Canonical Isoform Research Articles

Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells

Stretch is known to stimulate myometrial hyperplasia and hypertrophy in early pregnancy and uterine contraction at term. We propose that transduction of the stretch signal involves alteration of intracellular calcium signalling, including changes in transient receptor potential canonical (TRPC) isoform expression. The aim of the present study was to investigate the effect of prolonged mechanical (tonic) stretch in vitro on human myometrial smooth muscle cell calcium signalling and TRPC expression. Cells were cultured from myometrial biopsies, obtained from women undergoing elective Caesarean section at term, grown on Flexiplates and subjected to 25% tonic mechanical stretch for 1, 4 and 14 h. Time-matched control cells were not stretched. Mechanical stretch (14 h) increased basal calcium entry and cyclopiazonic acid (CPA)-induced calcium/Mn(2+) entry (P < 0.05) in Fura-2 loaded cells. The calcium selectivity of CPA-thapsigarin induced inward currents, measured by patch clamp electrophysiology, was also increased in stretched cells compared with control cells (P < 0.05). Real time PCR and Western blot data demonstrated that TRPC3 and TRPC4 mRNA and TRPC3 protein expression were increased by stretch (P < 0.05), respectively. These data support the hypothesis that uterine stretch modulates uterine growth and contractility in pregnancy via alterations in calcium signalling.

Noncanonical RNAs From Transcripts of the Drosophila muscleblind Gene

It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. Here we describe new cDNAs from the Drosophila melanogaster muscleblind (mbl) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2') or having exons with a different order compared to the corresponding genomic DNA (mblE2E3'E2'; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomplete exon 2 tandem repetition was confirmed by sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products, rapid amplification of cDNA ends, and detection of a band consistent with cDNA sizes in total RNA northern blots. RT-PCRs with exon-specific primers downstream of exon 2 were unable to amplify products other than those expected from canonical mbl isoforms, thus indicating that no other exons were efficiently spliced downstream of exon 2. Moreover, mblE2E2' transcripts seem to be poorly polyadenylated, if at all, and behave aberrantly in a polyacrylamide gel electrophoresis (PAGE) mobility assay. Taken together, lack of polyadenylation, lack of downstream splicing events, small size of mblE2E2', and PAGE behavior all suggest that these noncanonical transcripts may be circular RNAs. The functional implications for these noncanonical transcripts are unclear. A developmental expression profile of mblE2E2' revealed an almost constant expression except during early embryogenesis and early adulthood. The protein putatively encoded is unlikely to be functional because an in-frame stop codon occurs almost immediately after the splice site. Such noncanonical transcripts have previously been observed in vertebrates, and these data provide the first experimental evidence for similar phenomena in invertebrates.

Calmodulin/Ca2+‐ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform‐specific Ca2+ dependencies

Arabidopsis thaliana plasma membrane (PM) Ca2+‐ATPase is a type IIB P‐type ATPase, which binds calmodulin (CaM) to an autoinhibitory N‐terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At‐ACA8, the first cloned A. thaliana PM Ca2+‐ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At‐ACA8 N‐terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide 41I‐T63 had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µM free Ca2+ about 25 nM], thus localizing At‐ACA8 CaM‐binding site within this sequence. The interaction of At‐ACA8 N‐terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca2+‐ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca2+‐ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca2+ concentration, suggesting that enzyme conformation is affected by Ca2+. Binding of CaM isoforms to At‐ACA8 N‐terminus was affected differently by free Ca2+ concentration, suggesting that plant CaMs may have different affinities for Ca2+.

Alternative Splicing of Human Insulin-Degrading Enzyme Yields a Novel Isoform with a Decreased Ability To Degrade Insulin and Amyloid β-Protein

Deletion of insulin-degrading enzyme (IDE) in mice causes accumulation of cerebral amyloid beta-protein (Abeta), hyperinsulinemia, and glucose intolerance. Together with genetic linkage and allelic association of IDE to Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), these findings suggest that IDE hypofunction could mediate human disease. To date, no coding mutations have been found in the canonical isoform of IDE, suggesting that pathological mutations could exist in undiscovered exons or regulatory regions, including untranslated regions (UTRs). However, neither isoforms arising from alternative splicing nor the UTRs have been described. Here, we systematically characterize human IDE mRNAs, identify a novel splice form, and compare its subcellular distribution, kinetic properties, and ability to degrade Abeta to the known isoform. Six distinct human IDE transcripts were identified, with most of the variance attributable to alternative polyadenylation sites. In the novel spliceoform, an exon we designate "15b" replaces the canonical exon "15a", and the resultant variant is widely expressed. Subcellular fractionation, immunofluorescent confocal microscopy, and immunogold-electron microscopy reveal that the 15b-IDE protein occurs in both cytosol and mitochondria. Organelle targeting of both isoforms is determined by which of two translation start sites is used, and only those isoforms utilizing the second site regulate levels of secreted Abeta. 15b-IDE can exist as a heterodimer with the 15a isoform or as a homodimer. The apparent K(m) values of recombinant 15b-IDE for both insulin and Abeta are significantly higher and the k(cat) and catalytic efficiency markedly lower than those of 15a-IDE. In accord, cells coexpressing beta-amyloid precursor protein (APP) and 15b-IDE accumulated significantly more Abeta in their media than those expressing APP and 15a-IDE. Our results identify a novel, catalytically inefficient form of IDE expressed in brain and non-neural tissues and recommend novel regions of the IDE gene in which to search for mutations predisposing patients to AD and DM2.

Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G.

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca2+ stores and/or diacylglycerol and/or Ca2+/calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). We found that the overexpressed TRPC3 mediated store-operated Ca2+ influx and that this type of Ca2+ influx was inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels could be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolished the store-operated Ca2+ influx mediated by TRPC3 channels in HEK293 cells.

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCα and PKCϵ—a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCα and PKCϵ activities; 2) whereas PKCϵ activity is induced by cytokine stimulation, PKCα activity is inhibited; and 3) both the induction of PKCϵ and the inhibition of PKCα functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation. Anat Rec 266:87–92, 2002. © 2002 Wiley-Liss, Inc.

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.

Revised genomic structure of the human MAGP1 gene and identification of alternate transcripts in human and mouse tissues.

The human MAGP1 (or MFAP2) and mouse Magp1 genes code for the microfibril-associated glycoprotein-1 (MAGP-1), an extracellular matrix protein of microfibrillar structures. We report a revised 5' genomic structure including the use of a single transcription start site that gives rise to a 32-bp 5' exon spanning a segment of the previously described exon B. No evidence of heterogeneous 5' ends from the use of alternative promoters was found in human tissues and cell lines. We located the genetic marker D1S170 to a position 3 kb downstream of the polyadenylation site. Large-scale comparison of the human and mouse genes revealed conservation of sequence outside the coding exons. Although the 5' flanking regions were found to be divergent certain cis-elements for transcription factors are conserved, including Sp1, AP-2, AP-4, NF-kappaB, and c-ETS motifs. We identified a total of five splice variants in addition to the canonical MAGP1A/Magp1A form. These transcripts are species-specific and are generated by different processing mechanisms. The alternate forms MAGP1A', MAGP1B, and MAGP1C are expressed in human tissues; and the two variants Magp1A" and Magp1D were found only in mouse. The alternatively spliced forms show restricted patterns of expression relative to the canonical isoform.

Alteration of the expression of human estrogen receptor gene by distamycin

The effects of distamycin on the expression of the estrogen receptor gene were determined in the MCF7 human breast cancer cell line. Estrogen receptor (ER) RNA transcripts were analyzed by Northern blotting and RT-PCR using specific oligonucleotides for the 5′ upstream region and for ER cDNA. After ex vivo distamycin treatment of the cells the expression of the canonical ER mRNA isoform of 6.3 kb is strongly inhibited, without appreciable alteration of the accumulation of 5′ upstream ER mRNA isoforms. These results suggest that distamycin alters the transcriptional activity of the ER gene causing a change in the ratio between the canonical transcript and other isoforms containing 5′ upstream regions.

The Epping jaundice outbreak: a 24 year follow up.

The aim was to trace 84 cases of jaundice that occurred following accidental ingestion of methylene dianiline (MDA) in Epping in 1965, and to look at long term health effects. The original case notes of the cases were used to identify the patients. Subsequent tracing procedures included local general practitioners, the Central NHS Registry, electoral rolls, and company records. This was a community based survey. The health status of 68 (81%) of the group was established with 18 deaths. Of the 50 cases known to be alive, 58% completed a health questionnaire. The causes of death were unremarkable except for one case of carcinoma of the biliary tract. Two surviving cases had suffered retinal pathology. Four other surviving cases had had a further, perhaps unrelated, episode of jaundice. Although the dose and route of administration in the epidemic differed from occupational exposure, this follow up study a generation on provides little, if any, evidence of long term health sequelae. Nevertheless, in the absence of well documented exposure and health effects data, such accidental poisonings with proven animal carcinogens warrant long term follow up. The identified cohort will be the subject of further study.