Additional Isoforms Research Articles

The Influence of Troponin C, Isoform 4 on Drosophila Development, Stretch Activation, and Power Generation

Drosophila indirect flight muscle (IFM) is one of the most rapidly contracting muscle types known. To increase IFM efficiently and power generation many insect species have evolved high degrees of stretch activation and shortening deactivation in their IFMs. In addition to expressing TnC1, a typical calcium binding troponin C, these IFMs express an unusual unique, additional isoform, TnC4. TnC1 has two calcium binding sites, a high-affinity structural site and a lower-affinity regulatory site. TnC4 has only the high-affinity structural binding site. It is hypothesized to respond to stretch rather than calcium concentration to further activate the thin filament. We are investigating the roles of TnC4 and TnC1 in Drosophila using RNAi and by creating a TnC4 null mutant. Using RNAi, we eliminated TnC1 expression, which resulted in upregulation of TnC4. Power generation and stretch activation of IFMs without TnC1 were statistically identical to wild-type. IFMs with undetectable levels of TnC4 expression produced by RNAi do not generate power or display stretch-activation, suggesting that only TnC4 is necessary for normal muscle function. We have also created a TnC4 null, which will allow us to mechanically evaluate transgenically expressed TnC isoforms and mutants in IFM. Preliminary characterization of the TnC4 null indicates that it is homozygous lethal, but viable as a heterozygote. Flight ability of the heterozygotes is reduced, displaying a 10% reduction in wing-beat frequency than wild-type. The homozygous nulls die at the first molt suggesting that TnC4 is also expressed in some larval muscle types and that it plays a vital role in fly development.

Abstract 18433: A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Activates Innate Immunity and Leads to Cardiac Failure

Modulation of cardiac structure and function by matrix metalloproteinases (MMP) is an area of intense interest. Experimental and clinical evidence has pinpointed a critical role for a specific enzyme, MMP-2, in ventricular remodeling and systolic failure. Transgenic expression of active 68 kDa MMP-2 yields multiple defects, including systolic failure and mitochondrial dysfunction. We hypothesized that an additional intracellular isoform of MMP-2 must exist that initiates mitochondrial dysfunction and inflammation. Western blots of mitochondria from ageing mice and a transgenic model of atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cardiomyoblast cells subjected to transient inhibition of oxidative phosphorylation also generated the mitochondrial 65 kDa MMP-2 isoform. MMP-2 contains three in-frame methionines in the N-terminus, each with a Kozak sequence supporting translational initiation. The 65 kDa MMP-2 isoform is an N-terminal truncation variant generated by alternative translational start site selection at M 77 , thereby deleting the signal sequence and the inhibitory prodomain: thus the 65 kDa MMP-2 is intracellular and enzymatically active. A portion of the 65 kDa MMP-2 localizes in the mitochondrial intramembranous space, triggers mitochondria-to-nuclear stress signaling and actvitates NF-κB and NFAT. Microarray analysis showed that 65 kDa MMP-2 induces a restricted innate immune response transcriptome, including viral stress response genes (OAS1, IFIT, PRKRA), the crucial innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes. Cardiac-specific transgenic expression of the truncated MMP-2 isoform induced progressive cardiomyocyte and ventricular hypertrophy, followed by mononuclear inflammatory cell infiltration and cardiomyocyte apoptosis, as predicted from the microarray results. Functionally, the transgenics showed cardiomyocyte hypertrophy, contractile defects, systolic failure and enhanced injury following ex vivo ischemia/reperfusion. Conclusion: a novel intracellular isoform of MMP-2 is generated by acute or chronic oxidative stress, initiates inflammatory signaling and drives cardiomyocyte apoptosis, features directly relevant to clinical cardiac disease.

Novel Hyperactive Glucocorticoid Receptor Isoform Identified Within a Human Population

Glucocorticoids serve as important therapeutic agents in diseases of inflammation, but clinical use, especially in advanced septic shock, remains controversial because of the unpredictable response. Prior studies correlate human glucocorticoid receptor (hGR) isoforms with a decreased response to steroid therapy. Further analysis of additional hGR isoforms may improve the understanding of the steroid response. Ninety-seven human volunteers' blood samples were surveyed for hGR isoforms. An isoform matching National Center for Biotechnology Informatics (NCBI) hGRα (hGR NCBI) served as a reference. Two isoforms were of particular interest-one isoform had three nonsynonymous single-nucleotide polymorphisms (SNPs) (hGR NS-1), and the second had a single-nucleotide deletion (hGR DL-1) resulting in a truncated protein. Transactivation potentials were measured using a luciferase reporter assay. Human glucocorticoid receptor NS-1 had activity more than twice of hGR NCBI, whereas hGR DL-1 demonstrated less than 10% of the activity of hGR NCBI. Cotransfection of two isoforms revealed that the presence of hGR NS-1 increased transactivation potential, whereas hGR DL-1 decreased activity. Synthetic constructs isolating individual and paired SNPs of hGR NS-1 were created to identify the SNP responsible for hyperactivity. Transactivation studies revealed a SNP within the ligand-binding domain exerted the greatest influence over hyperactivity. In evaluating the response to hydrocortisone, hGR NCBI and hGR NS-1 displayed an increased dose-dependent response, but hGR NS-1 had a response more than twice hGR NCBI. Characterization of the novel hyperactive hGR NS-1 provides insight into a possible mechanism underlying the unpredictable response to steroid treatment.

Identification, Tissue Distribution, and Molecular Modeling of Novel Human Isoforms of the Key Enzyme in Sialic Acid Synthesis, UDP-GlcNAc 2-Epimerase/ManNAc Kinase

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE isoforms (hGNE1-hGNE3), our database and polymerase chain reaction analysis yielded five additional human isoforms (hGNE4-hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-hGNE8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue N-terminal extension compared to hGNE1. On the basis of similarities to kinases and helicases, this extension does not seem to hinder the epimerase enzymatic active site. hGNE3 and hGNE8 contain a 55-residue N-terminal deletion and a 50-residue N-terminal extension compared to hGNE1. The size and secondary structures of these fragments are similar, and modeling predicted that these modifications do not affect the overall fold compared to that of hGNE1. However, the epimerase enzymatic activity of GNE3 and GNE8 is likely absent, because the deleted fragment contains important substrate binding residues in homologous bacterial epimerases. hGNE5-hGNE8 have a 53-residue deletion, which was assigned a role in substrate (UDP-GlcNAc) binding. Deletion of this fragment likely eliminates epimerase enzymatic activity. Our findings imply that GNE is subject to evolutionary mechanisms to improve cellular functions, without increasing the number of genes. Our expression and modeling data contribute to elucidation of the complex functional and regulatory mechanisms of human GNE and may contribute to further elucidating the pathology and treatment strategies of the human GNE-opathies sialuria and hereditary inclusion body myopathy.

The plasma membrane calcium pump in the hearing process: physiology and pathology

Mammalian cells express four different plasma membrane Ca(2+) ATPases. Two of them (PMCA1 and PMCA4) are expressed ubiquitously, and are considered housekeeping isoforms. Two (PMCA2 and PMCA4) have tissue restricted distribution. They are abundantly expressed in the brain and in nervous tissue-derived cell types. The primary transcripts of all PMCAs undergo alternative splicing, generating a large number of additional isoforms. Splicing occurs at site A, in the N-terminal moiety of the pump, and at site C, within the C-terminal calmodulin binding domain: The pumps are canonical targets of calmodulin stimulation. The site C insertion leads to a truncation of the pump about 50 residues short of the original C-terminal. One of the pumps (PMCA2) has special properties: It displays high activity even in the absence of the natural activator calmodulin, and has a particularly complex pattern of alternative splicing at both sites A and C. A variant of the PMCA2 pump containing an insert at site A and truncated C-terminally is the resident isoform of the pump in the stereocilia of hair cells of the inner ear. It exports Ca(2+) to the endolymph that bathes the stereocilia less efficiently than the full length, non-inserted PMCA2 pump. The proper functioning of hair cells demands the precise maintenance of the Ca(2+) balance between hair cells and the endolymph. Disturbances in the balance affect the process of mechano-electrical transduction, which depends on the ability of the stereociliar bundle to deflect in response to sound waves. The tip links that organize the bundle are formed by the Ca(2+) binding protein cadherin 23 and by protocadherin 15: Disturbances of the Ca(2+) binding by cadherin 23 and/or of the ability of the PMCA2 variant of the stereocilia to export Ca(2+) to the endolymph generate hearing loss phenotypes. Such phenotypes have now been described in mice and humans. In some cases they are linked to mutations of both cadherin 23 and the PMCA2 pump, but in other cases they may be generated by mutations of particular severity in only one of the two proteins. The PMCA2 defect that leads to deafness has now been analyzed molecularly: It affects the long range, unstimulated ability of PMCA2 to export Ca(2+).

Abstract 3089: The role of p63 regulation during epithelial to mesenchymal transition (EMT) and subsequent accumulation of malignant features of primary immortalized prostate cells

Abstract A family of transcription factors is constituted by p53, p63 and p73. Unlike p53, which is expressed in response to environmental stress, p63 (TP73L) is constitutively expressed at high levels in a variety of epithelial tissues including prostate. Our genome-wide gene expression analysis identified p63 as one of the most consistently under-expressed genes in prostate cancer samples compared to matched benign prostate tissue. All benign prostate samples including benign prostate hyperplasias (BPH) exhibited high p63 expression. Two alternative p63 promoters generate two different N-terminal variants, TA or ΔN which contains and lacks the transactivating domain, respectively. Differential splicing generates multiple additional isoforms. The activity and interactions of the different isoforms remain unresolved. Using isoform-specific TaqMan real-time quantitative PCR assays, we have found that ΔNp63α is the predominant isoform in prostate tissues, but the other isoforms are detectable. Our group has established a new experimental cell culture model based upon primary, immortalized prostate epithelial cells (EP156T cells), where p63 was found to be shut-down as EP156T cells underwent epithelial-to-mesenchymal transition (EMT) to become EPT1 cells. Subsequently, EPT2 cells were derived from EPT1 cells and p63 stayed shut-down. EPT2 cells had acquired several malignant features in contrast to the progenitors, including anchorage independent growth in soft agar and ability to grow at much higher density in monolayers. To dissect the role of p63 in EMT and in gene expression modules associated with carcinogenesis, we have stably re-expressed the predominant isoform ΔNp63α in both EPT1 and EPT2 cells using retroviral vector transductions. Microarray analysis shows that p63 re-expression leads to re-expression of genes participating in several cell-junction components, such as hemidesmosomes, focal adhesions and gap junctions. These genes were down-regulated during EMT. There were also morphological changes and a decreased ability to migrate following p63 re-expression. This indicates that ΔNp63α re-expression might result in a partial mesenchymal-to-epithelial transition (MET). The present focus is to identify co-factors that may act complementary or synergetically with p63 to induce a complete MET in EPT1 cells and to identify p63 target genes and regulatory cross-talk with relevance for anti-cancer strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2011-3089

The alternative protein isoform NK2B, encoded by the vnd/NK-2 proneural gene, directly activates transcription and is expressed following the start of cells differentiation

NK-2 is a homeodomain protein essential for the development of the central nervous system in the Drosophila embryo. Here, we show that the vnd/NK-2 gene encodes an additional protein isoform (NK-2B) that differs from the known one (NK-2A) in its N-terminal domain. While NK-2A is a transcription repressor, NK-2B directly activates transcription from promoters containing NK-2 binding sites, with its N-terminal domain possessing a strong transcription activation potency. The transcription of NK-2B starts at the onset of metamorphosis. Its expression is observed in precursors of differentiating photoreceptors and in photoreceptors of the adult eye. Both NK-2B and NK-2A are expressed in the lamina. However, the expression of NK-2A is mostly associated with the undifferentiated state of nervous cells.

The Influence of Troponin C Isoforms on Drosophils Stretch Activation and Power Generation

Drosophila indirect flight muscle (IFM) is one of the most rapidly contracting muscle types known. To efficiently generate power for flight at such high speeds, insects have evolved stretch activation and shortening deactivation. In IFM of some insects, in addition to a typical calcium binding troponin C, TnC1, there is a unique, additional isoform, TnC4. TnC1 has 2 calcium binding sites, a high-affinity structural site and a lower-affinity regulatory site. TnC4 has only the high-affinity structural binding site. It is hypothesized to respond to stretch rather than Ca2+ concentration to further activate the thin filament. We are investigating TnC4 in Drosophila via two different routes. The first is to chemically remove TnC from IFM using either o-vanadate or trifluoperazine which eliminates power generation and stretch activation. Bacterially-expressed TnC1 and TnC4 are reincorporated into the fiber in physiological proportions, which restores power-generating ability and stretch activation to the IFM. We are currently optimizing the extraction and reintroduction protocol to determine the effects on stretch activation of varying the ratio of TnC1 and TnC4. The second method is to breed a TnC4 null using p-element mediated male homologous recombination. We are performing this by crossing flies containing identical p-elements in positions which bracket the TnC4 gene on chromosome 2R. When the p-elements are mobilized, some of the offspring will have the region between the p-elements excised. We are currently screening offspring of the final cross for null TnC4 alleles. These two methods of investigation into TnC4 function will allow modified TnC4 to either be introduced or expressed in IFM to help elucidate TnC4's structural mechanism.

Biochemical Characterization, Kinetic Analysis and Molecular Modeling of Recombinant Vegetative Lipoxygenases from Soybean

Plants in general have multiple lipoxygenase isoforms that exhibit distinct biochemical attributes and discrete cellular and sub-cellular localization patterns. This suggests that plant lipoxygenases are multifunctional and contribute to several biochemical and physiological processes during plant growth and development. Soybeans have at least eight isoforms and the three that are localized to the seed (L-1; L-2; L-3) have been characterized extensively and L-1 serves as a model. The vegetative organs, however, have five additional isoforms that are not well characterized at present. To better understand the cellular function of these vegetative lipoxygenases, kinetic parameters and pH profiles for the recombinant VLXs were determined for three free fatty acids above and below the corresponding critical micelle concentrations, including free fatty acids and the triglyceride trilinolein. The kinetic data using trilinolein as a substrate from two isoforms, VLX-C & VLX-D, supports a potential role for VLX-C & -D in the metabolism of lipids in soybean leaves as suggested by the significantly higher kcat/km and turnover of lipid substrates for these two isoforms. In addition, structural studies indicate clear differences exist between VLX-A, -B & -E and VLX-C & -D in the exposed loops of the ?-barrel, O2 cavity and entry site of the subcavity IIa. Therefore, based on distinct patterns of activity and structure, the existence of functional subgroups among the five VLX isoforms was confirmed.

Two novel human NUMB isoforms provide a potential link between development and cancer

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

Intron 4 Containing Novel GABAB1 Isoforms Impair GABAB Receptor Function

BackgroundGamma-aminobutyric acid type B (GABAB) receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants.Methodology/Principal FindingsBased on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF) and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K+ channel (GIRK) currents at Xenopus oocyte two-electrode voltage clamp system.Conclusions/SignificanceThis study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice variants.

S31 Characterisation of cell adhesion molecule-1 in lung mast cells

Introduction and ObjectivesCell adhesion molecule 1 (CADM1) is implicated in several diseases and a prognostic factor for lung cancer. In human lung mast cells (HLMC) it contributes to cell-cell adhesion...

Antioxidant metabolism of grapefruit infected with Xanthomonas axonopodis pv. citri

Grapefruit is one of the most susceptible citrus genotypes to Asiatic Citrus Canker, caused by Xanthomonas axonopodis pv. citri (Xac), that can cause severe losses in citrus yield and quality. Although much is known about citrus response to Xac, little is known of the role of antioxidant metabolism. Grapefruit leaves were artificially injected with a strain of Xac obtained from a commercial grove in Florida and components of oxidative metabolism were measured. Symptoms observed included water soaking (2dai; days after inoculation), raised and ruptured epidermis (6–8dai), formation of necrotic lesions (16dai), and leaf abscission (21dai). The Xac population increased to a maximum (≈109CFU/cm2) 8dai and then declined to ≈107CFU/cm2 by 20dai. Lipid peroxidation was higher in infected leaves than uninoculated controls from 4 to 21dai indicating greater oxidative stress. H2O2 concentration demonstrated a biphasic pattern with peak concentrations at 4 and 13dai and minimum concentrations that were lower than the controls at 10 and 20dai. The H2O2 concentration somewhat corresponded with superoxide dismutase (SOD) activity, which generates H2O2 via dismutase of superoxide ions. Total SOD activity in Xac-infected leaves increased to a maximum at 4dai, the day of highest H2O2 concentration, and then declined and remained at or below controls. Mn-SOD and Fe-SOD activities both increased to maximum activities at 4dai. Mn-SOD had four isoforms in Xac-infected leaves but only three in the controls. Fe-SOD had three isoforms in both infected and control plants. Suppression of H2O2 in Xac-infected leaves also corresponded to higher activities of the H2O2 catabolising enzymes catalase (CAT), ascorbate peroxidase (APOD), and peroxidase (POD). Two additional CAT isoforms were detected in infected leaves and not the controls. Three POD isoforms were detected in both control and infected leaves. Previous research has shown that Xac is sensitive to intraplant H2O2 concentration, however, the pattern of Xac in this study did not correspond to H2O2 concentration, which initially increased due to enhanced SOD activity, but was later suppressed apparently with the aid of peroxidases. In conclusion, Xac infection altered H2O2 metabolism in grapefruit leaves by changes in the activities and isoforms of SODs, CATs, PODs and APOD.

The role of alternative mRNA splicing in chromosome instability

Chromosomal instability (CIN) involves the gain or loss of complete or partial chromosomes during cellular division, and it is a common characteristic of tumors that have aneuploidy. In addition, CIN is considered to be a closely related event to carcinogenesis. The mechanisms that lead to CIN include defects in the cohesion of sister chromatids, mitotic spindle checkpoint, and regulation of the number of centrosomes. Different studies have found that transcription variants, also known as isoforms, which are generated by the alternative splicing of exons and introns in mRNA that encodes many of the regulator proteins of chromosomal segregation, have an important role in mechanisms that lead to CIN. The majority of these isoforms are newly described. The discovery of additional isoforms and the study of their mechanisms of action allow a more integrated view of how cells regulate the segregation of their genetic material, and of how errors occur in chromosomal segregation.

Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

BackgroundPathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.Methodology/Principal FindingsIn a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.Conclusions/SignificanceOur study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.

Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis

Plants challenged by pathogens, especially necrotrophic fungi such as Botrytis cinerea, produce high levels of ethylene. At present, the signaling pathways underlying the induction of ethylene after pathogen infection are largely unknown. MPK6, an Arabidopsis stress-responsive mitogen-activated protein kinase (MAPK) was previously shown to regulate the stability of ACS2 and ACS6, two type I ACS isozymes (1-amino-cyclopropane-1-carboxylic acid synthase). Phosphorylation of ACS2 and ACS6 by MPK6 prevents rapid degradation of ACS2/ACS6 by the 26S proteasome pathway, resulting in an increase in cellular ACS activity and ethylene biosynthesis. Here, we show that MPK3, which shares high homology and common upstream MAPK kinases with MPK6, is also capable of phosphorylating ACS2 and ACS6. In the mpk3 mutant background, ethylene production in gain-of-function GVG-NtMEK2(DD) transgenic plants was compromised, suggesting that MPK6 and MPK3 function together to stabilize ACS2 and ACS6. Using a liquid-cultured seedling system, we found that B.cinerea-induced ethylene biosynthesis was greatly compromised in mpk3/mpk6 double mutant seedlings. In contrast, ethylene production decreased only slightly in the mpk6 single mutant and not at all in the mpk3 single mutant, demonstrating overlapping roles for these two highly homologous MAPKs in pathogen-induced ethylene induction. Consistent with the role of MPK3/MPK6 in the process, mutation of ACS2 and ACS6, two genes encoding downstream substrates of MPK3/MPK6, also reduced B.cinerea-induced ethylene production. The residual levels of ethylene induction in the acs2/acs6 double mutant suggest the involvement of additional ACS isoforms, possibly regulated by MAPK-independent pathway(s).

The activity pattern and gene expression profile of aldehyde oxidase during the development of Pisum sativum seeds

Aldehyde oxidase (AO, EC 1.2.3.1) is a molybdenohydroxylase that is considered to catalyze the final step in the synthesis of abscisic acid (ABA) and possibly of indole-3-acetic acid (IAA). Five AO activity bands were detected after native PAGE with indole-3-aldehyde (PsAO-α, -β, -γ, -δ, -κ) and three with abscisic aldehyde (PsAO-γ, -δ, -κ) in developing seeds of Pisum sativum. At early and mid-development, PsAO-α, -β, -γ and only PsAO-γ were observed, respectively, and their localization as well as the expression of PsAOs genes was almost exclusively restricted to the maternal fruit tissues, the seed coat and pericarp. Towards the end of rapid reserve synthesis, two additional isoforms (PsAO-δ, -κ) appeared in cotyledons, coinciding with a high transcript level of PsAO2. At this developmental stage, the activity level of PsAO-γ, was still considerable in the testa, and was higher than at earlier stages in the embryonic axis, which correlated with the PsAO3 transcript level. In mature dry seeds, AO activity and the expression of PsAOs became restricted to the embryonic tissues. The possible involvement of AO isoforms in ABA or IAA synthesis during pea seed development as well as the contribution of particular PsAO genes to the formation of the dimeric pea AO isoforms is discussed.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

Molecular coevolution of kisspeptins and their receptors from fish to mammals

Kisspeptin and its receptor, GPR54, play a pivotal role in vertebrate reproduction. Recent advances in bioinformatic tools combined with comparative genomics have led to the identification of a large number of kisspeptin and GPR54 genes in a variety of vertebrate species. Genome duplications may have produced at least two isoforms of both ligand (KiSS1 and KiSS2) and receptor (GPR54-1 and GPR54-2). Additional isoforms of kisspeptin (KiSS1b) and GPR54 (GPR54-1b) have been found in an amphibian species, Xenopus (Silurana) tropicalis. Here, we describe the evolutionary lineages of these kisspeptin and GPR54 isoforms using genome synteny and phylogenetic analyses, and possible molecular interactions between kisspeptin and GPR54 subtypes based on ligand-receptor selectivity. Together, kisspeptin and GPR54 provide an excellent model for understanding molecular coevolution of the peptide ligand and GPCR pairs.

Identification of novel transcripts from the porcine MYL1 gene and initial characterization of its promoters

The fast skeletal alkali myosin light polypeptide 1 (MYL1) gene is one of three mammalian alkali MLC genes and encodes two isoforms, 1f and 3f, which play a vital role in embryonic, fetal, and adult skeletal muscle development. We isolated the MYL1 gene from a pig BAC library with the goal of characterizing its promoter and identifying its transcripts. Genes and isoforms were identified by reverse transcriptase-PCR, northern blot and RACE (Rapid Amplification of cDNA Ends). Potential MYL1 gene promoters were characterized using a luciferase reporter assay and electrophoretic mobility shift assays (EMSA). MLC1f, MLC3f, and three additional isoforms of porcine MYL1, MLC5f-A, -B, and -C were identified. Up to now, the three novel isoforms had not been reported in human or mouse. Northern blot analysis indicated that MLC1f, MLC3f, and MLC5fs were expressed only in longissimus dorsi muscles. Two transcription initiation and termination sites were identified by RACE. Promoter analysis and EMSA demonstrated the presence of a MEF3 (skeletal muscle-specific transcriptional enhancer) binding site (+384 to +481), which might be essential for porcine MYL1 transcription. Our results suggested that five transcript variants were generated using alternative promoters, two transcription start sites, and polyA sites, as well as variable splicing of the pig MYL1 exon 5. The identification of alternative promoters and splice variants, the expression of the splice variants in different muscle tissues, and the definition of regulatory elements provide important molecular genetic knowledge concerning the MYL1 gene.