type VI mRNA expression suggests, strongly, that this isoform is expressed only by adult-type Leydig cells in the mouse testis and that this development starts shortly before day 11. The limited expression of the type VI isoform means that it will be a useful marker in studies of adult Leydig cell development.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Increase of brain-derived neurotrophic factor gene expression in NG108-15 cells by the nuclear isoforms of Ca2+/ calmodulin-dependent protein kinase II.

We have reported that the delta3 isoform of Ca2+/ calmodulin-dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain-derived neurotrophic factor (BDNF), we transiently overexpressed the delta3 isoform in NG108-15 cells. The quantitative RT-PCR analysis revealed that rat cerebellum and NG108-15 cells expressed the exon IV-containing mRNA of BDNF (exon IV-BDNF mRNA) more than the exon III-BDNF mRNA. Treatment of NG108-15 cells with Bay K 8644 increased both exon III- and exon IV-BDNF mRNAs, and overexpression of the 83 isoform potentiated the expression of the exon IV-BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the 61 isoform, a nonnuclear isoform, or the inactive mutant of the delta3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the delta3 isoform increased luciferase gene expression. Double-immunostaining of NG108-15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the delta3 isoform or the alphaB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF.

Animals↗
PubMed1998

A dominant negative isoform of the long QT syndrome 1 gene product.

Mutations in the KvLQT1 gene are the cause of the long QT syndrome 1. KvLQT1 gene product is associated with the regulator protein IsK to produce a component of the delayed rectifier K+ current in cardiac myocytes. We identified an N-terminal truncated isoform of the KvLQT1 gene product, referred to as isoform 2. In RNase protection assays, isoform 2 represented 28.1 +/- 0.6% of the total KvLQT1 expression in the human adult ventricle. COS-7 cells injected intranuclearly with KvLQT1 isoform 1 cDNA exhibited a fast-activating K+ current, whereas those injected with a KvLQT1 isoform 1 plus IsK cDNA showed a slow-activating K+ current. Cells injected with KvLQT1 isoform 2 plasmid showed no detectable K+ current. Those injected with a 1/1 isoform 2/isoform 1 ratio showed no detectable K+ current. Those injected with 1/5 and 2/5 ratios showed a K+ current with markedly reduced amplitude. Coexpression of the IsK regulator consistently reduced the dominant negative effects of isoform 2. Our results indicate that KvLQT1 isoform 2 exerts a pronounced negative dominance on isoform 1 channels and that the cardiac KvLQT1 K+ channel complex is composed of at least three different proteins as follows: isoform 1, isoform 2, and IsK.

Animals↗
PubMed1998

Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family.

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

Amino Acid Sequence↗