Major Isoform (major + isoform)

Distribution by Scientific Domains

Selected Abstracts

Deficient long-term synaptic depression in the rostral cerebellum correlated with impaired motor learning in phospholipase C ,4 mutant mice

Mariko Miyata
Abstract Long-term depression (LTD) at parallel fibre,Purkinje cell synapse of the cerebellum is thought to be a cellular substrate for motor learning. LTD requires activation of metabotropic glutamate receptor subtype 1 (mGluR1) and its downstream signalling pathways, which invariably involves phospholipase C,s (PLC,s). PLC,s consist of four isoforms (PLC,1,4) among which PLC,4 is the major isoform in most Purkinje cells in the rostral cerebellum (lobule 1 to the rostral half of lobule 6). We studied mutant mice deficient in PLC,4, and found that LTD was deficient in the rostral but not in the caudal cerebellum of the mutant. Basic properties of parallel fibre,Purkinje cell synapses and voltage-gated Ca2+ channel currents appeared normal. The mGluR1-mediated Ca2+ release induced by repetitive parallel fibre stimulation was absent in the rostral cerebellum of the mutant, suggesting that their LTD lesion was due to the defect in the mGluR1-mediated signalling in Purkinje cells. Importantly, the eyeblink conditioning, a simple form of discrete motor learning, was severely impaired in PLC,4 mutant mice. Wild-type mice developed the conditioned eyeblink response, when pairs of the conditioned stimulus (tone) and the unconditioned stimulus (periorbital shock) were repeatedly applied. In contrast, PLC,4 mutant mice could not learn the association between the conditioned and unconditioned stimuli, although their behavioural responses to the tone or to the periorbital shock appeared normal. These results strongly suggest that PLC,4 is essential for LTD in the rostral cerebellum, which may be required for the acuisition of the conditioned eyeblink response. [source]

The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation

FEBS JOURNAL, Issue 1 2007
Mirko Zaffagnini
In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post-translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A4 -glyceraldehyde-3-phosphate dehydrogenase (A4 -GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2. The formation of a mixed disulfide between glutathione and A4 -GAPDH resulted in the inhibition of enzyme activity. A4 -GAPDH was also inhibited by oxidants such as H2O2. However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the AnBn -GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3-bisphosphoglycerate. In the presence of 1,3-bisphosphoglycerate, A4 -GAPDH was fully protected from either oxidation or glutathionylation. Site-directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active-site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A4 -GAPDH from irreversible oxidation under stress. [source]

The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris

FEBS JOURNAL, Issue 20 2005
Daniel Kolarich
Hyaluronidase (E.C., one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43,45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms. [source]

Functional dissection of two Arabidopsis PsbO proteins

FEBS JOURNAL, Issue 9 2005
PsbO protein is an extrinsic subunit of photosystem II (PSII) and has been proposed to play a central role in stabilization of the catalytic manganese cluster. Arabidopsis thaliana has two psbO genes that express two PsbO proteins; PsbO1 and PsbO2. We reported previously that a mutant plant that lacked PsbO1 (psbo1) showed considerable growth retardation despite the presence of PsbO2 [Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett523, 138,142]. In the present study, we characterized the functional differences between PsbO1 and PsbO2. We found that PsbO1 is the major isoform in the wild-type, and the amount of PsbO2 in psbo1 was significantly less than the total amount of PsbO in the wild-type. The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; howeVER, it neVER reached the total PsbO level observed in the wild-type, suggesting that the poor activity of PSII in psbo1 was caused by a shortage of PsbO. In addition, an in vitro reconstitution experiment using recombinant PsbOs and urea-washed PSII particles showed that oxygen evolution was better recoVERed by PsbO1 than by PsbO2. Further analysis using chimeric and mutated PsbOs suggested that the amino acid changes Val186,Ser, Leu246,Ile, and Val204,Ile could explain the functional difference between the two PsbOs. Therefore we concluded that both the lower expression level and the inferior functionality of PsbO2 are responsible for the phenotype observed in psbo1. [source]

Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin,

HUMAN MUTATION, Issue 6 2008
Shweta Singh
Abstract Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20,LD families from the Indian population. We identified 12 distinct mutations in 15,LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210,K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype. © 2008 Wiley-Liss, Inc. [source]

Suppression of cyclic GMP-specific phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cells

Bing Zhu
Abstract Phosphodiesterase 5 (PDE5) is a major isoform of cGMP phosphodiesterase in a variety of human tumor cell lines and plays a key role in regulating intracellular cGMP concentrations ([cGMP]i). Here, we demonstrate that suppression of PDE5 gene expression by antisense pZeoSV2/ASP5 plasmid transfection results in a sustained increase in [cGMP]i, growth inhibition, and apoptosis in human colon tumor HT29 cells. With stable transfection, antisense transcripts exhibited a specific suppression in PDE5 activity, mRNA levels, and a 93 kDa hPDE5A1 protein. In cloned antisense cells, prolongation of the cell growth doubling times correlate positively with suppressed PDE5 activity and increased [cGMP]i. The growth inhibition in PDE5 antisense clones is due to an increased apoptotic rate and delayed cell-cycle progression. These results corroborate previous findings with the PDE5 inhibitor exisulind and its derivatives showing that sustained [cGMP]i induces apoptosis and growth inhibition in tumor cells. Furthermore, an inducible mitotic inhibitor p21WAF1/CIP1 has been found to account for the delay of cell-cycle progression in PDE5 antisense clones at G2/M phase. A proteolytic cleavage of p21WAF1/CIP1 in the antisense clones is also increased at the later stage of serum stimulation. The protein kinase G (PKG) inhibitor, KT5823, can prevent the cleavage of p21WAF1/CIP. These data substantiate a pivotal role for PDE5 as a modulator of apoptosis and cell-cycle progression for human carcinoma via a mechanism involving the activation of [cGMP]i/PKG signaling pathways. © 2004 Wiley-Liss, Inc. [source]

Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MS

Alexander Plematl
Abstract The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of ,-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of ,-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of ,-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than ,-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance. [source]

Decreased activity of the smooth muscle Na+/Ca2+ exchanger impairs arteriolar myogenic reactivity

Hema Raina
Arteriolar myogenic vasoconstriction occurs when stretch or increased membrane tension leads to smooth muscle cell (SMC) depolarization and opening of voltage-gated Ca2+ channels. While the mechanism underlying the depolarization is uncertain a role for non-selective cation channels has been demonstrated. As such channels may be expected to pass Na+, we hypothesized that reverse mode Na+/Ca2+ exchange (NCX) may act to remove Na+ and in addition play a role in myogenic signalling through coupled Ca2+ entry. Further, reverse (Ca2+ entry) mode function of the NCX is favoured by the membrane potential found in myogenically active arterioles. All experiments were performed on isolated rat cremaster muscle first order arterioles (passive diameter ,150 ,m) which were pressurized in the absence of intraluminal flow. Reduction of extracellular Na+ to promote reverse-mode NCX activity caused significant, concentration-dependent vasoconstriction and increased intracellular Ca2+. This vasoconstriction was attenuated by the NCX inhibitors KB-R7943 and SEA 04000. Western blotting confirmed the existence of NCX protein while real-time PCR studies demonstrated that the major isoform expressed in the arteriolar wall was NCX1. Oligonucleotide knockdown (24 and 36 h) of NCX inhibited the vasoconstrictor response to reduced extracellular Na+ while also impairing both steady-state myogenic responses (as shown by pressure,diameter relationships) and acute reactivity to a 50 to 120 mmHg pressure step. The data are consistent with reverse mode activity of the NCX in arterioles and a contribution of this exchanger to myogenic vasoconstriction. [source]

Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma,

Joseph C. Sniezek MD
Abstract Objectives/Hypothesis: p63, a member of the p53 family of genes, is vital for normal epithelial development and may play a critical role in epithelial tumor formation. Although p63 has been identified in various head and neck malignancies, a detailed analysis of which of the six isoforms of the p63 gene is present in normal mucosa and head and neck malignancies has not yet been performed. The study analyzed p63 isoform expression on the RNA and protein level in normal, diseased, and malignant mucosa of the head and neck to examine the differential expression of p63 isoforms in head and neck tumors versus adjacent nonmalignant tissue and to identify the predominant p63 isoform expressed in head and neck squamous cell carcinoma (HNSCC). Study Design: Three experiments were performed. In experiment 1, p63 expression was analyzed by immunohistochemical analysis in 36 HNSCC specimens and matched normal tissue control specimens harvested from the same patient. Western blot analysis was also performed on matched specimens to confirm the identity of the p63 isoforms that were found. In experiment 2, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on matched normal and tumor specimens to analyze and quantitatively compare p63 isoform expression at the RNA level. In experiment 3, p63 expression was evaluated by immunohistochemical analysis in oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. Methods: Immunohistochemical analysis, RT-PCR, and Western blot analysis of p63 were performed on HNSCC specimens and matched normal tissue control specimens. p63 expression in oral lichen planus specimens was also examined by immunohistochemical analysis. Results: In experiment 1, analysis of 36 HNSCC specimens from various head and neck subsites showed p63 expression in all tumors and matched normal tissue specimens (36 of 36). Western blot analyses indicated that dominant negative (,N) isoform p63, (,Np63,) is the major isoform expressed at the protein level in tumors and adjacent normal tissue. In experiment 2, RT-PCR analyses of 10 matched specimens confirmed that, although all three ,Np63 isoforms (,Np63,, ,Np63,, and ,Np63,) are expressed in normal and malignant mucosa of the head and neck, ,Np63, is the predominant transcript expressed. In experiment 3, immunohistochemical analysis of p63 in the pro-apoptotic condition of lichen planus indicated that p63 is underexpressed as compared with normal mucosal specimens. Conclusion: Although all three ,Np63 isoforms are present in HNSCC, ,Np63, protein is the predominant isoform expressed in these malignancies. ,Np63, is also overexpressed in tumors compared with matched normal tissue specimens and is underexpressed in the pro-apoptotic condition of lichen planus. These findings suggest that ,Np63, plays an anti-differentiation and anti-apoptotic role in the mucosal epithelium of the head and neck, possibly playing a pivotal role in the formation of HNSCC. Currently, ,Np63, is an attractive target for mechanistic study aimed at therapeutic intervention. [source]

Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigra

Akifumi Kamata
Abstract Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, ,, ,, , and ,, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT,PCR analyses revealed that the , and , isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were ,A, ,C, ,1 and ,3. An immunohistochemical study also confirmed the preferential localization of , and , isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKII,1,4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of , isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKII,3 isoform is involved in the expression of BDNF in the SN. [source]

Local isoform-specific NOS inhibition: A promising approach to promote motor function recovery after nerve injury

Bernardo Moreno-López
Abstract Physical injury to a nerve is the most frequent cause of acquired peripheral neuropathy, which is responsible for loss of motor, sensory and/or autonomic functions. Injured axons in the peripheral nervous system maintain the capacity to regenerate in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Therefore, the identification of molecules that regulate degenerative and regenerative processes is indispensable in developing therapeutic tools to accelerate and improve functional recovery. Here, I consider the role of nitric oxide (NO) synthesized by the three major isoforms of NO synthases (NOS) in motor neuropathy. Neuronal NOS (nNOS) seems to be the primary source of NO that is detrimental to the survival of injured motoneurons. Endothelial NOS (eNOS) appears to be the major source of NO that interferes with axonal regrowth, at least soon after injury. Finally, NO derived from inducible NOS (iNOS) or nNOS is critical to the process of lipid breakdown for Wallerian degeneration and thereby benefits axonal regrowth. Specific inhibitors of these isoforms can be used to protect injured neurons from degeneration and promote axonal regeneration. A cautious proposal for the treatment of acquired motor neuropathy using therapeutic tools that locally interfere with eNOS/nNOS activities seems to merit consideration. © 2010 Wiley-Liss, Inc. [source]

Phosphorylation of the neural cell adhesion molecule on serine or threonine residues is induced by adhesion or nerve growth factor

Stephanie Matthias
Abstract The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily and plays a crucial role during development and regeneration. It is expressed in three major isoforms; two of them with intracellular domains of different length and one without any intracellular domain. NCAM is known to be phosphorylated and contains up to 49 serine or threonine residues, which could be phosphorylated. However, the impact of NCAM phosphorylation is still unclear. Here we describe NCAM being phosphorylated during neuronal differentiation of PC12 cells. We provide evidence that protein kinase C is involved in the phosphorylation of NCAM. In agreement with our earlier observation that the protein phosphatase 1 is associated with NCAM, we additionally found that NCAM is a substrate for the protein phosphatase 1 but not for the protein phosphatase 2A. © 2006 Wiley-Liss, Inc. [source]