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At least 1,531 records · Page 85Linked to original sources
PubMed2003

A single amino acid change (substitution of the conserved Glu-590 with alanine) in the C-terminal domain of rat liver carnitine palmitoyltransferase I increases its malonyl-CoA sensitivity close to that observed with the muscle isoform of the enzyme.

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme.

Alanine↗
PubMed2004

A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb.

LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domain-containing protein that functions as an intrinsic determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing. Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded. The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.

Amino Acid Substitution↗
PubMed2004

A protein phosphatase-1gamma1 isoform selectivity determinant in dendritic spine-associated neurabin.

Protein phosphatase-1 (PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical (R/K)(V/I)XF motif. Despite sharing approximately 90% amino acid sequence identity, PP1beta and PP1gamma1 have distinct subcellular localizations that may be determined by selective interactions with PP1-binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1gamma1 selectively interacts with spinophilin and neurabin, F-actin-targeting proteins, whereas PP1beta selectively interacted with G(M)/R(GL), the striated-muscle glycogen-targeting subunit. Glutathione S-transferase (GST) fusion proteins containing residues 146-493 of neurabin (GST-Nb-(146-493)) or residues 1-240 of G(M)/R(GL) (GST-G(M)-(1-240)) recapitulated these isoform selectivities in binding and phosphatase activity inhibition assays. Site-directed mutagenesis indicated that this isoform selectivity was not due to sequence differences between the canonical PP1-binding motifs (neurabin, (457)KIKF(460); G(M)/R(GL), (65)RVSF(68)). A chimeric GST fusion protein containing residues 1-64 of G(M)/R(GL) fused to residues 457-493 of neurabin (GST-G(M)/Nb) selectively bound to and inhibited PP1gamma1, whereas a GST-Nb/G(M) chimera containing Nb-(146-460) fused to G(M)-(69-240) selectively interacted with and weakly inhibited PP1beta, implicating domain(s) C-terminal to the (R/K)(V/I)XF motif as determinants of PP1 isoform selectivity. Deletion of Pro(464) and Ile(465) in neurabin (deltaPI) to equally space a conserved cluster of amino acids from the (R/K)(V/I)XF motif as in G(M)/R(GL) severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1gamma1 selectivity. Further analysis of a series of C-terminal truncated GST-Nb-(146-493) proteins identified residues 473-479 of neurabin as containing a crucial PP1gamma1-selectivity determinant. In combination, these data identify a novel PP1gamma1-selective interaction domain in neurabin that may allow for selective regulation and/or subcellular targeting of PP1 isoforms.

Actins↗
PubMed2004

The three isoforms of the light-harvesting complex II: spectroscopic features, trimer formation, and functional roles.

The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE. Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms. We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes. The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.

Amino Acid Sequence↗
PubMed2006

The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) Ca2+ pump isoforms 1, 2, and 3 to thapsigargin and other inhibitors.

Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K(i) values being 0.21, 1.3, and 12 nm for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca(2+)-ATPase by binding to sites on SERCA distinct from that of thapsigargin.

Adenosine Triphosphate↗
PubMed2006

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana.

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.

Arabidopsis↗
PubMed2006

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration.

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Cell Line, Tumor↗
PubMed2003

Expression of vascular endothelial growth factor isoforms in the Japanese quail embryo.

Vascular endothelial growth factor (VEGF) i s required for vascular development. In the quail, four VEGF isoforms formed by alternative splicing, are translated into proteins with 122, 146, 166, and 190 amino acids. VEGF isoforms differ biochemically, with variable affinities for heparan sulfate proteoglycans, the extracellular matrix and VEGF receptors. There are few data on the functional significance of VEGF isoforms. RT-PCR was used to examine isoform expression during quail vascular development. Our results suggest that all quail isoforms are expressed during establishment of the vascular pattern in whole embryos and extraembryonic tissues at apparently equal levels. No isoform-specific expression patterns were detected in isolated endoderm or between embryo halves.

Alternative Splicing↗
PubMed2006

Transforming growth factor-beta as a key molecule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells.

Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.

Becaplermin↗
PubMed2005

Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles.

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.

Animals↗
PubMed2002

Molecular analysis of collagen XVIII reveals novel mutations, presence of a third isoform, and possible genetic heterogeneity in Knobloch syndrome.

Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy.

Adolescent↗
PubMed2002

Alternative splicing regulates the subcellular localization of divalent metal transporter 1 isoforms.

Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3' exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3' untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type-specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y(555)XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type-specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types.

Alternative Splicing↗
PubMed2005

Proteolytic cleavage and phosphorylation of a tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth.

The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-alpha converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by gamma-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.

ADAM Proteins↗
PubMed2006

Conditional dominant mutations in the Caenorhabditis elegans gene act-2 identify cytoplasmic and muscle roles for a redundant actin isoform.

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes.

Actin Cytoskeleton↗
PubMed1999

Bioactivity of prolactin isoforms: lactation and recovery of menses in nursing women.

To assess whether plasma prolactin (PRL) characteristics relate to lactogenesis and absence or presence of menstrual cycles, we measured bioactive PRL (BIO-PRL) using the Nb2 assay, immunoreactive PRL (IR-PRL) by radio-immunoassay, calculated equations describing the BIO-PRL-IR-PRL relationship and separated charged PRL isoforms (by chromatofocusing) in five amenorrhoeic and five cycling nursing women at 6 months postpartum and in 10 cycling non-nursing women. Plasma samples were drawn before and 30 min after a suckling episode at 0800, 1600 and 2400 h in nursing women and at the same hours in non-nursing women. BIO-PRL and IR-PRL concentrations were highest in amenorrhoeic nursing women, intermediate in cycling nursing women and lowest in cycling non-nursing women. The BIO-PRL-IR-PRL relationship shows that a given amount of IR-PRL corresponds to equivalent amounts of BIO-PRL in cycling nursing and cycling non-nursing women, and to a larger extent in amenorrhoeic nursing women. IR-PRL was present in plasma as several charge isoforms. Bioactive isoforms eluting at pH 6.0-5.1 were found in amenorrhoeic and cycling nursing women, reaching similar concentrations after suckling. Bioactive isoforms eluting at pH 7.0-6.1 were found only in amenorrhoeic nursing women. We speculate that isoforms eluting at pH 6.0-5.1 may play a role in lactation and isoforms eluting at pH 7.0-6.1, in lactational amenorrhoea.

Adult↗
PubMed2006

Effect of activase level and isoform on the thermotolerance of photosynthesis in Arabidopsis.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate heat stress. This decrease is caused by an impairment of activase function, which is exacerbated by faster rates of Rubisco deactivation at elevated temperatures. To determine if stromal oxidation causes inhibition of activase, transgenic Arabidopsis plants expressing suboptimal amounts of either the redox-regulated 46 kDa alpha- or non-redox regulated 43 kDa beta-isoform of activase were examined. Photosynthesis, as measured by gas exchange and chlorophyll fluorescence, and Rubisco activation were inhibited to a much greater extent by moderately high temperatures in the two transgenic lines expressing suboptimal levels of the individual isoforms of activase compared with wild-type plants or transgenic plants expressing levels of the beta-isoform sufficient for wild-type rates of photosynthesis. Net photosynthesis and Rubisco activation in transgenic plants expressing suboptimal amounts of the beta-isoform of activase from the Antarctic hairgrass were even more sensitive to inhibition by moderate heat stress than in the transgenic plants containing Arabidopsis activase. The results demonstrate that photosynthesis exhibits a similar sensitivity to inhibition by moderately high temperature in plants expressing either of the two different isoforms of activase. Thus, impairment of activase function under heat stress is not caused by oxidation of the redox-sensitive sulphydryls of the alpha-isoform of activase. Instead, the results are consistent with thermal denaturation of activase under moderate heat stress, the effects of which on Rubisco activation would be enhanced when activase levels are suboptimal for photosynthesis.

Arabidopsis↗
PubMed2002

Novel hepatocyte growth factor/scatter factor isoform transcripts in the macaque endometrium and placenta.

Hepatocyte growth factor/scatter factor (HGF/SF) induces proliferation, motility and morphogenesis of cells that express the proto-oncogene for the tyrosine kinase receptor, c-Met. Because these cellular events occur in the endometrium during the menstrual cycle and in placenta during development, we have initiated studies of this growth factor in these tissues from macaques. Several HGF/SF alternatively spliced transcripts have been previously reported in other tissues. However, expression of HGF/SF isoforms in the endometrium has not been studied. Here we describe the relative transcript amounts of HGF/SF isoforms in the endometrium and placenta using RNase protection analyses. During these analyses, we discovered two unexpected protected bands that were found through sequence analyses to represent isoforms similar to the previously reported NK1 and NK2 except that they encode a five amino acid deletion in the first kringle domain. We designated these two isoforms as dNK1 and dNK2. Endometrium expressed all of the isoforms; however, dNK2 was consistently expressed at higher levels than NK2 transcripts. In contrast, placenta expressed NK2 and dNK2 mRNA at equal levels, and both NK1 and dNK1 were undetectable in placenta. HGF/SF function in endometrium and placenta may involve complex interactions between the isoforms of HGF/SF and those of c-Met.

Alternative Splicing↗
PubMed1999

CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms.

The transcription factor CCAAT/enhancer binding protein beta, C/EBPbeta, plays a significant role in the regulation of hepatocyte growth and differentiation. A single mRNA coding for C/EBPbeta produces several protein isoforms. Two pathways for generation of low molecular weight C/EBPbeta isoforms have been described: specific proteolytic cleavage and initiation of translation from different AUG codons of C/EBPbeta mRNA. A truncated C/EBPbeta isoform, LIP, is induced in rat livers in response to partial hepatectomy (PH) via the alternative translation mechanism. Here we present evidence that CUG repeat binding protein, CUGBP1, interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Two binding sites for CUGBP1 are located side by side between the first and second AUG codons of C/EBPbeta mRNA. One binding site is observed in an out of frame short open reading frame (sORF) that has been previously shown to regulate initiation of translation from different AUG codons of C/EBPbeta mRNA. Analysis of cytoplasmic and polysomal proteins from rat liver after PH showed that CUGBP1 is associated with polysomes that translate low molecular weight isoforms of C/EBPbeta. The binding activity of CUGBP1 to the 5' region of C/EBPbeta mRNA shows increased association with these polysomal fractions after PH. Addition of CUGBP1 into a cell-free translation system leads to increased translation of low molecular weight isoforms of C/EBPbeta. Our data demonstrate that CUGBP1 protein is an important component for the regulation of initiation from different AUG codons of C/EBPbeta mRNA.

5' Untranslated Regions↗