Tissue Culture Assay Research Articles

Examination of the Neuroprotective effects of Liposome Encapsulated Curcumin Against Methylmercury induced Neurotoxicity in SH‐SY5Y Cells

Curcumin has been reported to produce a spectrum of effects, including antioxidant and anti‐inflammatory activity. However, a major limitation to the use of curcumin as a therapeutic agent is its limited absorption and rapid metabolism resulting in low bioavailability. One possible strategy would be to encapsulate curcumin within a liposome, allowing greater penetrance of the drug into the target tissue. This study was conducted to examine the neuroprotective effects of liposomal curcumin (LC) and unencapsulated curcumin (UC) against methylmercury (MeHg), an established neurotoxin. A cell viability tissue culture assay was utilized to examine the neuroprotective effects of both LC and UC. When neuroblastoma (NB) cells were incubated in culture with MeHg (50 nM), cell viability was found to be 61 ± 1.2 % at 48 hours of incubation. The minimal concentration found to produce complete protection against the neurotoxic effects of MeHg for LC and UC was 625nM and 2.5μM, respectively. Blank liposome control did not significantly alter the neurotoxicity of MeHg. These results indicate that curcumin is able to produce a neuroprotective effect against MeHg in NB cells and in the liposomal form, it is significantly more potent than the unencapsulated form. Such a finding supports the hypothesis that LC may have greater penetrance into the cell and thus may be a useful tool to treat methylmercury induced neurological diseases.

Attenuation of Methylmercury‐induced Neurotoxicity by N‐methyl‐D‐aspartate Antagonist, MK801 in SH‐SY5Y Cells

Methylmercury (MeHg), a well known neurotoxin, has been reported to alter glutamate homeostasis in the neuronal environment resulting in excitotoxicity. The present study was conducted to investigate whether, and if so, under what conditions, MeHg induced excitotoxicity may be attenuated by N‐methyl‐D‐aspartate (NMDA) receptor blockade. A cell viability tissue culture assay was utilized to study the effects of MeHg, glutamate (glu) and a non‐competitive NMDA antagonist, MK‐801 on a human neuroblastoma cell line, SH‐SY5Y. Treatment of cells with MeHg (50nM) and glu (1mM) alone demonstrated a decrease of cell viability from control of 64%±6.5 and 79%±8.5, respectively. When glu and MeHg were added to cells in combination there was a 45% decrease in cell viability (p<0.005 n=3), as compared to treatment of glu alone. MK‐801 (4μM) significantly protected the cells from the effects of glu alone (p<0.005 n=3) as well as from combination treatments with glu and MeHg (p<0.0001 n=3). This study indicates that MeHg may induce an increase in number of NMDA receptors on the cell surface of neuroblastoma cells, and thus enhance the toxicity of glutamate. This apparent blockade of NMDA receptor with MK801 demonstrated a significant attenuation of the toxicity of both MeHg and glutamate. These results support the hypothesis that NMDA receptor antagonists may provide a potential treatment for MeHg‐induced neurological diseases.

Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits

The RNA processing exosome complex was originally defined as an evolutionarily conserved multisubunit complex of ribonucleases responsible for the processing and/or turnover of stable RNAs. The exosome complex is also involved in the surveillance of mRNAs in both the nucleus and the cytoplasm, including nonsense-mediated decay (NMD) targets. The detailed mechanisms for how individual exosome subunits participate in each of these RNA metabolic pathways remains unclear. Here, we use RNAi to deplete exosome subunits, the exonucleases Rrp6 and Dis3, and an exosome cofactor in Drosophila melanogaster S2 tissue culture cells and assay the effects on global mRNA levels using gene expression microarrays. Consistent with the RNA degradative activities ascribed to the exosome, most mRNAs are increased. Notably, these stabilized mRNAs possess 3' untranslated regions that are longer than the representative transcriptomic average. Moreover, our results reveal substantial differences in the pools of affected mRNAs for each depleted subunit. For example, approximately 25% of the affected transcripts in Rrp6 depleted cells represent NMD substrates. While the affected mRNAs were dissimilar, they encode proteins that function in similar cellular pathways. We conclude that individual exosome subunits are largely functionally independent at the transcript level, but are interdependent on a transcriptomic level.

Activation of ExoU Phospholipase Activity Requires Specific C-Terminal Regions

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A(2) domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication.

Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India

Members of the genus Aeromonas (family Aeromonadaceae) are medically important, Gram-negative, rod-shaped micro-organisms and are ubiquitous in aquatic environments. Aeromonas species are increasingly recognized as enteric pathogens; they possess several virulence factors associated with human disease, and represent a serious public health concern. In the present study, putative virulence traits of Aeromonas hydrophila isolates collected from different natural surface waters of Kolkata, India, were compared with a group of clinical isolates from the same geographical area using tissue culture and PCR assays. Enteropathogenic potential was investigated in the mouse model. Of the 21 environmental isolates tested, the majority showed cytotoxicity to HeLa cells (81 %), haemolysin production (71 %) and serum resistance properties (90 %), and they all exhibited multi-drug resistance. Some of the isolates induced fluid accumulation (FA ratio>or=100), damage to the gut and an inflammatory reaction in the mouse intestine; these effects were comparable to those of clinical strains of A. hydrophila and toxigenic Vibrio cholerae. Interestingly, two of the isolates evoked a cell vacuolation effect in HeLa cells, and were also able to induce FA. These findings demonstrate the presence of potentially pathogenic and multi-drug-resistant A. hydrophila in the surface waters, thereby indicating a significant risk to public health. Continuous monitoring of surface waters is important to identify potential water-borne pathogens and to reduce the health risk caused by the genus Aeromonas.

Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells

To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts. A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor. Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction. Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG x CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.

Roles of Putative Type II Secretion and Type IV Pilus Systems in the Virulence of Uropathogenic Escherichia coli

BackgroundType II secretion systems (T2SS) and the evolutionarily related type IV pili (T4P) are important virulence determinants in many Gram-negative bacterial pathogens. However, the roles of T2SS and T4P in the virulence of extraintestinal pathogenic Escherichia coli have not been determined.Methodology/Principal FindingsTo investigate the functions of putative T2SS and T4P gene clusters present in the model uropathogenic E. coli (UPEC) strains UTI89 and CFT073, we deleted the secretin gene present in each cluster. The secretin forms a channel in the outer membrane that is essential for the function of T2S and T4P systems. We compared the secretin deletion mutants with their wild type counterparts using tissue culture assays and the CBA/J mouse model of ascending urinary tract infection. No deficiencies were observed with any of the mutants in adherence, invasion or replication in human bladder or kidney cell lines, but UTI89 ΔhofQ and UTI89 ΔgspD exhibited approximately 2-fold defects in fluxing out of bladder epithelial cells. In the mouse infection model, each of the knockout mutants was able to establish successful infections in the bladder and kidneys by day one post-infection. However, UTI89 ΔhofQ and a CFT073 ΔhofQ ΔyheF double mutant both exhibited defects in colonizing the kidneys by day seven post-infection.Conclusions/SignificanceBased on our results, we propose that the putative T4P and T2S systems are virulence determinants of UPEC important for persistence in the urinary tract, particularly in renal tissues.

Characterization of Virulence Genes of Non-O157 Shiga Toxin-Producing <i>Escherichia coli</i> Isolates from Two Provinces of Iran

Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types among non-O157 isolates. PCR-RFLP results for the fliC gene and classical serology examination with H antisera indicated the presence of nine different H types. In this study, three new serotypes of non-O157:H7 (i.e., O25:H3, O85:H32, and O162:H21) were found to be Shiga toxin producers. These findings reconfirm the results of our previously reported studies showing that non-O157:H7 serotypes are more prevalent under the present conditions. More detailed characterization of these isolates will require additional genetic studies.

A mixture containing galactooligosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-16E cells, the presence of approximately 2 mM Bimuno significantly reduced the invasion of S. Typhimurium SL1344nal(r) (P<0.0001). In the murine ligated ileal gut loops, the presence of Bimuno prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse model, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, <0.0001, respectively). Collectively, the results indicate that Bimuno significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

In vitro assessment of the effectiveness of powder disinfectant (Stalosan ® F) against Lawsonia intracellularis using two different assays

The objective of this study was to determine the in vitro efficacy of Stalosan ® F, a mixed chemical and heavy metal disinfectant, against two strains of Lawsonia intracellularis using both a modified tissue culture and a direct count method. For testing as a powder, 1 g, 0.5 g, or 0.25 g of Stalosan F was applied to bacterial solutions spread into sterile dishes. For use as an aqueous suspension, Stalosan F was prepared to final concentrations of 1%, 4%, 8%, 16%, and 32%. In both applications, L. intracellularis was exposed to Stalosan F for 0.5 h, 1 h, 2 h, and 4 h. The results showed that both strains were similar in their susceptibilities to Stalosan F. The modified tissue culture assay showed no detectable L. intracellularis in cell culture after exposure to all levels of Stalosan F powder for 0.5 h. Furthermore, the number of viable bacteria was markedly reduced in the aqueous concentration of 4% and no L. intracellularis was detected at concentrations of ≥8% for 0.5 h. Using the direct count method, detection of live bacteria was less than 1% after exposure to the powder for 0.5 h. After exposure to the aqueous form, the number of viable bacteria killed was over 99% in concentrations of ≥16% compared to controls. Our results indicate that Stalosan F in both powder and suspension forms is able to inactivate over 99% of L. intracellularis after 30 min of exposure. Furthermore, both laboratory methods can be used to determine the effect of disinfectants on L. intracellularis viability.

Meniere’s Attacks Occur in the Inner Ear with Excessive Vasopressin Type‐2 Receptors

Meniere's disease is peculiar to humans and is characterised by episodic vertigo, fluctuating hearing loss and tinnitus, and attacks of the affliction occurring under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although subsequently proposed as a disorder of water metabolism in the inner ear, its pathogenesis remains unsolved. The present study aimed to assess the link between the inner ear pathology in Meniere's disease and vasopressin, an anti-diuretic stress hormone with a potential role in inner ear fluid homeostasis. Blood samples were obtained from Meniere's disease patients in the morning, before any surgical treatment, to examine plasma vasopressin (pAVP) levels, and then from inner ear tissue during surgical treatment, to examine vasopressin type-2 receptor (V2R) in the endolymphatic sac. pAVP and the relative V2R mRNA expression in the endolymphatic sac were examined using a real-time polymerase chain reaction. Relative cAMP activity in the endolymphatic sac was also examined using tissue culture and cAMP assay. Both pAVP (1.6-fold versus controls; P = 0.048) and inner ear V2R mRNA expression (41.5-fold versus controls; P = 0.022) were significantly higher in Meniere's patients. cAMP activity was basally up-regulated (2.1-fold versus controls) and cAMP sensitivity to vasopressin application was largely elevated (4.9-fold versus controls) in Meniere's patients. We conclude that, in the pathogenesis of inner ear hydrops, resulting in Meniere's attacks, elevation of pAVP levels (probably as a result of stress) may present as a matter of consequence, but susceptibility of the V2R-overexpressed and cAMP-hypersensitized inner ear to pAVP elevation might be essential as the basis of this disease. Further experimental and clinical studies are needed to better clarify the relationship between Meniere's disease and stress.

Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.

Effects, in an in-vivo model system, of 1,2,3,4-tetrahydroisoquinoline on glioma

The effects of 1-(biphenyl-4-ylmethyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (EDL-155) on the growth of glioma was tested in vitro and in vivo. Normal cultured rat astrocytes and C6 rat glioma were used as a differential screen to test the effects of EDL-155. The compound was preferentially cytotoxic for C6 glioma (EC50=1.5 micromol/l) relative to cultured neonatal astrocytes (EC50=27.4 micromol/l). When compared with a standard chemotherapeutic agent, carmustine (1,3-bis[2-chloroethyl]-1-nitrosourea), or temozolomide, EDL-155 was more selective and more potent in our differential tissue culture assay. The effect of EDL-155 was also tested in an animal model in which C6 glioma was transplanted into the brains of Sprague-Dawley rats. EDL-155 was delivered directly onto the tumor by an osmotic minipump directly into the brain or by intraperitoneal injection. Animals treated with EDL-155 had significantly smaller tumors than did control animals treated with carrier solution. We observed anatomical changes in cultured glioma cells treated with EDL-155 that were consistent with selective destruction of mitochondria and the induction of autophagy. These changes were not observed in normal astrocytes cultured from rat pups. The selective killing of glioma in tissue culture and in the rat brain models indicates that EDL-155 has potential therapeutic value in treating this type of brain cancer.

Evaluation of the tolerogenic effects of donor bone marrow cells using a severe combined immunodeficient mouse–human islet transplant model

The immunoregulatory role of human donor bone marrow cells (DBMC) has been studied extensively in our laboratory using in vitro and ex vivo assays. However, new experimental systems that can overcome the limitations of tissue culture assays but with more clinical relevance than purely animal experimentation, needed to be generated. Therefore we have developed a new human peripheral blood lymphocyte (PBL) severe combined immunodeficient (SCID) mouse islet transplantation model without the occurrence of graft-versus-host disease (GvHD) and have used it to evaluate the tolerogenic effects of DBMC. Nonobese diabetogenic (NOD)-SCID mice were transplanted with human deceased donor islets and were reconstituted with human PBL (allogeneic to islets; denoted as recipient) with or without DBMC from the islet donor. It was observed that the most cellularly economical dose was 3000 islets per animal and that injection into the portal vein was better than implantation under the kidney capsule. Even though maximal lymphoid reconstitution was observed with 40-million fresh and anti-CD3 activated recipient PBL (conventional method), the mice developed severe graft GvHD. However, with the new method of reconstitution where animals were injected with 20-million anti-CD3-activated plus 40-million anti-donor-activated recipient PBL, no discernible GvHD was observed. More importantly, this latter method was associated with islet transplant rejection, which in turn could be abrogated by co-injection of the animals with DBMC. These in vivo results confirmed our previous in vitro observations that human DBMC have regulatory activity.

Methoxychlor and Estradiol Induce Oxidative Stress DNA Damage in the Mouse Ovarian Surface Epithelium

Estrogenic compounds such as 17beta-estradiol (E(2)) and methoxychlor (MXC) induce oxidative stress damage in breast cells and mouse ovarian follicles, respectively. However, little is known about whether estrogenic compounds cause oxidative stress in the ovarian surface epithelium (OSE). Thus, this work tested the hypothesis that E(2) and MXC cause oxidative stress in the OSE. To test this hypothesis, we employed an improved mouse tissue culture assay in which OSE cells were treated with hydrogen peroxide (H2O2; positive control), MXC, or E(2) +/- the anti-oxidant vitamin E, or progesterone. The cells then were subjected to a novel direct immunofluorescent assay in which cells in the microtiter plate were reacted with antibodies that detect oxidative damage to DNA (8-hydroxy-2'-deoxyguanosine). The signal was identified with a tyramide Alexa Fluor fluorescent probe and quantified by microfluorimetry. Correction for cellularity was carried out for each well with a fluorescent DNA dye system (CyQuant) at a different wavelength. After 24 h, the mean Alexa Fluor CyQuant ratio was 11.3 +/- 0.9 for controls, 132 +/- 15 for H2O2 treated positive control cells (p < or = 0.01 from control), 105 +/- 6.6 for E(2) treated cells (p < or = 0.01 from control), and 64 +/- 5.1 for MXC-treated cells (p < or = 0.01 from control). After 72 h, the mean ratio was 121 +/- 10.6 for controls, 391 +/- 23 for H2O2 treated cells (p < or = 0.01 from control), 200 +/- 15 for E(2) treated cells (p < or = 0.03), and 228 +/- 21 for MXC-treated cells (p < or = 0.01). Further, vitamin E, but not progesterone, protected OSE cells from E(2)- and MXC-induced oxidative damage. This study demonstrates the feasibility of direct immunofluorescent quantitation of DNA adducts in cell cultures without DNA extraction. Moreover, these data indicate that E(2) and MXC produce oxidative DNA damage in the OSE, and that this damage is prevented by the anti-oxidant vitamin E.

Translation from nonautonomous type IAP retrotransposon is a critical determinant of transposition activity: Implication for retrotransposon-mediated genome evolution

Retrotransposons constitute a major component of the genome and their proliferation significantly impacts genome evolution. Retrotransposons can propagate autonomously or nonautonomously. Nonautonomous type transposition occurs through trans-complementation by autonomous type retrotransposons. While autonomous type retrotransposons have been studied extensively, the translation products from nonautonomous type retrotransposons are not well characterized. In a previous study, we isolated both autonomous and nonautonomous type intracisternal A particle (IAP) elements from the mouse genome and established a tissue culture assay to examine trans-complementation of nonautonomous type IAP element. Using this system in the present study, we determined an active role for the translation product from nonautonomous type IAP element. Point mutations that either eliminated or truncated the IAP protein were introduced and their effects on trans-complementation were examined. Trans-complementation efficiency correlated with the expression of nonautonomous type IAP protein. The effect of nonautonomous type IAP protein was observed only when it was provided in cis, suggesting an interaction of nonautonomous type IAP protein and its transcript immediately after transcription. Interaction of autonomous and nonautonomous type IAP proteins was demonstrated by immunostaining and coimmunoprecipitation assay. Based on these findings, we propose a model in which nonautonomous type IAP protein associates with its transcript, recruits autonomous type IAP protein, and promotes the assembly of transposition competent IAP particle. The active role of the nonautonomous type IAP protein revealed in this study may provide a new insight into retrotransposon proliferation within the genome.

LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1 RP and LRE3) and rodent (L1 A102 and L1 spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

Erythropoietin: A Potent Inducer of Peripheral Immuno/Inflammatory Modulation in Autoimmune EAE

BackgroundBeneficial effects of short-term erythropoietin (EPO) therapy have been demonstrated in several animal models of acute neurologic injury, including experimental autoimmune encephalomyelitis (EAE)-the animal model of multiple sclerosis. We have found that EPO treatment substantially reduces the acute clinical paralysis seen in EAE mice and this improvement is accompanied by a large reduction in the mononuclear cell infiltration and downregulation of glial MHC class II expression within the inflamed CNS. Other reports have recently indicated that peripherally generated anti-inflammatory CD4+Foxp3+ regulatory T cells (Tregs) and the IL17-producing CD4+ T helper cell (Th17) subpopulations play key antagonistic roles in EAE pathogenesis. However, no information regarding the effects of EPO therapy on the behavior of the general mononuclear-lymphocyte population, Tregs or Th17 cells in EAE has emerged.Methods and FindingsWe first determined in vivo that EPO therapy markedly suppressed MOG specific T cell proliferation and sharply reduced the number of reactive dendritic cells (CD11c positive) in EAE lymph nodes during both inductive and later symptomatic phases of MOG35–55 induced EAE. We then determined the effect in vivo of EPO on numbers of peripheral Treg cells and Th17 cells. We found that EPO treatment modulated immune balance in both the periphery and the inflamed spinal cord by promoting a large expansion in Treg cells, inhibiting Th17 polarization and abrogating proliferation of the antigen presenting dendritic cell population. Finally we utilized tissue culture assays to show that exposure to EPO in vitro similarly downregulated MOG-specific T cell proliferation and also greatly suppressed T cell production of pro-inflammatory cytokines.ConclusionsTaken together, our findings reveal an important new locus whereby EPO induces substantial long-term tissue protection in the host through signaling to several critical subsets of immune cells that reside in the peripheral lymphatic system.

Serum or Plasma Samples?

Dysregulations of the synthesis and breakdown of the extracellular matrix (ECM) is a key processes in the physiopathology of vascular remodeling and the development of atherothrombotic syndromes.1–4 Matrix metalloproteinases (MMPs) are endopeptidases, belonging to the matrixin family that can degrade almost all the ECM components.5 Although there is little doubt that MMPs are an important components in cancer invasion and metastasis,6 they are also regarded as key regulatory molecules in degenerative processes and inflammation,5,7 as playing roles also in physiopathological remodeling of the vascular wall2,8 and as the pathogenesis of cardio- and cerebrovascular diseases.9,10 Thus, MMPs have emerged as potential biomarkers of atherothrombotic risk and predictors of coronary and cerebrovascular disease recurrence.8 ATVB 2008;28:e15–e16. Normal and diseased blood vessel wall cells may upregulate and activate MMPs in a multistep fashion, driven in part by soluble cytokines and cell–cell interactions with platelets and white blood cells. Enhanced MMP activation (alone or in concert with the fibrinolytic system) contributes to increased matrix turnover, which may have beneficial effects (eg, in vascular repair and atherosclerotic plaque stabilization)11 but also detrimental effects (eg, in vascular intimal thickening, atherosclerosis, and in thrombotic syndromes).12 Because the changes on the cellular level are reflected in body fluids, determination of MMPs in blood have been recommended as noninvasive tools in the diagnosis and monitoring of several diseases.13 It has been demonstrated repeatedly that both the MMP/tissue inhibitor of matrix metalloproteinase (TIMP) assay and zymography essentially depend on the blood sampling procedures, with some MMPs and TIMPs having higher concentrations in serum than in plasma.14–41 Although assays for an ever-increasing number of MMPs are now commercially available, the …