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At least 811 records · Page 45Linked to original sources
PubMed1998

Determination of the specificity of CD45 and CD45R monoclonal antibodies through the use of transfected hamster cells producing individual porcine CD45 isoforms.

The exclusive presence of the tyrosine phosphatase, CD45, on the surface of hematopoietic cells coupled with the differential expression of its various isoforms has enabled the selection of lymphocyte subsets based on their reactivity with monoclonal antibodies (mAbs) specific for one or more CD45 species. As a prelude to defining the specificity of anti-porcine CD45 mAbs for this purpose, Chinese hamster ovary cells were transfected with constructs containing cDNAs encoding the extracellular and transmembrane domains of four pig CD45 isoforms. Cells expressing only one of the three predominant types (CD45RO, CD45RC, and CD45RAC) or the minor species (CD45RA) of porcine CD45 on their surface were sorted based on positive reactivity with the CD45 mAb K252.1E4. Initially, these CD45+ cells were used as a source of antigen when determining the specificities of nine mAbs, which had been identified during the First and Second International Swine CD Workshops as being reactive with porcine CD45. Later, cloned cell lines were established and phenotypically verified for the production of the correct CD45 transcript by RT-PCR. Binding of two more mAbs (74-9-3 and 10-14-1) in addition to the original mAb panel to these cell lines was assessed by using a cell ELISA in lieu of one-color flow cytometry. Despite differences in detection methodology, identical mAb binding results were obtained. As anticipated, CD45 mAbs K252.1E4, MAC 323, and 74-9-3 which recognize an epitope(s) in the common portion of porcine CD45, reacted with cells expressing any one of the four isoforms but not with the parental CHO cells. In contrast, none of the restricted (CD45R) mAbs bound to cells producing the CD45RO isoform which lacks any of the alternate extracellular regions. However, three of these mABs (6E3/7, FG2F9 and STH267) did react specifically with the CD45RA and CD45RAC isoforms, indicating their specificity for an epitope(s) encoded by the CD45 A exon. The other four CD45R mAbs (MAC326, 3a56, MIL5, and -a2) recognized the CD45RC isoform. Interestingly, only CD45R mAb MAC326 also bound to cells expressing the CD45RAC isoform, suggesting that the epitope(s) recognized by the other three may have arisen due to the juncture of the invariant 5' leader sequence with the CD45C exon. The eleventh mAb (10-14-1) was unique in that it did not react with any of the expressed CD45 isoforms. This inability coupled with the previously demonstrated recognition of a 240 kDa protein suggests that it may be specific for CD45RABC. Overall, this panel of CD45R mAbs should prove useful for obtaining functionally distinct subpopulations of B and T lymphocytes.

Animals↗
PubMed2000

Expression of two vacuolar-type ATPase B subunit isoforms in swimbladder gas gland cells of the European eel: nucleotide sequences and deduced amino acid sequences.

The poly(A)(+) RNA of swimbladder gas gland cells of the European eel Anguilla anguilla was isolated and used for cDNA synthesis. Using a pair of degenerate PCR primers directed towards the evolutionary highly conserved central part of the B subunit of vacuolar type H(+)-ATPase (V-ATPase) a fragment of 388 bp was amplified. By sequencing the cloned PCR products two different amplicons with a sequence identity of about 86% were obtained. BLASTN searches revealed a high degree of similarity of both to V-ATPase B subunits of other species. The sequences were completed by performing rapid amplification of cDNA ends PCR, subsequent cloning, and sequencing of the obtained products. The expression of two different isoforms of the V-ATPase B subunit is already demonstrated for Homo sapiens and Bos taurus. This is the first report that attributes the same phenomenon to a non-mammalian species, A. anguilla. The first isoform found in eel (vatB2) shows the highest degree of amino acid sequence homology with the human brain isoform (98.2%), the second one (vatB1) with the B subunit sequence of rainbow trout (Oncorhynchus mykiss) gill and kidney (98, 6%). The alignment of the deduced amino acid sequences of vatB1 and vatB2 shows that the highest sequence variation between these two isoforms is found at the amino-terminus, where vatB1 is nine amino acids shorter than vatB2, while at the carboxy-terminus it is two amino acids longer than vatB2. This has also been reported for the human and bovine kidney isoforms when compared with the brain isoforms. Northern blot analysis using specific hybridization probes revealed the expression of two mRNA's with lengths of about 2.9 kb and 3.5 kb for vatB1 and vatB2, respectively. For mammals, it is well known that V-ATPases containing the kidney isoforms of the B subunit are responsible for the extrusion of protons across the plasma membranes of several cell types. The fact that eel vatB1 seems to share structural features with the kidney isoforms in mammals supports the hypothesis that in gas gland cells a V-ATPase contributes to the acidification of the blood in the swimbladder.

Adenosine Triphosphatases↗
PubMed2000

Immunohistochemical localization of adenylyl cyclase isoforms in the lateral wall of the rat cochlea.

The enzymatic activity of adenylyl cyclase (AC) is attributable to nine isoforms with individual pharmacology and tissue distribution. Polyclonal antibodies for AC isoforms I-IV, VII and VIII were applied to sections of cochlear lateral wall, a tissue involved in ion transport contributing to the unique ion content of endolymph and electrical potential of scala media. Within the stria vascularis, immunoreactivity primarily to Ca(2+)/calmodulin-independent isoforms II, IV and VII was localized to sites consistent in position to the basolateral extensions of marginal cells. Little immunoreactivity was observed in the stria vascularis for Ca(2+)/calmodulin-dependent isoforms I, III and VIII. Within the spiral ligament, type II and type IV fibrocytes exhibited moderate staining for ACII, IV and VII, less staining for VIII and little for I and III. Immunoreactivity to ACII, IV, VII and VIII was observed in type I fibrocytes. The outer sulcus cells and root processes were highly immunoreactive for isoforms I and VIII, but not for III or the Ca(2+)/calmodulin-independent isoforms. The differential pattern of immunoreactivity in the lateral wall overall appears to reflect subfamily-specific expression with Ca(2+)/calmodulin-independent isoforms expressed in the stria vascularis and Ca(2+)/calmodulin-dependent isoforms expressed in the outer sulcus cells and root processes. cAMP-mediated modulation of ion transport by marginal cells is predicted to exhibit, in the microenvironment of basolateral membrane infoldings, pharmacological characteristics of the AC type II subfamily (II, IV and VII), including activation by protein kinase C (II and VII).

Adenylyl Cyclases↗
PubMed1997

Regional differences in expression of transcripts for Na+/Ca2+ exchanger isoforms in rat brain.

The Na+/Ca2+ exchanger has a primary role in maintaining intraneuronal Ca2+ homeostasis. There are three distinct Na+/Ca2+ exchanger isoforms cloned from rat brain, NCX1, NCX2 and NCX3, which are the products of three different genes. In the present study, isoform expression in different regions of rat brain was determined by using reverse transcription PCR (RT-PCR) and Northern analysis. RT-PCR detected all three Na+/Ca2+ exchanger isoforms in each region studied (brainstem/spinal cord, cerebellum, cerebral cortex, striatum/septum and hippocampus). Northern analysis was performed to determine the steady-state mRNA levels of each isoform. NCX1 had two transcripts, 14 and 7 kb, and the 7-kb transcript was predominant in brainstem/spinal cord, cerebellum and hippocampus. NCX2 expression (4.8-kb transcript) was an order of magnitude higher than NCX1 or NCX3 expression in all the five areas except brainstem/spinal cord where the 4.8-kb transcript was nearly absent. The third isoform (NCX3) had two transcripts, one was 6 kb and the other was 4 kb. The 6-kb transcript was predominant in brainstem/spinal cord and cerebellum. The results suggest that Na+/Ca2+ exchanger isoforms are expressed ubiquitously in rat brain but that each isoform shows a unique distribution within the brain. The exchanger probably participates in the regulation of intracellular calcium homeostasis in a wide range of cell types within the brain. Furthermore, individual cells may contain more than one type of exchanger isoform with distinct subcellular distributions.

Animals↗
PubMed2002

Thyroid hormone up-regulates Na+/K+ pump alpha2 mRNA but not alpha2 protein isoform in cultured skeletal muscle.

Thyroid hormone (T(3)) is known to up-regulate the physiological expression of the Na(+)/K(+) pump in cultured skeletal muscle. We recently reported that primary cultured rat skeletal muscle expresses only the alpha(1), beta(1) and beta(2) protein isoforms of Na(+)/K(+) pump. Interestingly, alpha(2) mRNA is detectable while the alpha(2) protein isoform is not. We therefore examined whether T(3) might up-regulate the expression of Na(+)/K(+) pump alpha(2) isoform at the protein and mRNA level. We also examined the regulation by this hormone of the other isoforms of the pump. Primary cultures were treated with T3 for 48 h from day 4 to day 6 of differentiation. Protein and mRNA isoforms of Na(+)/K(+) pump were identified by Western blotting and Northern blotting, respectively. T(3) induced a marked increase in the beta(1) protein and a slight increase in the alpha(1) protein. T(3) did not affect expression of the beta(2) protein. The alpha(2) protein was not detected in either untreated or T(3)-treated cells. In contrast, alpha(2) mRNA was highly up-regulated by T(3) treatment compared to the other isoforms. The lack of expression of the alpha(2) protein isoform following T(3) treatment suggests that posttranscriptional events related to this isoform may be dependent on other growth factors or hormones.

Animals↗
PubMed1997

Myosin isoforms and functional diversity in vertebrate smooth muscle.

The expression of fast and slow myosin isoforms in individual cells is associated with differences in shortening velocities and power output in fully differentiated vertebrate striated muscle. This paradigm in which shortening velocity is determined by the myosin isoform (and load) is inappropriate for smooth muscle. Smooth muscle tissues express multiple myosin heavy and light chain isoforms, and it is not currently possible to separate and identify chemically distinct native myosin hexamers (i.e., isoforms). It is not known if different isoforms are localized in subpopulations of cells or in specific cellular domains nor whether they combine preferentially to form a small number of native myosin hexamer isoforms. Potentially, thick filaments are aggregates of many different combinations of heavy and light chain isoforms that may or may not exhibit different kinetics. Shortening velocities in smooth muscle are regulated by Ca(2+)-dependent crossbridge phosphorylation of the myosin regulatory light chains. Much of the observed diversity in power output in smooth muscle may be attributed to regulatory mechanisms modulating crossbridge cycling rates rather than contractile protein isoform expression.

Animals↗
PubMed1998

Evidence for multiple protein kinase C isoforms in the leukocytes of a marine teleost, Sciaenops ocellatus.

The protein kinase C (PKC) family of isozymes mediates a diverse range of cellular functions, including activation of vertebrate lymphocytes through membrane-bound antigen receptors. The complex role of PKC in mammalian cells may be orchestrated in part by the presence of multiple isoforms, each of which displays a distinctive tissue distribution, substrate specificity and pattern of regulation. In the present study, PKC isoforms were identified in peripheral blood leukocytes of the marine teleost fish Sciaenops ocellatus by immunoprecipitation and Western blot using antibodies to mammalian isoforms. Functional activity was monitored by evaluating translocation of the teleost isoforms from membrane to cytosol in response to phorbol ester treatment. Teleost conventional isoforms PKC alpha and PKC beta (82 kDa) completely translocated out of the cytosol in response to phorbol ester. Phorbol ester did not induce translocation of teleost atypical isoform PKC zeta (67 kDa), as has been shown for its mammalian homologue. Although their identity as distinct isoforms is less clear, proposed teleost novel PKC delta (84, 86 kDa) and PKC eta (83, 85 kDa) also translocated out of the cytosol. The presence of multiple isoforms representing each of the three major classes of PKC in red drum leukocytes implies that the complexity of signal transduction pathways in vertebrates is highly conserved.

Animals↗
PubMed2000

Multiple isoforms of porcine aromatase are encoded by three distinct genes.

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.

Animals↗
PubMed1988

Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum.

The differential availability of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of [125I]T4 and [125I]T3 to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, [125I]T4 was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. [125I]T3 was localized to only two isoforms of TBG, TBG-III and -IV, with pI's that were identical with those for [125I]T4. In normal female serum, [125I]T4 was localized to the same four isoforms of TBG as those of normal male serum, while [125I]T3 was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, [125I]T4 was localized to five isoforms, whereas [125I]T3 was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T3. The Ki values of T3 binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and chromatofocusing (pH range of 3.5-5.0). T4 preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T4 and T3. In conclusion, this study shows that T4 and T3 do not bind to a single competitive binding site on TBG. Instead, T4 is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T3 for these proteins.

Humans↗
PubMed2000

Isoform patterns of chitinase and beta-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection.

Isoform patterns of chitinase and beta-1,3-glucanase of maturing kernels of yellow dent corn (Pioneer 3394) infected with Aspergillus flavus at the milk stage were investigated through polyacrylamide gel electrophoresis (PAGE). Proteins on the sodium dodecyl sulfate (SDS) gel with an apparent molecular mass range of 23-46 kDa were differentially present in the kernels infected with both aflatoxin-producing and non-aflatoxin-producing strains of A. flavus. From in-gel (native PAGE) enzyme activity assays, three bands corresponding to chitinase isoforms and two bands corresponding to beta-1,3-glucanase isoforms were detected in the infected kernels. One chitinase isoform of 29 kDa was present only in the infected kernels, and another one of 28 kDa was present in both infected and noninfected kernels. They were judged to be acidic on the basis of their migration on an acrylamide isoelectric focusing (IEF) gel. For the beta-1,3-glucanase, one isoform of 35 kDa was present in both infected and noninfected kernels, but another one, a 33 kDa isoform, was present only in the infected kernels. Both acidic and basic beta-1,3-glucanase isoforms were detected in the IEF gel. The results of this study are the first to demonstrate patterns of enhanced or inducible proteins in maturing corn kernels in response to A. flavus infection at the milk stage. The results also indicate that only particular isoforms of the two hydrolytic enzymes are involved in the maturing corn kernels infected at the milk stage with A. flavus.

Aspergillus↗
PubMed1997

Coordinated fast-to-slow transitions of myosin and SERCA isoforms in chronically stimulated muscles of euthyroid and hyperthyroid rabbits.

Changes in the patterns of myosin heavy chain (MHC) isoforms, isomyosins, and Ca(2+)-ATPase (SERCA) isoforms were studied in long-term (72 d) stimulated fast-twitch extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of euthyroid and hyperthyroid rabbits. The chronic low-frequency stimulation-induced fast-to-slow transitions in MHC isoforms, isomyosins and SERCA isoforms were pronounced in muscles from euthyroid rabbits, but less pronounced in muscles from hyperthyroid rabbits. Thus, hyperthyroidism counteracted to same extent the stimulation-induced fast-to-slow transition. Analyses of all parameters were performed on the same individual muscles, providing information on the co-ordinated expression of SERCA and myosin isoforms. A high correlation (r = 0.97) was detected between relative concentrations of slow SERCA2a and slow MHCI isoforms. This correlation persisted under all experimental conditions, suggesting a co-ordinated expression of slow myosin and Ca(2+)-ATPase isoforms. Conversely, fast SERCA1a was correlated to fast myosin isoforms as a whole.

Animals↗
PubMed1995

High glucose concentrations and protein kinase C isoforms in vascular smooth muscle cells.

High extracellular glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. However, which PKC isoforms are involved and where in the cell they operate is unclear. We tested the hypothesis that only those PKC isoforms binding to diacylglycerol (DAG) are activated by high glucose. We also reasoned that the isoforms would translocate to different parts of the cell, where they presumably serve different functions. The PKC isoforms alpha, beta, delta, epsilon, and zeta were studied. Twenty mM glucose caused an increase in total PKC activity at six hours, which was maintained at 24 hours. High glucose decreased the angiotensin II-induced calcium signal. This effect was reversed by preincubating the cells with the PKC inhibitor staurosporine. Glucose induced a translocation of all PKC isoforms except PKC zeta by Western blot. Confocal microscopy showed that PKC alpha, beta, and epsilon were translocated into the nucleus. PKC delta showed strong association with cytoskeletal structures. The effects were sustained at 24 hours for PKC isoform beta and to a lesser extent for PKC delta and epsilon, but not for PKC alpha. Thus, PKC isoforms differ in their propensity to be activated by high glucose. Those isoforms binding to DAG are activated. Both cytoskeletal and nuclear signaling may be involved.

Alkaloids↗
PubMed1996

Glomerular up-regulation of EIIIA and V120 fibronectin isoforms in proliferative immune complex nephritis.

Fibronectin: (FNs) comprise a family of adhesive glycoproteins that are prominent components of mesangial extracellular matrix and accumulate during glomerular injury. By alternative splicing of an unique mRNA precursor, various FN isoforms can be originated. In rat, three regions of the molecule are involved: EIIIA, EIIIB and V. Because specific FN isoforms are expressed in embryogenesis and wound healing, conditions characterized by cell migration and adhesion, we examined the pattern of FN isoforms in the mild and severe phases of a progressive immune complex proliferative nephritis in rats. We constructed specific probes to analyze the splicing pattern of FN pre-mRNAs by ribonuclease protection assays. FN mRNAs containing EIIIA, EIIIB and V regions increased along, the progression of nephritis, though the increment of EIIIB-FN mRNA was modest. However, different regulation of all these isoforms was observed. The percentage of FN mRNA containing the EIIIA exon versus total FN increased with the severity of the disease, while the percentage of FN mRNA containing the EIIIB exon decreased. Relative V-FN mRNA expression versus total FN mRNA increased only in the severe phase. By means of specific antibodies we also studied the presence of EIIIA, EIIIB and V-FN proteins in the kidney. In the normal glomerutus, EIIIA-FN protein was barely detectable in the mesangium, increasing in the mild phase of nephritis. In the severe phase of nephritis, increased EIIIA-FN was localized in the mesangium, in Bowman's capsule and in crescents. By contrast, EIIIB-FN protein in the glomerulus was absent even in the severe phase. V120-FN protein, an isoform that mediates the attachment of leukocytes through the VLA-4 integrin, was present in the mesangium and glomerular capillary loops in control animals, and increased in the severe phase of nephritis, coinciding with a strong leukocyte infiltration. In conclusion, our results show that during immune glomerular injury there were marked changes in the pattern of FN isoforms expression. Since those isoforms, particularly V120 isoform, are important in cell adhesion and migration, their up-regulation may facilitate the recruitment of cells into the injured glomeruli. The blockade of the interaction between V120-FN and infiltrating leukocytes may represent a new approach to the treatment of nephritis.

Alternative Splicing↗
PubMed2002

Human PDE5A gene encodes three PDE5 isoforms from two alternate promoters.

Sildenafil improves erectile function by inhibiting the cGMP-catalytic activity of phosphodiesterase type V (PDE5). We used rapid amplification of cDNA Ends-polymerase chain reaction (RACE-PCR) to isolate three PDE5 isoforms from human corpus cavernosum. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis on eight human cavernous tissue samples showed that all samples expressed the PDE5A1 at a lower level than the PDE5A2 isoform. Five samples expressed the PDE5A3 isoform at various levels while the other three did not. Analysis on non-penile tissues showed that all tissues expressed the A1 and A2 isoforms while only those that have substantial amounts of smooth muscle expressed the A3 isoform. Cloning and sequencing of the PDE5A gene showed that the isoform-specific 5'-ends of the PDE5 mRNAs are encoded from three alternative first exons arranged in the order of A1-A3-A2. Promoter activities were detected upstream from the A1-specific exon and in the intron preceding the A2-specific exon. The upstream PDE5A promoter is expected to direct the expression of all three PDE5 isoforms while the intronic PDE5A2 promoter only the A2 isoform. Both promoters were upregulated by increasing concentrations of either cAMP or cGMP. Several transcription factor AP2 and Sp1-binding sequences identified in the promoters are likely to be the mediators of cAMP/cGMP-responsiveness.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Increased expression of the differentiation-defective granulocyte colony-stimulating factor receptor mRNA isoform in acute myelogenous leukemia.

Granulocyte colony-stimulating factor (G-CSF) critically affects all stages of granulopoiesis by activating a signaling cascade initiated by dimerization of its receptor (G-CSFR). Five human G-CSFR isoforms have been identified (classes I-V). A quantitative polymerase chain reaction (Q-PCR) technique was used to examine the expression of these five isoforms in normal and leukemic myeloid cells. We demonstrated that neutrophils expressed predominantly the class I isoform and low levels of class IV isoform (IV/I = 0.037 +/- 0.005). No expression of the class II, class III, or class V isoform was detected. In contrast, all AML cell lines and acute myelogenous leukemia (AML) patient samples expressed increased relative amounts of the class IV isoform (IV/I = 0.047-0.350). When compared to normal immature myeloid cells, as represented by the CD34+ fraction of adult bone marrow (ABM) cells, three of eight AML cell lines and three of six AML patient samples expressed significantly increased levels of the class IV isoform relative to class I. This suggests that the increase in the relative expression of the class IV isoform seen in a considerable portion of AML cell samples is related to their leukemic phenotype. Given the inability of the class IV G-CSFR to drive myeloid maturation, the relative increase in class IV expression in AML cells may contribute to their aberrant response to G-CSF.

Adult↗
PubMed1998

Grb2 binding to the different isoforms of Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.

3T3 Cells↗
PubMed2000

Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones.

The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3alpha-HSD (AKR1C4), type 2 3alpha(17beta)-HSD (AKR1C3), type 3 3alpha-HSD (AKR1C2) and 20alpha(3alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K(m) values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one; 5alpha-DHT) to yield 5alpha-androstane-3alpha,17beta-diol (3alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxy-pregn-4-ene-3,20-dione (20alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3alpha-androstanediol to the active hormone 5alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxidized 17beta-oestradiol to oestrone, and 20alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K(m) allows this enzyme to form 5alpha/5beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17beta-oestradiol), oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3alpha-hydroxysteroids exists.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas.

This paper describes some properties of glutamine oxidation and glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas. The slow-growing adenoma-derived cell line AA/C1, and the rapidly proliferating carcinoma cell line HT29, both required glutamine for growth. The rate of (14)CO(2) production from [U-(14)C]glutamine was faster in AA/C1 cells than in HT29 cells. Conversely HT29 cells showed faster rates of glucose oxidation and lactate production. The activity of glutaminase was 3 times higher in AA/C1 cell extracts than in extracts of HT29 cells. Glutaminase activity in the two cell lines had similar K(m) values for glutamine, but the activity in AA/C1 cells had a higher K(0.5) for activation by phosphate. Glutaminase activity in extracts of both cells was inhibited by glutamate. Western blotting showed the presence, in both cell lines, of isoform(s) of glutaminase with an molecular mass of 63 kDa, intermediate between that of kidney glutaminase and liver glutaminase. PCR-based analysis showed that an mRNA species identical to the kidney-type isoform glutaminase C was present in both cell types as was an additional mRNA species identical to the liver-type glutaminase isoform from human breast tumour cells. Northern blotting using isoform-specific cDNA probes demonstrated that mRNA for both glutaminase isoforms was expressed at significant levels in both cell types. Similar results to those in AA/C1 cells and HT29 cells were obtained in two further adenoma and carcinoma cell lines respectively. These results contrast with those reported previously in hepatocyte/hepatoma model systems with respect to fuel selection, glutaminase activity and isoform expression. They also constitute the first demonstration of simultaneous expression of two glutaminase isoforms in a single cell type.

Cell Division↗