Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes.

In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.

Disassembly of shank and homer synaptic clusters is driven by soluble beta-amyloid(1-40) through divergent NMDAR-dependent signalling pathways.

Disruption of the postsynaptic density (PSD), a network of scaffold proteins located in dendritic spines, is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer's disease (AD). Extending our previous demonstration that derangement of the PSD by soluble amyloid-beta (Abeta) involves proteasomal degradation of PSD-95, a protein important for ionotropic glutamate receptor trafficking, we now show that Abeta also disrupts two other scaffold proteins, Homer1b and Shank1, that couple PSD-95 with ionotropic and metabotropic glutamate receptors. Treatment of fronto-cortical neurons with soluble Abeta results in rapid (within 1 h) and significant thinning of the PSD, decreased synaptic levels of Homer1b and Shank1, and reduced synaptic mGluR1 levels. We show that de novo protein synthesis is required for the declustering effects of Abeta on Homer1b (but not Shank1) and that, in contrast to PSD-95, Abeta-induced Homer1b and Shank1 cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Abeta diverges in two other respects: i) whereas the activity of both NMDAR and VDCC is required for Abeta-induced declustering of Homer1b, Abeta-induced declustering of Shank1 only requires NMDAR activity; and ii) whereas the effects of Abeta on Homer1b involve engagement of the PI-3K pathway and calcineurin phosphatase (PP2B) activity, those on Shank1 involve activation of the ERK pathway. In summary, soluble Abeta recruits discrete signalling pathways to rapidly reduce the synaptic localization of major components of the PSD and to regulate the availability of mGluR1 in the synapse.

Tumor suppressor KAI1 affects integrin alphavbeta3-mediated ovarian cancer cell adhesion, motility, and proliferation.

The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin alphavbeta3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin alphavbeta3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with beta1-integrins, also colocalizes with integrin alphavbeta3. Functionally, elevated KAI1 levels drastically increased integrin alphavbeta3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin alphavbeta3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin alphavbeta3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

Imaging of placental transport mechanisms: a review.

Functional analysis of material transfers requires precise statement of residence times in each tissue compartment. For the placenta, neither extractive biochemistry, isotope partitioning, nor mass-based quantitative assays provide adequate spatial resolution to allow the necessary precision. Dual-perfusion assays of material transfer in isolated placental cotyledons provide time-series data for two compartments, the maternal and fetal blood, but fail to distinguish the two cellular compartments (syncytiotrophoblast, fetal endothelium) which actively regulate rates of transfer in each direction for essentially every important molecule type. At present, no definitive technology exists for functional analysis of placental transfer functions. The challenge in developing such a technology lies in the exquisitely small and delicate structures involved, which are scaled at cellular and subcellular sizes (between 50 nm and 50 microm). The only available technologies attaining this high spatial resolution are imaging technologies, primarily light and electron microscopy. To achieve the high-quality images necessary, confocal laser scanning microscopy (CLSM) is required, to provide a uniform optical sectioning plane. In turn, this requires relatively high fluorescence intensities. Design of an adequate technology therefore bases on CLSM imaging fluorochrome-tagged tracers. The temporal resolution necessary to analyse placental material transfers is expected to be of the order of a few seconds, so that conventional wet-fixation protocols are too slow. For adequately rapid fixation, snap-freezing is required. As part of this review we report results obtained from an appropriately designed experimental protocol, analysed by CLSM and transmission electron microscopy (TEM). The images acquired were tested for uniformity of illumination and fluorescence emission strength. Relevant data was encoded in the green channel of the trichrome images obtained, and this was thresholded by application of strict quantitative criteria. The thresholding procedure is suitable for automation and produces reproducible, objectifiable results. Thresholded images were subjected to image calculation procedures designed to highlight image elements (pixels) containing (green) fluorescence associated with the tracer protein; all other sources of fluorescence were visualised in the final images only if no green fluorescence was detectable in that pixel. The resulting images were maps, showing the distribution of tracer molecules at a predefined time interval after perfusion of the tracer into the vital (term) cotyledon. Spatial resolution was routinely better than 1 microm and temporal resolution was approximately 5s. At timepoints up to 10 min after intravital application into the fetal vascular circulation, tracer was associated with capillaries in the villous structures, and no tracer was observed in the syncytiotrophoblast. Clear distinction was achieved between the four tissue compartments relevant to placental transfers, thus providing a novel technology capable of generating high-quality data concerning the regulation of transfers of any molecule that can bear a fluorescent tag. The potential applications of this methodology lie in analyses of factors influencing the rates of fetomaternal and maternofetal exchanges (for example, drugs), and of functional responses of the placental regulation to pathophysiological conditions such as hypoxia.

Cerebral ischemia and neurogenesis: a two-time comparison.

BACKGROUND: This study compares the effect of mild and severe cerebral ischemia on neuronal damage and neurogenesis. METHODS: Sixteen Sprague-Dawley rats, anesthetized with 0.8 vol% halothane in O(2)/air, were subjected to forebrain ischemia by bilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure = 40 mmHg) for 8 (mild) or 13 (severe) min. Four non-ischemic animals were investigated as naïve controls. Bromodeoxyuridine (50 mg/kg), a marker of new cells, was administrated for seven consecutive postischemic days. After 28 days, animals were perfused with 4% paraformaldehyde and the brains were sliced. Histopathological damage of the hippocampus and the volume of the dentate gyrus were assessed by HE-staining. With immunohistochemistry BrdU-positve cells were detected in the dentate gyrus. The amount of new generated neurons was identified by double-immunofluorescence-staining of BrdU and neuronal marker (NeuN). RESULTS: In the CA-1 region of the hippocampus, mild ischemia induced damage up to 10% (HE-index 0.8 +/- 1.2) and severe ischemia up to 50% (HE-index 2.1 +/- 1.4). There was no histopathological damage in naïve control animals. The amount of new neurons was increased by 250% after mild insult and by 160% after severe insult compared to the naïve control animals. CONCLUSIONS: These data indicate that histopathological damage depends on the severity of the ischemic insult and that forebrain ischemia activates generation of new neurons. A mild ischemic challenge appears to be a more potent neurogenic stimulus than severe ischemia. The new neurons survive at least 28 days. This may relate to delayed histopathological and functional recovery after cerebral ischemia.

Primary extramedullary plasmacytoma: similarities with and differences from multiple myeloma revealed by interphase cytogenetics.

Primary extramedullary plasmacytoma is an indolent neoplasm that infrequently converts to multiple myeloma. Since cytogenetic data on extramedullary plasmacytoma are lacking, we studied 38 cases of this type of neoplasm by fluorescence in situ hybridization. Fourteen cases (37%) contained IGH breaks, including six with a t(4;14) translocation. No translocations t(11;14), t(14;16), t(8;14), nor breaks involving MALT1, BCL6 or FOXP1 were found. Loss of 13q (40%), as well as chromosomal gains (82%) were common. There was no correlation between chromosomal alterations and clinical features or local relapse. Cytogenetically, extramedullary plasmacytoma and multiple myeloma are closely related. However, the distribution of IGH translocation partners, with the notable absence of t(11;14), is different. Key words: extramedullary plasmacytoma, multiple myeloma, cytogenetics, IGH translocation, fluorescence in situ hybridization.

Deletion of exon 8 increases cisplatin-induced E-cadherin cleavage.

E-Cadherin-mediated cell-cell adhesion plays a key role in epithelial cell survival and loss of E-cadherin or beta-catenin expression is associated with invasive tumor growth. Somatic E-cadherin mutations have been identified in sporadic diffuse-type gastric carcinoma. Here, we analysed the fate of E-cadherin with an in frame deletion of exon 8 compared to wild-type E-cadherin and the involved signalling events during cisplatin-induced apoptosis. We report that mutant E-cadherin was more readily cleaved during apoptosis than the wild-type form. Also beta-catenin, an important binding partner of E-cadherin, was processed. E-cadherin cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin and beta-catenin in the cytoplasm. Inhibitor studies demonstrated that E-cadherin cleavage was caused by a caspase-3-mediated mechanism. We identified the Akt/PKB and the ERK1/2 signalling pathways as important regulators since inhibition resulted in increased E-cadherin cleavage and apoptosis. In summary, we clearly demonstrate that somatic E-cadherin mutations affect apoptosis regulation in that way that they can facilitate the disruption of adherens junctions thereby possibly influencing the response to cisplatin-based chemotherapy. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design.

Significance of HER2 low-level copy gain in Barrett's cancer: implications for fluorescence in situ hybridization testing in tissues.

PURPOSE: HER2 may be a relevant biomarker in Barrett's cancer. We compared three HER2 laboratory methods, standard fluorescence in situ hybridization (FISH), image-based three-dimensional FISH in thick (16 microm) sections, and immunohistochemistry, to predict patient outcome. EXPERIMENTAL DESIGN: Tissue microarray sections from 124 Barrett's cancer patients were analyzed by standard FISH on thin (4 microm) sections and by image-based three-dimensional FISH on thick (16 microm) sections for HER2 and chromosome-17, as well for p185(HER2) by immunohistochemistry. Correlations with clinical and follow-up data were examined. RESULTS: Only three-dimensional FISH on thick (16 microm) sections revealed HER2 gene copy gain to be associated with increased disease-specific mortality (relative risk, 2.1; 95% confidence interval, 1.06-4.26; P = 0.033). In contrast, standard FISH on thin (4 microm) sections and immunohistochemistry failed to predict clinical outcome. Low-level gain of HER2 occurred frequently in Barrett's cancer (>or=2.5-4.0 HER2 copies, 59.7%; HER2-to-chromosome-17 ratio, >or=1.1-2.0; 61.2%) and defined a subpopulation for patient outcome as unfavorable as HER2 gene amplification [disease-free survival, P = 0.017 (HER2 copies)]. This low-level group was neither definable by standard FISH nor immunohistochemistry. No prognostic significance was found for chromosome-17 aneusomy. CONCLUSIONS: Low-level copy gains of HER2 define a biologically distinct subpopulation of Barrett's cancer patients. Importantly, these subtle copy number changes are not reliably detected by standard FISH in thin (4 microm) tissue sections, highlighting a thus far unrecognized weakness in HER2 FISH testing. These results should be taken into account for accurate evaluation of biomarkers by FISH and for HER2 FISH testing in tissue sections.

Sevoflurane affects neurogenesis after forebrain ischemia in rats.

BACKGROUND: The effect of sevoflurane on the neuroregenerative potential after neuronal injury is unclear. We investigated the effect of low and high concentrations of sevoflurane on endogenous neurogenesis after cerebral ischemia. METHODS: Anesthetized and ventilated rats were randomized to four different treatment groups. Groups 1 and 2: 1.4% sevoflurane; Groups 3 and 4: 2.8% sevoflurane. In Groups 1 and 3, no cerebral ischemia was induced (sham-operated). In Groups 2 and 4, 10 min of forebrain ischemia was induced by bilateral carotid artery occlusion plus hemorrhagic hypotension. Physiological variables were maintained constant. Bromodeoxyuridine was given as a marker of neurogenesis. After 28 days brains were perfused. Histopathological damage of the hippocampus was evaluated in hematoxylin and eosin (HE) stained sections using the HE-index (0 = no damage; 1 = 1%-10% damage; 2 = 11%-50% damage; 3 = 51%-100% damage). Immunohistochemistry was used to detect bromodeoxyuridine-positive neurons. Eight untreated rats were investigated as naive controls (Group 5). RESULTS: In neither sham-operated group was histopathological damage or change in neurogenesis observed compared to naive controls. In rats anesthetized with 1.4% sevoflurane, cerebral ischemia caused mild neuronal damage (HE-index of 0.64 +/- 0.84) and increased neurogenesis by 60% when compared with respective sham-operated animals; with 2.8% sevoflurane, the HE-index was 1.22 +/- 1.14, and the number of newly generated neurons increased by 230% when compared with respective sham-operated animals. CONCLUSION: The present data suggest that high concentrations of sevoflurane stimulate neurogenesis in the dentate gyrus after cerebral ischemia.

Induction of systemic resistance in tomato by N-acyl-L-homoserine lactone-producing rhizosphere bacteria.

N-acyl-L-homoserine lactone (AHL) signal molecules are utilized by Gram-negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density-dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL-negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL-negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA- and ethylene-dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.

The long-term effect of sevoflurane on neuronal cell damage and expression of apoptotic factors after cerebral ischemia and reperfusion in rats.

We investigated the long-term effects of sevoflurane on histopathologic injury and key proteins of apoptosis in a rat hemispheric ischemia/reperfusion model. Sixty-four male Sprague-Dawley rats were randomly assigned to Group 1 (fentanyl and N2O/O2; control) and Group 2 (2.0 vol% sevoflurane and O2/air). Ischemia (45 min) was produced by unilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure 40 mm Hg). Animals were killed after 1, 3, 7, and 28 days. In hematoxylin and eosin-stained brain sections eosinophilic hippocampal neurons were counted. Activated caspase-3 and the apoptosis-regulating proteins Bax, Bcl-2, Mdm-2, and p53 were analyzed by immunostaining. No eosinophilic neurons were detected in sevoflurane-anesthetized rats over time, whereas 9%-38% of the hippocampal neurons were eosinophilic (days 1-28) in control animals. On days 1 and 3, the concentration of Bax was 140%-200% larger in fentanyl/N2O-anesthetized animals compared with sevoflurane. Bcl-2 was 100% less in control animals during the first 3 days. Activated caspase-3 was detected in neurons of both groups (0.75%-2.2%). These data support a sustained neuroprotective potency of sevoflurane related to reduced eosinophilic injury after cerebral ischemia/reperfusion.

In situ quantitation of the spatial scale of calling distances and population density-independent N-acylhomoserine lactone-mediated communication by rhizobacteria colonized on plant roots.

We used computer-assisted microscopy at single cell resolution to quantify the in situ spatial scale of N-acylhomoserine lactone (AHL)-mediated cell-to-cell communication of Pseudomonas putida colonized on tomato and wheat root surfaces. The results of this in situ quantification study on close-to-natural surfaces challenge the conventional view of a quorum group requirement of high cell densities for this type of bacterial communication. In situ image analysis indicated that the effective 'calling distance' on root surfaces was most frequent at 4-5 microm, extended to 37 microm in the root tip/elongation zone and further out to 78 microm in the root hair zone. The spatial scale of these calling distances is very long-range in proportion to the size of individual bacteria. Geostatistical modeling analysis implicated the importance of AHL-gradients mediating effective communication between remote cells. We conclude that AHL-mediated cell-to-cell communication occurs not only within dense populations, but also in very small groups and over long ranges between individual bacteria, and therefore this cellular activity is more commonplace and effective than hitherto predicted. We propose that this cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL-gradients than the requirement for a quorum group of high population density.

The gene ENHANCER OF PINOID controls cotyledon development in the Arabidopsis embryo.

During Arabidopsis embryo development, cotyledon primordia are generated at transition stage from precursor cells that are not derived from the embryonic shoot apical meristem (SAM). To date, it is not known which genes specifically instruct these precursor cells to elaborate cotyledons, nor is the role of auxin in cotyledon development clear. In laterne mutants, the cotyledons are precisely deleted, yet the hypocotyl and root are unaffected. The laterne phenotype is caused by a combination of two mutations: one in the PINOID (PID) gene and another mutation in a novel locus designated ENHANCER OF PINOID (ENP). The expression domains of shoot apex organising genes such as SHOOT MERISTEMLESS (STM) extend along the entire apical region of laterne embryos. However, analysis of pid enp stm triple mutants shows that ectopic activity of STM does not appear to cause cotyledon obliteration. This is exclusively caused by enp in concert with pid. In pinoid embryos, reversal of polarity of the PIN1 auxin transport facilitator in the apex is only occasional, explaining irregular auxin maxima in the cotyledon tips. By contrast, polarity of PIN1:GFP is completely reversed to basal position in the epidermal layer of the laterne embryo. Consequently auxin, which is believed to be essential for organ formation, fails to accumulate in the apex. This strongly suggests that ENP specifically regulates cotyledon development through control of PIN1 polarity in concert with PID.

Simultaneous over-expression of the Her2/neu and PTK6 tyrosine kinases in archival invasive ductal breast carcinomas.

Expression of eight tumour-relevant genes was studied in formalin-fixed, paraffin-embedded tissue from 54 invasive ductal breast carcinomas using quantitative reverse transcription PCR (Q-RT-PCR). Seven of the genes map to chromosome 20q and are potential candidates for gene amplification and over-expression. The Her2/neu oncogene, on chromosome 17q, was investigated in the same tumours. Increased expression was most frequent for PTK6, Her2/neu, and ADA. No other 20q candidate gene (AIB1, PTPN1, ZNF217, and PFDN4) was prominent. A significant correlation between the expression of the tyrosine kinases PTK6 and Her2/neu was detected. The frequent elevation of PTK6 expression (in 43/54 tumours), and its correlation with Her2/neu oncogene over-expression, suggests a clinically relevant link between these two over-expressed tyrosine kinases.

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3.

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.

ADAM15 decreases integrin alphavbeta3/vitronectin-mediated ovarian cancer cell adhesion and motility in an RGD-dependent fashion.

We have recently described that integrin alphavbeta3 upon interaction with its major extracellular matrix ligand vitronectin induces adhesion, motility, and proliferation of human ovarian cancer cells. Due to the important function of alphavbeta3 in cancer cell biology, it has been the effort of many scientific approaches to specifically target alphavbeta3-mediated cell adhesion and tumorbiological effects arising thereof by synthetic integrin antagonists. More recently, proteins of the ADAM family have been recognized as naturally occurring integrin ligands. Among those, human ADAM15 which encompasses the integrin binding RGD motif was shown to interact with integrin alphavbeta3. Thus, we investigated in human ovarian OV-MZ-6 cancer cells, expressing both ADAM15 and alphavbeta3, whether ADAM15 might affect alphavbeta3-mediated tumorbiological effects. We stably (over)expressed ADAM15 or its extracellular domain in OV-MZ-6 cells as well as respective ADAM15 mutants containing the tripeptide SGA instead of RGD. Cells (over)expressing ADAM15-RGD exhibited a significantly reduced alphavbeta3-mediated adhesion to vitronectin. Also, a significant time-dependent decline in numbers of cells cultivated on vitronectin was noticed. This effect was found to be rather due to impaired alphavbeta3-mediated cell adhesion than decreased cell proliferation rates, since de novo DNA synthesis was not significantly altered by elevated ADAM15 expression. Moreover, a substantially decreased random cellular motility was noticed as a function of ADAM15 encompassing an intact RGD motif. In conclusion, our results point to a physiological role of ADAM15 as a natural binding partner of integrin alphavbeta3 thereby loosening tumor cell adhesion to the underlying matrix and regulating tumor cell migration and invasion.

Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes.

Lipopolysaccharides (LPS) are cell-surface components of Gram-negative bacteria and are microbe-/pathogen-associated molecular patterns in animal pathosystems. As for plants, the molecular mechanisms of signal transduction in response to LPS are not known. Here, we show that Arabidopsis thaliana reacts to LPS with a rapid burst of NO, a hallmark of innate immunity in animals. Fifteen LPS preparations (among them Burkholderia cepacia, Pseudomonas aeruginosa, and Erwinia carotovora) as well as lipoteichoic acid from Gram-positive Staphylococcus aureus were found to trigger NO production in suspension-cultured Arabidopsis cells as well as in leaves. NO was detected by confocal laser-scanning microscopy in conjunction with the fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, by electron paramagnetic resonance, and by a NO synthase (NOS) assay. The source of NO was addressed by using T-DNA insertion lines. Interestingly, LPS did not activate the pathogen-inducible varP NOS, but AtNOS1, a distinct NOS previously associated with hormonal signaling in plants. A prominent feature of LPS treatment was activation of defense genes, which proved to be mediated by NO. Northern analyses and transcription profiling by using DNA microarrays revealed induction of defense-associated genes both locally and systemically. Finally, AtNOS1 mutants showed dramatic susceptibility to the pathogen Pseudomonas syringae pv. tomato DC3000. In sum, perception of LPS and induction of NOS contribute toward the activation of plant defense responses.

Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas.

The nuclear disaster that occurred in Chernobyl in 1986 offered the unique opportunity to study the molecular genetics of one human tumor type, papillary carcinoma of the thyroid gland, associated with a specific etiology. We have analyzed RET rearrangements in post-Chernobyl papillary thyroid carcinomas (n = 29), follicular thyroid adenomas (n = 2), and follicular thyroid carcinoma (n = 1) by interphase fluorescence in situ hybridization (FISH) analysis on paraffin-embedded tissue sections. Paraffin sections were microdissected before use to ensure that only tumor was present. Cell nuclei were scored for the presence of a split FISH signal (separated red and green signal) in addition to an overlapping signal. Only cells with either two overlapping signals or one split and one overlapping signal were counted to ensure that only complete cell nuclei had been scored. In total, 23 of 32 cases (72%) showed RET rearrangements diagnosed by FISH interphase analysis. In all cases, the tumors were composed of a mixture of cells with and without ret rearrangement on FISH. In some cases, this distribution was clearly nonrandom because clustering of rearranged cells was detected within the same tumor nodule. Accordingly, only 31% of the cases positive for rearrangement on FISH also scored positive using RT-PCR. These findings suggest that because RET/PTC rearrangements are not present in a majority of tumor cells, either a fraction of post-Chernobyl papillary thyroid tumors are of multiclonal origin, or ret rearrangement is a later, subclonal event.

Influence of propofol on neuronal damage and apoptotic factors after incomplete cerebral ischemia and reperfusion in rats: a long-term observation.

BACKGROUND: Propofol reduces neuronal damage from cerebral ischemia when investigated for less than 8 postischemic days. This study investigates the long-term effects of propofol on neuronal damage and apoptosis-related proteins after cerebral ischemia and reperfusion. METHODS: Male Sprague-Dawley rats were randomly assigned as follows: group 1 (n = 32, control): fentanyl and nitrous oxide-oxygen; group 2 (n = 32, propofol): propofol and oxygen-air. Ischemia (45 min) was induced by carotid artery occlusion and hemorrhagic hypotension. Pericranial temperature and arterial blood gases were maintained constant. After 1, 3, 7, and 28 postischemic days, brains were removed, frozen, and sliced. Hippocampal eosinophilic cells were counted. The amount of apoptosis-related proteins Bax, p53, Bcl-2, and Mdm-2 and neurons positive for activated caspase-3 were analyzed. RESULTS: In propofol-anesthetized rats, no eosinophilic neurons were detected, whereas in control animals, 16-54% of hippocampal neurons were eosinophilic (days 1-28). In control animals, the concentration of Bax was 70-200% higher after cerebral ischemia compared with that in animals receiving propofol over time. Bcl-2 was 50% lower in control animals compared with propofol-anesthetized rats during the first 3 days. In both groups, a maximal 3% of the hippocampal neurons were positive for activated caspase-3. CONCLUSIONS: These data show sustained neuroprotection with propofol. This relates to reduced eosinophilic and apoptotic injury. Activated caspase-3-dependent apoptotic pathways were not affected by propofol. This suggests the presence of activated caspase-3-independent apoptotic pathways.

Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein.

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.