Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos.

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

Production of IVF transgene-expressing bovine embryos using a novel strategy based on cell cycle inhibitors.

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.

Incidence of intracranial hypertension related to jugular bulb oxygen saturation disturbances in severe traumatic brain injury patients.

OBJECTIVE: To analyze the Intracranial Hypertension (IH) development related to jugular bulb oxygen saturation (SjO2) disturbances in severe traumatic brain injury patients (sTBI). MATERIALS AND METHODS: One hundred and thirty-five sTBI patients were reviewed. Those without IH at admission (n = 116) were included. All patients underwent ICP and SjO2 continuous monitoring. Two groups were distinguished according to the SjO2 values during the first 24 hours. Group A: those with abnormal SjO2 (SjO2 more than 75% or less than 55%) and Group B: those with normal SjO2 (55-75%). Differences in IH development and outcome between groups were analyzed. Causes of abnormally low SjO2 were identified. RESULTS: IH developed in 56.9% of patients, between 12 and 48 hours from admission. Group A had a significantly higher incidence of IH than Group B (p < 0.001) and it also had a worse outcome than Group B (GOS 1-2) (p < 0.005). Patients from Group A had a risk of IH 4.5 fold higher than Group B. Considering only patients who developed IH, an abnormal SjO2 value increased 2.3 fold the risk of death compared to those without SjO2 disturbances. Main causes of SjO2 desaturation were hyperventilation (40.7%), hypovolemia (28.4%) and anemia (21%). CONCLUSIONS: Early detection of disturbances in oxygen supply-demand relationship and prevention or resolution of the secondary insults which produce these disturbances, might lead to a reduction in the incidence of intracranial hypertension.

Early effects of insulin-like growth factor-1 in activated human T lymphocytes.

This study evaluates the effects of insulin-like growth factor (IGF)-1 receptor (IGF-1R) down-regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)-2. We found that IGF-1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL-2. The lowest level of IGF-1R detected after 15 min of activation suggested that the effects of IGF-1 occur at the initiation of cell activation. The activation of IGF-1R was confirmed by IGF-1R phosphorylation and increased phosphorylation of microtubule-associated protein kinase. We also detected the alternative IGF-1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF-1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF-1 on T lymphocytes include both autocrine/paracrine and endocrine processes.

APC senses cell-cell contacts and moves to the nucleus upon their disruption.

The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.

Differential expression of CPD1 during postnatal development in the mouse cerebellum.

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

Specific oligobodies against ERK-2 that recognize both the native and the denatured state of the protein.

Oligonucleotide aptamer(s), obtained by using the SELEX procedure, has been used as reagents to recognize different molecules with high affinity and specificity. However, until recently, it was not possible to obtain oligonucleotide-based reagents able to recognize proteins with high specificity in assays typical of antibodies, such as immunohistochemistry, Western blotting and immunoprecipitations. Here, we show the results obtained by applying the strategy of "target switching" to obtain specific polyclonal and monoclonal oligobodies against the protein ERK2. We were able to develop highly specific polyclonal oligobodies by using only one selection step with a temporary target and one selection step with the final target (ERK2). Since only two selection steps were required, these results demonstrate that it is possible to obtain specific reagents against a protein without a need for an "in vitro evolution" using many selection steps, or error-prone polymerases. After one additional selection step, the polyclonal oligobodies were cloned to obtain a highly specific monoclonal oligobody.

The rate of Tau synthesis is differentially regulated during postnatal development in mouse cerebellum.

1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced afte the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum. 2. We found four [35S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9-P12), and decreased by P20. 3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12. 4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.

Development of monoclonal oligobodies and chemically synthesized oligobodies.

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.

Development of monoclonal oligobodies and chemically synthesized oligobodies

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.

Development of monoclonal oligobodies and chemically synthesized oligobodies.

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.

Oligobodies: bench made synthetic antibodies.

Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

Oligobodies: bench made synthetic antibodies.

Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as [quot ]synthetic antibodies[quot ]. The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named [quot ]oligobodies[quot ], has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

HIV/gp120 and PMA/ionomycin induced apoptosis but not activation induced cell death require PKC for Fas-L upregulation.

HIV protein gp120 in combination with T cell antigen receptor (TCR) triggering induces apoptosis (gp120-apoptosis) in Th1 cells. Gp120-apoptosis occurs by induction of Fas-L and subsequent triggering of the Fas apoptotic pathway. Here, through the use of several compounds inhibiting induction of Fas-L, we show that, in a Th1 clone, a protein kinase C (PKC) independent pathway activated by TCR stimulation is distinguishible from a PKC dependent pathway activated by either phorbol 12-myristate 13-acetate (PMA)/ionomycin or asynchronous stimulation of TCR and CD4 as occurs in gp120-apoptosis.

Human melanoma-reactive CD4+ and CD8+ CTL clones resist Fas ligand-induced apoptosis and use Fas/Fas ligand-independent mechanisms for tumor killing.

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Thl clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Thl lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.

Apoptosis induced by HIV-gp120 in a Th1 clone involves the generation of reactive oxygen intermediates downstream CD95 triggering.

HIV-gp120 sensitizes Th1 clones from seronegative donors to apoptosis, which occurs through two distinct events: expression of CD95L followed by its interaction with CD95 to trigger cell death. gp120-apoptosis of the Th1 clone 103 was inhibited by Cyclosporin A, the PTK inhibitors Genistein and PNU152518, as well as the anti-oxidants Ascorbic Acid and Glutathione. Cyclosporin A interfered with CD95L expression, Ascorbic Acid and Glutathione inhibited cell death triggered by CD95/CD95L interaction; Genistein and PNU152518 acted on both steps. The occurrence of oxidative stress during CD95-dependent apoptosis was supported by the direct evidence of ROI production.