Soft Electroporation Through 3D Hollow Nanoelectrodes.

Generally, electroporation of in vitro cells is performed under very high electric fields to overcome the physical barrier of plasma membrane. Since traditional electroporation techniques make use of very high voltages, which is critical to cell viability, this study presents a microfluidic platform able to perform cell membrane electroporation with the application of low voltages (1.5-2 V). The platform is manufactured based on the milling by mean of focused ionic beam, which offers an established approach to fabricate ordered arrays of 3D gold hollow nanoelectrodes protruding from an insulating substrate. The novelty of this fabrication relies on the fact that the nanoelectrodes used for electroporation are simultaneously metallic, hollow and communicate through its nanochannels with an isolated microfluidic chamber beneath the device. Adherent cultured cells on the nanoelectrodes can be electroporated in this platform, and molecules can be selectively delivered only inside the porated cells.

Chemical imaging of live fibroblasts by SERS effective nanofilm.

Reliable and strong surface enhanced Raman scattering (SERS) signatures of intracellular compartments in live NIH3T3 fibroblasts are collected in real time by means of SERS active thin nanofilm (30 nm) on colloidal silica (1.5 µm). Nanofilm is composed of preformed silver nanoparticles in the matrix of polyacrylic acid, protecting against heating (37 °C) in water, or culture medium or phosphate buffered saline aqueous solution. The SERS enhancement factors (EFs) of the order 10(8) allow single biomolecule detection in the native environment of a single live cell. Primary and secondary SERS hot spots of nanofilm are responsible for such high EFs. A slow SERS EF intensity decay occurs over a broader distance of micron silica with nanofilm, not achievable in a common core-shell model (silver nanoparticle coated with a thin silica layer). Extensive local field EFs and SERS EFs are mainly delivered by prolate silver nanoparticles ("rugby-like" shape). This is achieved if an incident field is polarized along the z-axis and the direction of incident polarization and main axis (z) are perpendicular to each other, not observable in water or on gold.

Vascular injury induces expression of periostin: implications for vascular cell differentiation and migration.

OBJECTIVE: Periostin mRNA is among the most strongly upregulated transcripts in rat carotid arteries after balloon injury. The goal of the present study was to gain insight into the significance of periostin in the vasculature. METHODS AND RESULTS: Periostin expression after injury was localized to smooth muscle cells of the neointima and the adventitia. The expression of periostin in smooth muscle cells in vitro was not regulated by cytokines such as fibroblast growth factor-2 (FGF-2). In contrast, stimulation of MC3T3-E1 osteoblastic cells, NIH3T3 fibroblasts, or mesenchymal C3H10T1/2 cells with FGF-2 reduced periostin mRNA levels to <5% of controls, whereas conversely bone morphogenetic protein-2 (BMP-2) increased periostin mRNA levels. Periostin expression was induced and maintained during retinoic acid-induced smooth muscle cell differentiation in A404 cells. In addition, overexpression of periostin in C3H10T1/2 cells caused an increase in cell migration that could be blocked with an anti-periostin antibody. CONCLUSIONS: Periostin expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Unlike other mesenchymally derived cell lines, periostin expression is not regulated by FGF-2 in smooth muscle cells. This distinction may be useful in discriminating smooth muscle and fibroblast lineages.

Over expression of the selectable marker blasticidin S deaminase gene is toxic to human keratinocytes and murine BALB/MK cells.

BACKGROUND: The blasticidin S resistance gene (bsr) is a selectable marker used for gene transfer experiments. The bsr gene encodes for blasticidin S (BS) deaminase, which has a specific activity upon BS. Therefore, its expression is supposed to be harmless in cells. The work reported on herein consisted of experiments to verify a possible toxicity of bsr on mammalian cells, which include several cell lines and primary cultures. RESULTS: Murine keratinocyte BALB/MK and human primary keratinocyte cells transduced with the retroviral vector LBmSN, which has an improved expression system of bsr, namely bsrm, died in five days after the transduction. Meanwhile the control vector LBSN, which expresses bsr, did not provoke cell death. The lethal activity of bsrm was observed only in human keratinocytes and BALB/MK cells among the cell types tested here. Death appears to be mediated by a factor, which is secreted by the BALB/MK transduced cells. CONCLUSION: By our study we demonstrated that the expression of bsrm gene is toxic to human keratinocytes and BALB/MK cells. It is likely over expression of BS deaminase gene is responsible for the death.

Transcriptional oscillation of canonical clock genes in mouse peripheral tissues.

BACKGROUND: The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes. RESULTS: We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (mBmal1, mNpas2, mRev-erbalpha, mDbp, mRev-erbbeta, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements (RORE, DBPE, and E-box), which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes. CONCLUSIONS: The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE.

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis.

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death - Fas (CD95)/FasL - in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor alpha (TNF-alpha) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-alpha and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.

BACKGROUND: Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity. RESULTS: We have constructed and compared different retroviral vectors containing two co-stimulatory molecules (CD70, CD80) and selectable marker genes linked to different IRES sequences (IRES from EMCV, c-myc, FGF-2 and HTLV-1). The tricistronic recombinant amphotropic viruses containing the IRES from EMCV, FGF-2 or HTLV-1 were equally efficient in inducing the expression of an exogenous gene in the transduced murine or human cells, without displaying any cell type specificity. The simultaneous presence of several IRESes on the same mRNA, however, can induce the differential expression of the various cistrons. Here we show that the IRESes of HTLV-1 and EMCV interfere with the translation induced by other IRESes in mouse melanoma cells. The IRES from FGF-2 did however induce the expression of exogenous cDNA in human melanoma cells without any positive or negative regulation from the other IRESs present within the vectors. Tumor cells that were genetically modified with the tricistronic retroviral vectors, were able to induce an in vivo anti-tumor immune response in murine models. CONCLUSION: Translation of the exogenous gene is directed by the IRES and its high level of expression not only depends on the type of cell that is transduced but also on the presence of other genetic elements within the vector.

Spermidine/spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine and is not involved in polyamine metabolism.

Spermidine/spermine-N1-acetyltransferase (SSAT1) is a short-lived polyamine catabolic enzyme inducible by polyamines and polyamine analogues. Induction of SSAT1 plays an important role in polyamine homoeostasis, since the N1-acetylated polyamines can be excreted or oxidized by acetylpolyamine oxidase. We have purified a recombinant human acetyltransferase (SSAT2) that shares 45% identity and 61% homology with human SSAT1, but is only distally related to other known members of the GNAT (GCN5-related N-acetyltransferase) family. Like SSAT1, SSAT2 is widely expressed, but did not turn over rapidly, and levels were unaffected by treatments with polyamine analogues. Despite similarity in sequence to SSAT1, polyamines were found to be poor substrates of purified SSAT2, having K(m) values in the low millimolar range and kcat values of <0.01 s(-1). The kcat/K(m) values for spermine and spermidine for SSAT2 were <0.0003% those of SSAT1. Expression of SSAT2 in NIH-3T3 cells was not detrimental to growth, and did not reduce polyamine content or increase acetylpolyamines. These results indicate that SSAT2 is not a polyamine catabolic enzyme, and that polyamines are unlikely to be its natural intracellular substrates. A promising candidate for the physiological substrate of SSAT2 is thialysine [S-(2-aminoethyl)-L-cysteine], which is acetylated predominantly at the epsilon-amino group with K(m) and kcat values of 290 muM and 5.2 s(-1). Thialysine is a naturally occurring modified amino acid that can undergo metabolism to form cyclic ketimine derivatives found in the brain and as urinary metabolites, which can undergo further reaction to form antioxidants. SSAT2 should be renamed 'thialysine N(epsilon)-acetyltransferase', and may regulate this pathway.

Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system.

Dec2, a member of the basic helix-loop-helix superfamily, is a recently confirmed regulatory protein for the clockwork system. Transcripts of Dec2, as well as those of its related gene Dec1, exhibit a striking circadian oscillation in the suprachiasmatic nucleus, and Dec2 inhibits transcription from the Per1 promoter induced by Clock/Bmal1 [Honma, Kawamoto, Takagi, Fujimoto, Sato, Noshiro, Kato and Honma (2002) Nature (London) 419, 841-844]. It is known that mammalian circadian rhythms are controlled by molecular clockwork systems based on negative-feedback loop(s), but the molecular mechanisms for the circadian regulation of Dec2 gene expression have not been clarified. We show here that transcription of the Dec2 gene is regulated by several clock molecules and a negative-feedback loop. Luciferase and gel retardation assays showed that expression of Dec2 was negatively regulated by binding of Dec2 or Dec1 to two CACGTG E-boxes in the Dec2 promoter. Forced expression of Clock/Bmal1 and Clock/Bmal2 markedly increased Dec2 mRNA levels, and up-regulated the transcription of the Dec2 gene through the CACGTG E-boxes. Like Dec, Cry and Per also suppressed Clock/Bmal-induced transcription from the Dec2 promoter. Moreover, the circadian expression of Dec2 transcripts was abolished in the kidney of Clock/Clock mutant mice. These findings suggest that the Clock/Bmal heterodimer enhances Dec2 transcription via the CACGTG E-boxes, whereas the induced transcription is suppressed by Dec2, which therefore must contribute to its own rhythmic expression. In addition, Cry and Per may also modulate Dec2 transcription.

Alternative reading frame protein (ARF)-independent function of CARF (collaborator of ARF) involves its interactions with p53: evidence for a novel p53-activation pathway and its negative feedback control.

CARF, a collaborator of ARF (alternative reading frame protein), was cloned as a novel ARF-binding protein from a yeast-interaction screen. It potentiated ARF-mediated p53 function, and also caused a moderate increase in p53 activity in the absence of ARF. We herein report the molecular mechanism of ARF-independent function of CARF. By employing a variety of approaches, including overexpression of CARF, its suppression by small interfering RNA and use of protease inhibitors, we demonstrate that: (i) CARF directly interacts with wild-type p53, causing its stabilization and functional activation; and (ii) CARF and p53 levels show an inverse relationship that is instigated by a negative-feedback control via a proteasome-mediated degradation pathway.

Epigenetic modifications affect Dnmt3L expression.

Imprinted genes are expressed from a single allele due to differential methylation of maternal or paternal alleles during gametogenesis. Dnmt3L (DNA cytosine-5-methyltransferase 3 like), a member of de novo methyltransferase Dnmt3 protein family, is a regulator of maternal imprinting. In the present study, we have characterized the promoter region of the mouse Dnmt3L gene. Transient transfection assays performed with 5'-deletion promoter constructs indicated a minimal promoter area within 440 bp upstream from the translational start site. Longer promoter constructs showed decreased activity, suggesting the presence of repressor elements within the upstream sequences. According to electrophoretic mobility-shift assays and mutation analysis, the minimal promoter region contained four functional binding sites for the Sp1 (specificity protein 1) family of transcription factors, Sp1 and Sp3. In vitro methylation of Dnmt3L promoter constructs decreased the transcriptional activity significantly, demonstrating down-regulation by cytosine methylation. This was supported by the results from bisulphite sequencing and real-time quantitative reverse transcriptase-PCR analysis of different mouse cell lines and tissues. In testis and embryonic stem cells showing strong Dnmt3L expression, all CpG sites studied were fully unmethylated, whereas non-expressive cell lines and tissues with lesser Dnmt3L expression showed complete or diverse CpG methylation levels. Treatment of Dnmt3L non-expressive cell lines with deacetylase inhibitor trichostatin A and methyltransferase inhibitor 5-aza-2'-deoxycytidine induced the expression of Dnmt3L mRNA. Furthermore, we show that the repressional effect of longer promoter fragments was also relieved by these inhibitors, altogether indicating an epigenetic control for Dnmt3L gene regulation.