Expression In COS-7 Cells Research Articles

Decreasing Activity and Altered Protein Processing of Human Iduronate-2-sulfatase Mutations Demonstrated by Expression in COS7 Cells

Hunter syndrome, or mucopolysaccharidosis type II (MPS II; MIM:309900), is a rare X-linked recessive lysosomal storage disorder caused by defects of the gene coding for iduronate-2-sulfatase (IDS; EC 3.1.6.13) (Bach et al. 1973). IDS catabolizes dermatan sulfate and heparan sulfate in the lysosome. Lack of this enzyme leads to the systemic accumulation of these substances, eventually causing characteristic clinical

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.

Update of the pompe disease mutation database with 60 novel GAA sequence variants and additional studies on the functional effect of 34 previously reported variants

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder, characterized by progressive muscle weakness. Deficiency of acid α-glucosidase (EC; 3.2.1.20/3) can be caused by numerous pathogenic variants in the GAA gene. The Pompe Disease Mutation Database at http://www.pompecenter.nl aims to list all variants and their effect. This update reports on 94 variants. We examined 35 novel and 34 known mutations by site-directed mutagenesis and transient expression in COS-7 cells or HEK293T cells. Each of these mutations was given a severity rating using a previously published system, based on the level of acid α-glucosidase activity in medium and transfected cells and on the quantity and quality of the different molecular mass species in the posttranslational modification and transport of acid α-glucosidase. This approach enabled to classify 55 missense mutations as pathogenic and 13 as likely nonpathogenic. Based on their nature and the use of in silico analysis (Alamut® software), 12 of the additional 25 novel mutations were predicted to be pathogenic including 4 splicing mutations, 6 mutations leading to frameshift, and 2 point mutations causing stop codons. Seven of the additional mutations were considered nonpathogenic (4 silent and 3 occurring in intron regions), and 6 are still under investigation.

Dissecting the Ca2+ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells

Rotavirus infection modifies Ca2+ homeostasis provoking an increase in Ca2+ permeation, cytoplasmic Ca2+ concentration ([Ca2+]cyto), total Ca2+ pools and, a decrease of Ca2+ response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca2+ homeostasis is not yet known. It has been proposed that the increase in [Ca2+]cyto is the result of Ca2+ release from intracellular stores, thereby activating store-operated Ca2+ entry (SOCE). We studied the mechanisms involved in the changes of Ca2+ permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca2+ stores induced by thapsigargin or ATP was not able to elicit Ca2+ entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca2+]cyto, is permeable to Mn2+ and inhibited by La3+ and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca2+ entry is small and not modified by infection. The plasma membrane permeability to Ca2+ in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na+/Ca2+ exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca2+ entry.

Short‐hairpin RNA‐mediated silencing of NKCC1 in COS7 cells and its effect on cell volume

Na+K+2Cl− cotransporters (NKCCs) effect the electroneutral movement of Na+, K+ and 2Cl− across the plasma membrane. Under physiological conditions NKCCs function as active, Na+‐dependent, Cl− uptake transporters. Two NKCC isoforms, encoded by two different genes have been identified: NKCC1 and NKCC2. NKCC1 is a ubiquitously expressed gene whereas NKCC2 is considered to be exclusive of the kidney. We describe for the first time a viable knock‐down strategy to silence endogenous NKCC1 expression in a widely used, easy to transfect cell line. NKCC1 mRNA expression in COS7 cells was eliminated transfecting these cells with a poly‐cistronic vector encoding for GFP and an shRNA directed against the third exon of human NKCC1. Stable transfectants were selected by continuous incubation in the presence of puromycin. Cells stably expressing GFP and resistant to the drug were selected and cloned. NKCC1 expression was followed by Western bolt, immunofluorescence and reverse transcription (RT) coupled to the polymerase chain reaction (PCR). The impact of NKCC1 depletion on cell volume was followed morphometrically. We found that cells lacking NKCC1 are shrunken suggesting that NKCC1 is the sole regulator of cell volume in these cells. This new cell system will open the possibility to study the functional, pharmacological and molecular properties of candidate genes involved in volume regulation.This work was supported by funds awarded to MDiF by the American Diabetes Association (Grant JF‐1001730).

Immunogenicity Study of Plasmid DNA Encoding Mouse Cysteine‐Rich Secretory Protein‐1 (mCRISP1) as a Contraceptive Vaccine

To examine the immunocontraceptive properties of the plasmid pcDNA-mCRISP1 and compare them to the corresponding recombinant mCRISP1 (r-mCRISP1). RT-PCR and indirect immunofluorescence were performed to observe the mCRISP1 protein expression in COS-7 cells. Three groups of mice received three injections of r-mCRISP1, pcDNA-mCRISP1 or pcDNA vector, respectively. ELISA and Western blot were used to examine the immune responses and immunoreactivity of antisera. Sperm-egg penetration assay was performed to examine the effect of anti-mCRISP1 antibodies in vitro fertilization of mouse oocytes. Fertility and mean litter size were analysed by natural mating. Histological analysis was carried out to look for potential immunopathologic effects of the antibodies. COS-7 cells transfected with pcDNA-mCRISP1 present the expression of mCRISP1. Both r-mCRISP1 and pcDNA-mCRISP1 raised an immune response against r-mCRISP1 protein and native CRISP1 in mouse sperm. The titres of anti-mCRISP1 antibodies from DNA immunized mice were significantly lower than that of r-mCRISP1 immunized mice, but it lasted relatively longer. Male and female pcDNA-mCRISP1 injected animals presented a statistically significant reduction in their fertility with no signs of immunopathologic effects. These studies demonstrated the feasibility of generating an immune response to mCRISP1 protein by DNA vaccine and pcDNA-mCRISP1 plasmid causing significant anti-fertility potential.

The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.

Experimental Mapping of the Canine KCNJ2 and KCNJ12 Gene Structures and Functional Analysis of the Canine KIR2.2 ion Channel

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

High allele frequency of the p.Q258X mutation and identification of a novel mis-splicing mutation in the STAR gene in Korean patients with congenital lipoid adrenal hyperplasia

Steroidogenic acute regulatory (STAR) protein plays a crucial role in steroidogenesis, and mutations in the STAR gene cause congenital lipoid adrenal hyperplasia (CLAH). This study investigated the STAR mutation spectrum and functionally analyzed a novel STAR mutation in Korean patients with CLAH. Mutation analysis of STAR was carried out in 25 unrelated Korean CLAH patients. A region of STAR comprising exons 4-7 was cloned from human genomic DNA into an expression vector, followed by site-directed mutagenesis and transient expression in COS7 cells. The splicing pattern was analyzed by in vitro transcription, and each transcript was functionally characterized by measuring pregnenolone production in COS7 cells cotransfected with the cholesterol side chain cleavage system. Mutation p.Q258X was identified in 46 of 50 alleles (92%); mutation c.653C>T was detected in two alleles (4%); and mutations p.R182H and c.745-6_810del were found in one allele (2%). Reverse transcriptase-PCR products amplified from a patient heterozygous for compound c.653C>T and c.745-6_810del mutation revealed multiple alternatively spliced mRNAs. In vitro expression analysis of a minigene consisting of exons 4-7 containing the c.653C>T yielded two transcripts in which exon 6 or exons 5 and 6 were skipped. The encoded proteins exhibited defective pregnenolone-producing ability. The c.745-6_810del mutation led to full and partial intron retention. p.Q258X is the most common STAR mutation in Korea. A previously reported c.653C>T variant was found to cause aberrant splicing at the mRNA level, resulting in perturbation of STAR function. The c.745-6_810del mutation also resulted in aberrant splicing.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Influence of Biological Media on the Structure and Behavior of Ferrocene-Containing Cationic Lipid/DNA Complexes Used for DNA Delivery

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li(2)SO(4)) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately -35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about -20 mV in OptiMEM and -10 mV in Li(2)SO(4)), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.

Functional effects of genetic polymorphisms in the N-acetyltransferase 1 coding and 3′ untranslated regions

The functional effects of N-acetyltransferase 1 (NAT1) polymorphisms and haplotypes are poorly understood, compromising the validity of associations reported with diseases, including birth defects and numerous cancers. We investigated the effects of genetic polymorphisms within the NAT1 coding region and the 3'-untranslated region (3'-UTR) and their associated haplotypes on N- and O-acetyltransferase catalytic activities, and NAT1 mRNA and protein levels following recombinant expression in COS-1 cells. 1088T>A (rs1057126; 3'-UTR) and 1095C>A (rs15561; 3'-UTR) each slightly reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. A 9-bp (TAATAATAA) deletion between nucleotides 1065 and 1090 (3'-UTR) reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. In contrast, a 445G>A (rs4987076; V149I), 459G>A (rs4986990; T153T), and 640T>G (rs4986783; S214A) coding region haplotype present in NAT1*11 increased NAT1 catalytic activity and NAT1 protein, but not NAT1 mRNA levels. A combination of the 9-bp (TAATAATAA) deletion and the 445G>A, 459G>A, and 640T>G coding region haplotypes, both present in NAT1*11, appeared to neutralize the opposing effects on NAT1 protein and catalytic activity, resulting in levels of NAT1 protein and catalytic activity that did not differ significantly from the NAT1*4 reference. Because 1095C>A (3'-UTR) is the sole polymorphism present in NAT1*3, our data suggest that NAT1*3 is not functionally equivalent to the NAT1*4 reference. Furthermore, our findings provide biologic support for reported associations of 1088T>A and 1095C>A polymorphisms with birth defects.

A functional tetranucleotide (AAAT) polymorphism in an Alu element in the NF1 gene is associated with mental retardation

Mental retardation (MR) is frequent in neurofibromatosis type 1 (NF1). Allele 5 of a tetranucleotide polymorphism in an Alu element (GXAlu) localized in intron 27b of the NF1 gene has previously been associated with autism. We considered that the microsatellite GXAlu could also represent a risk factor in MR without autism. We developed a rapid method for genotyping by non-denaturing HPLC and assayed the allelic variation of GXAlu marker on in vitro gene expression in Cos-7 cells. A French population of 157 individuals (68 non syndromic non familial MR (NS-MR) patients diagnosed in the University Hospital of Tours; 89 controls) was tested in a case-control assay. We observed a significant association ( χ 2 = 7.96; p = 0.005) between alu4 carriers (7 AAAT repeats) and MR (OR: 7.86; 95% C.I.: 2.13–28.9). The relative in vitro expression of a reporter gene encoding chloramphenicol acetyl transferase (CAT) was higher for alu4 and alu5, suggesting a regulation effect for these alleles on gene expression in vivo. Our results showed an association with a polymorphism regulating the NF1 gene or other genes during brain development.

Regulated Phosphorylation of a Major UDP-glucuronosyltransferase Isozyme by Tyrosine Kinases Dictates Endogenous Substrate Selection for Detoxification

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE1) with barely detectable 17β-estradiol (E2) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [33P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)−/− cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF−/− and SYF+/− cells metabolized 4-OHE1 at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E2 metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438–2B7 detected Tyr(P)-438–2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF−/−. 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE1 glucuronidation reaching 60% maximum while simultaneously increasing E2 metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E2 metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF−/− is more competent at metabolizing E2 in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E1-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE1, but not E2, in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.

Extracellular-Superoxide Dismutase Expression in COS7 Cells Exposed to Cadmium Chloride

Cadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactive oxygen species (ROS) and induces cytotoxicity. Extracellular-superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the cells from damaging effects of ROS; however, the effect of Cd on the expression of EC-SOD in COS7 cells remains unclear. In this study, exposure to cadmium chloride (CdCl₂) enhanced intracellular ROS generation and induced COS7 cell death. Moreover, exposure to Cd decreased the expression of EC-SOD at mRNA and protein levels, but not of other SOD isozymes, copper-and zinc-containing SOD and manganese-containing SOD. The reduction of EC-SOD and cell viability was partially attenuated by pretreatment with an antioxidant, N-acetylcysteine. Further, we determined the involvement of p38-mitogen-activated protein kinase (p38-MAPK) in the reduction of EC-SOD. From these observations, p38-MAPK signaling cascades activated by ROS play a pivotal role in the reduction of EC-SOD, and it is concluded that the reduction of EC-SOD leads to a decrease in the resistance to oxidative stress of Cd-exposed COS7 cells.

Clinical implications of novel activating EGFR mutations in malignant peritoneal mesothelioma

BackgroundThere is a paucity of information about the molecular perturbations involved in MPM tumor formation. We previously reported that EGFR-TK mutations in MPM were predictive of achieving optimal surgical cytoreduction, but the status of EGFR pathway activation potential of these mutations was not known. Here we present the mutant EGFR activating potential and the matured survival data of the EGFR mutant(mut+) relative to wild type EGFR(mut-) mesothelioma.MethodsTwenty-nine patients were evaluated and their tumors were probed for mutations in the catalytic TK-domain. Twenty-five patients were treated with cytoreductive surgery and complete clinical data was available for comparison of the mut+ and mut- groups. A COS-7 cell expression model was used to determine mutation activating profiles and response to erlotinib.ResultsFunctional mutations were found in 31%(9/29) of patients; 7 of these mutations were novel and another was the L858R mutation. All missense mutations were found to be activating mutations and responsive to erlotinib. Of the 25 patients managed surgically, there were 7 mut+ and 18 mut-. Two of 7 (29%) mut+ developed progressive disease and died with a median follow-up time of 22 months; while 13/18 (72%) mut- developed progressive disease and 10/18 (56%) died with median TTP of 12 months and median survival of 14 months.ConclusionsThe novel EGFR mutations identified are activating mutations responsive to erlotinib. The mut+ subset have a 'relative' improved outcome. Erlotinib may have a role in MPM and exploration for mutations in a larger patient cohort is warranted.

Construction and identification of recombinant plasmid pUIS3–BLC +

Objective To construct and identify recombinant plasmid p UIS3-BLC +. Methods The cDNA of B-lymphocyte chemoattractant (BLC) was amplified from the total RNA of spleen tissues by PCR method, and were inserted into plasmid of Plasmodium berghei with UIS3 knockout by digestion of restrictive endonuclease and T7 ligation. The recombinant plasmids were screened, and then underwent restriction enzymatic digestion and DNA sequencing. Then the confirmed plasmid was further transfected into COS-1 cells by lipofectamine and the BLC expression was tested by RT-PCR and Western blotting. Results The cDNA of BLC gene was correctively amplified by RT-PCR and the recombinant plasmid p UIS3-BLC + was constructed successfully, which was confirmed by restriction enzymatic digestion and DNA sequencing. RT-PCR and Western blotting also showed the BLC gene expression in COS-1 cells. Conclusions The recombinant plasmid p UIS3-BLC + has BLC expression in COS-1 cells, and is useful for further study on BLC transgene and UIS3 gene knockout in Plasmodium berghei.

SEDLIN forms homodimers: characterisation of SEDLIN mutations and their interactions with transcription factors MBP1, PITX1 and SF1.

BackgroundSEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT).Methodology/Principal FindingsWe investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions.Conclusions/SignificanceOur studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1.