Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “multimodal sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed2003

AMIDE: a free software tool for multimodality medical image analysis.

Amide's a Medical Image Data Examiner (AMIDE) has been developed as a user-friendly, open-source software tool for displaying and analyzing multimodality volumetric medical images. Central to the package's abilities to simultaneously display multiple data sets (e.g., PET, CT, MRI) and regions of interest is the on-demand data reslicing implemented within the program. Data sets can be freely shifted, rotated, viewed, and analyzed with the program automatically handling interpolation as needed from the original data. Validation has been performed by comparing the output of AMIDE with that of several existing software packages. AMIDE runs on UNIX, Macintosh OS X, and Microsoft Windows platforms, and it is freely available with source code under the terms of the GNU General Public License.

Costs and Cost Analysis↗
PubMed2000

P53 expression, p53 and Ha-ras mutation and telomerase activation during nitrosamine-mediated hamster pouch carcinogenesis.

Squamous cell carcinomas (SCC) induced in hamster buccal pouch (HBP) by 22 weeks of topical N-methyl-N-benzylnitrosamine (MBN) treatment (twice-weekly, 10 mg MBN/ml propylene glycol) were evaluated for: (i) altered expression of p53 using immunohistochemistry (IHC); (ii) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP); (iii) telomerase activity using the telomerase repeat amplification protocol (TRAP). Precancerous lesions were also evaluated using p53 IHC. Hamsters were killed for lesion analysis at either 3 days (group A, eight hamsters, 89 carcinomas) or 7 weeks (group B, six hamsters, 105 carcinomas) following the final MBN application. Between 3 days and 7 weeks post-treatment the proportion of tumors exhibiting p53 IHC activity (at least 10% of nuclei stained using D07 antibodies for detection of both mutant and wild-type p53) fell from 91 to 50%. However, during this same post-treatment period the frequency of tumors analyzed exhibiting confirmed sequence alterations in the conserved exons (E5-E8) of p53 remained constant (5/15 = 33% in group A versus 14/45 = 31% in group B). Heightened expression of wild-type p53 resulting from DNA damage in the immediate post-treatment period is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. Nearly 80% of the identified p53 mutations were G-->A and C-->T transitions. The identified p53 point mutations occurred at or near (within three codons) of the corresponding hot-spot codons (175, 245, 248 and 273) of human oral SCC. The proportion of group A and group B tumors analyzed exhibiting Ha-ras mutations was 1/15 (7%) and 7/45 (16%), respectively. Only four of the observed eight Ha-ras mutations occurred in codons known to result in activation of this gene. Telomerase activation was demonstrated in 11 of 13 group A tumors (85%) and in 23 of 24 (96%) group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in this HBP-MBN model are similar in many respects to those observed in the analogous human lesions of the head and neck. This model may be particularly useful for development of cancer chemoprevention regimens and multimodality cancer therapies.

Animals↗
PubMed1999

A space-time delay neural network for motion recognition and its application to lipreading.

Motion recognition has received increasing attention in recent years owing to heightened demand for computer vision in many domains, including the surveillance system, multimodal human computer interface, and traffic control system. Most conventional approaches classify the motion recognition task into partial feature extraction and time-domain recognition subtasks. However, the information of motion resides in the space-time domain instead of the time domain or space domain independently, implying that fusing the feature extraction and classification in the space and time domains into a single framework is preferred. Based on this notion, this work presents a novel Space-Time Delay Neural Network (STDNN) capable of handling the space-time dynamic information for motion recognition. The STDNN is unified structure, in which the low-level spatiotemporal feature extraction and high-level space-time-domain recognition are fused. The proposed network possesses the spatiotemporal shift-invariant recognition ability that is inherited from the time delay neural network (TDNN) and space displacement neural network (SDNN), where TDNN and SDNN are good at temporal and spatial shift-invariant recognition, respectively. In contrast to multilayer perceptron (MLP), TDNN, and SDNN, STDNN is constructed by vector-type nodes and matrix-type links such that the spatiotemporal information can be accurately represented in a neural network. Also evaluated herein is the performance of the proposed STDNN via two experiments. The moving Arabic numerals (MAN) experiment simulates the object's free movement in the space-time domain on image sequences. According to these results, STDNN possesses a good generalization ability with respect to the spatiotemporal shift-invariant recognition. In the lipreading experiment, STDNN recognizes the lip motions based on the inputs of real image sequences. This observation confirms that STDNN yields a better performance than the existing TDNN-based system, particularly in terms of the generalization ability. In addition to the lipreading application, the STDNN can be applied to other problems since no domain-dependent knowledge is used in the experiment.

Algorithms↗
PubMed2006

Acquisition of MR perfusion images and contrast-enhanced MR angiography in acute ischaemic stroke patients: which procedure should be done first?

Multimodal MRI for acute ischaemic stroke usually includes perfusion imaging (PI) and contrast-enhanced neck MR angiography (CE-MRA), as well as diffusion-weighted imaging and T2* weighted imaging. Because both PI and CE-MRA require the infusion of contrast medium, the likelihood exists that one study may conflict with the other due to the accumulation of previously injected contrast medium. The purpose of this study is to determine the appropriate order of PI and CE-MRA in this multimodal MRI protocol for evaluation of acute ischaemic stroke. We studied 35 patients with acute ischaemic stroke in the unilateral middle cerebral artery territory. 17 patients underwent CE-MRA following PI (group A) and 18 patients underwent PI following CE-MRA (group B). For qualitative analysis of the CE-MRA and colour-coded maps of the PI, two independent observers graded the image quality. Interobserver agreement was assessed using kappa statistics, and we assessed the statistical differences of imaging quality between groups A and B using the Mann-Whitney U-test). For the quantitative analysis of PI, two parameters--the maximum change in the transverse relaxation rate (DeltaR2(max)) and the relative signal drop (DeltaS/S(0))--were calculated from the time-signal intensity curve of an unaffected middle cerebral artery territory, and we compared the differences in the parameters of group A and B (t-test). Interobserver agreements for CE-MRA and PI were good. In the qualitative analysis of CE-MRA and PI, no significant difference was observed between groups A and B. In the quantitative analysis of PI, there were no relevant differences in DeltaR2(max) and DeltaS/S(0) between the two groups. In simultaneous CE-MRA and PI, there was no deterioration of diagnostic imaging quality with regard to the order of the two post-contrast sequences. They can be performed according to the preference of each institution.

Acute Disease↗
PubMed2003

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis.

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.

Base Sequence↗
PubMed1988

Separation of peptides by multimode chromatography on a column packed with vinyl alcohol copolymer gel.

Several mechanisms of peptide separation in high-performance liquid chromatography were observed to occur on the Asahipak GS-320 packed with vinyl alcohol copolymer. Neutral rather than acidic mobile phases were employed as they were found to result in higher retention of many peptides on the GS-320. For neutral peptides, the retention volume corresponded to the Rekker's hydrophobic fragmental constant, with a correlation coefficient of 0.71. Peptides with acidic residues eluted early, as an effect of ionic exclusion; those with basic residues were retained longer, owing to an ion-exchange effect. The ionic interactions were shown to involve the carboxylic group present on the gel polymer. The net result was found to be excellent separation of hydrophilic as well as hydrophobic peptides, related to differences in their isoelectric points. The combination of these complex mechanisms, together with the size-exclusion effect of the GS-320 gel for separation of proteins and other large molecules and for analysis with a mobile phase high in acetonitrile content, makes possible high-resolution isocratic analysis of peptides, which cannot be achieved on octadecylsilica or ion-exchange columns.

Amino Acid Sequence↗
PubMed2003

Phase II study of paclitaxel and carboplatin in patients with advanced gastric cancer.

5-Fluorouracil-based combination chemotherapy is commonly used in patients with advanced gastric cancer, but results with such therapy are fairly modest. Evaluation of newer agents is therefore required in this disease. Paclitaxel has shown promising activity as a single agent in gastric cancer. In vitro, paclitaxel exhibits sequence-dependent synergy with platinum compounds against gastric cancer. This study was conducted to evaluate the efficacy and toxicity of combination carboplatin and paclitaxel in patients with advanced gastric cancer. Twenty-seven patients with measurable or evaluable advanced gastric cancer were enrolled on the study from April 1996 to July 2000. Patients were treated with paclitaxel 200 mg/m intravenously during 3 hours followed by carboplatin at projected area under the curve 5 mg x h x ml (as per the Calvert formula). Twenty-six patients were assessable for toxicity, and 25 patients were assessable for objective response. Nine of the 27 enrolled patients had a major response for an objective response rate of 33% (95% CI 0.17-0.54) by intention-to-treat analysis. The median response duration was 4.9 months (95% CI 2.8-7.3), and median survival was 7.5 months. The 1-year survival rate was 23%. One hundred seventeen courses were administered with a median of four courses per patient administered. The major toxicity was neutropenia, with grade III to IV neutropenia observed in 9 patients (33%) and neutropenic fever in only 1 patient. Grade III peripheral neuropathy developed in two patients, and grade III myalgia and grade III fatigue developed in one patient each. There were no treatment-related deaths. The combination of carboplatin and paclitaxel is a highly tolerable, regimen with activity comparable to that of other regimens in advanced gastric cancer. This regimen needs to be further evaluated in combination with other agents and as a component of multimodality therapy in gastric cancer.

Adult↗
PubMed2004

Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach.

We tested the hypothesis that the effects on gene expression of altered DNA methylation by 5-aza-2'-deoxycytidine (5-aza-CdR) and genetic (DNMT knockout) manipulation of DNA are similar, and distinct from Trichostatin A (TSA)-induced chromatin decondensation. Surprisingly, the effects of 5-aza-CdR were more similar to those of TSA than to DNMT1, DNMT3B, or double DNMT somatic cell knockout. Furthermore, the effects of 5-aza-CdR were similar at one and five days exposure, suggesting active demethylation or direct influence of both drugs on the stability of methylation and/or chromatin marks. Agents that induce gene activation through hypomethylation may have unintended consequences, since nearly as many genes were downregulated as upregulated after demethylation. In addition, a 75 kb cluster of metallothionein genes was coordinately regulated.

Algorithms↗
PubMed2005

Temporal evolution of mouse striatal gene expression following MPTP injury.

The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

Multimodality expression profiling shows SEPT9 to be overexpressed in a wide range of human tumours.

Septins are an evolutionarily conserved family of GTPases with diverse functions including roles in cytokinesis that have been implicated in neoplasia. To address the potential role of SEPT9 in tumorigenesis, we assessed the expression of SEPT9 in 7287 fresh frozen human tissue samples and 292 human cell lines by microarray analysis. In addition, we used a sensitive RT-PCR strategy to define the expression of SEPT9 isoforms in archival formalin-fixed and paraffin-embedded normal human tissues. The mRNA data were further confirmed by immunohistological analyses of SEPT9 protein expression in normal human tissues using antisera that detect SEPT9 isoforms. Using these complementary approaches, we demonstrate that SEPT9 mRNA and protein are expressed ubiquitously, with the isoforms showing tissue-specific expression. The microarray analysis indicates that there is consistent overexpression of SEPT9 in diverse human tumours including breast, CNS, endometrium, kidney, liver, lung, lymphoid, oesophagus, ovary, pancreas, skin, soft tissue and thyroid. Since tumours are commonly associated with enhanced cell proliferation, we examined the possible correlation of Ki67 and SEPT9 expression in normal tissues and tumours. Our data indicate that the overexpression of SEPT9 in neoplasia is not simply a proliferation-associated phenomenon, despite its role in cytokinesis.

Cell Movement↗
PubMed1990

Trimodality therapy (drug/hyperthermia/radiation) with BCNU or mitomycin C.

To develop multimodality treatment combinations with high curative potential in advanced local disease, BCNU (N,N'-bis(2-chloroethyl)-N-nitro-sourea) and mitomycin C were tested with hyperthermia and radiation in the FSaIIC fibrosarcoma system. Growth delay experiments demonstrated that, while neither BCNU nor mitomycin C produced dose modification of the radiation response, and hyperthermia (43 degrees C, 30 min) produced only a moderate dose modification (1.4 +/- 0.2), the combination of BCNU plus hyperthermia resulted in a radiation dose modifying factor (DMF) of 1.9 +/- 0.3, and mitomycin C plus hyperthermia a dose modifying factor of 2.1 +/- 0.4. Tumor cell survival over a range of BCNU doses administered i.p. immediately before hyperthermia resulted in a dose modifying factor of 1.8 +/- 0.2 versus drug alone. With mitomycin C however, giving the drug immediately prior to heating produced a dose modifying factor due to hyperthermia of only 1.2 +/- 0.10. Hoechst 33342 diffusion was used to separate tumor cells into predominately oxic and hypoxic subpopulations. Administration of the single, double and trimodality therapies showed that BCNU was 3.1-fold more toxic to the oxic versus the hypoxic cells whereas mitomycin C was 3.5-fold more toxic to the hypoxic compared to the oxic cells. Hyperthermia was 1.4-fold more toxic to the hypoxic versus the oxic cells whereas 10 Gy of radiation was 2.0-fold more toxic to the oxic compared to the hypoxic cells. The combination of hyperthermia plus radiation increased killing in both Hoechst dye defined subpopulations but relatively more in the hypoxic cells in which killing was 1.8-fold greater than in the oxic cells. When heat was delivered immediately after i.p. administration of the anticancer drugs, hyperthermia increased BCNU killing in the oxic cells by 17.2-fold versus 4.4-fold in the hypoxic cells and increased mitomycin-killing by 2.6-fold in the oxic cells versus 17-fold in the hypoxic cells. Use of the full trimodality treatment, given in the sequence drug (BCNU, 50 mg/kg or mitomycin-C 5 mg/kg)----heat (43 degrees C, 30 min)----radiation (10 Gy) produced a 3 log kill in the oxic cells versus a 2 log kill in the hypoxic cells with BCNU and a 2 log kill in the oxic cells versus a 3 log kill in the hypoxic cells with mitomycin C. These results indicate that the use of selected anticancer drugs with hyperthermia and radiation can produce highly cytotoxic interactions which markedly modify the effect of radiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Retinoid receptors in ovarian cancer: expression and prognosis.

BACKGROUND: Ovarian cancer is frequently lethal despite aggressive multimodal therapy, and new therapies are therefore needed. Retinoids are potential candidate drugs: they prevent the development of ovarian carcinoma and enhance the efficacy of cytotoxic drugs in ovarian cancer cells. At present, little is known about the retinoid receptor expression in ovarian cancer. PATIENTS AND METHODS: The retinoid receptors comprise two classes, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with three subclasses, alpha, beta and gamma. We investigated the expression of the subtypes RARalpha, RARgamma, RXRalpha and RXRbeta by immunohistochemistry in ovarian cancers of 80 patients, and assessed their prognostic significance. In addition, we quantified the expression of retinoid receptor mRNA using real-time PCR and correlated the results with clinical characteristics. RESULTS: RARalpha and RXRbeta were highly expressed in a majority of ovarian cancers, particularly in advanced stages. High expression of RARalpha was an independent negative prognostic factor of survival in addition to FIGO stage, age and p53 accumulation. The mRNA expression of retinoid receptors did not correlate with clinical properties of the tumors. CONCLUSIONS: Retinoic acid receptors are frequently and strongly expressed in epithelial ovarian cancer and may be indicators of an adverse prognosis. This study provides the molecular basis for the therapeutic use of retinoids in ovarian cancer.

Aged↗
PubMed2005

pH dependence of extracellular calcium sensing receptor activity determined by a novel technique.

BACKGROUND: Increasing evidence points to the role of the extracellular Calcium Sensing Receptor (CaSR) as a multimodal receptor responding to diverse physiologic stimuli, such as extracellular divalent and polyvalent cations, amino acids, and ionic strength. Within the kidney, these stimuli converge on the CaSR to coordinate systemic calcium and water homeostasis. In this process, the impact of urinary pH changes on the activity of the CaSR has not yet been defined. We therefore performed the present study to analyze the pH sensitivity of the CaSR. METHODS: To assess the activation state of the CaSR, we developed a new method based on the functional coupling between CaSR activity and gating of calcium sensitive potassium currents mediated by SK4 potassium channels. Two-electrode voltage clamping was used to determine whole cell currents in Xenopus oocytes heterologously expressing rat CaSR and rat SK4 potassium channels. RESULTS: Coexpression of CaSR and SK4 gave rise to potassium currents that were dependent on CaSR-mediated intracellular calcium release, and thereby corresponded to the activation state of the CaSR. In presence of extracellular calcium, ambient alkalinization above pH 7.5 increased CaSR activity. Evaluation of the CaSR calcium sensitivity at various ambient proton concentrations revealed that this effect was due to a sensitization of the CaSR towards extracellular calcium. CONCLUSION: Coexpression with SK4 potassium channels provides a fast and sensitive approach to evaluate CaSR activity in Xenopus oocytes. As disclosed by this novel technique, CaSR activity is regulated by extracellular pH.

Animals↗
PubMed2005

Distinct molecular signature of inflammatory breast cancer by cDNA microarray analysis.

Inflammatory breast cancer (IBC) is a clinically distinct and aggressive form of locally advanced breast cancer with largely unknown genetic determinants. Overexpression of the RhoC GTPase and of HER2, and decreased ER-expression are involved in IBC. Multimodality treatment has increased survival but prognosis is still poor. Novel molecular targets for improved neoadjuvant treatment are necessary. Using cDNA microarrays, we performed genome-wide expression profiling of pre-treatment tumour samples of 16 patients with IBC and 18 patients with non-stage-matched non-IBC. Rigid clinical diagnostic criteria according to the TNM classification of the American Joint Committee on Cancer were adopted. Unsupervised hierarchical clustering accurately distinguished IBC and non-IBC samples. A set of 50 discriminator genes was identified in a learning group of tumour samples and was successful in diagnosing IBC in a validation group of samples (accuracy of 88%). Exclusion of ER-related or HER2-related genes did not alter this discriminatory accuracy, indicating that the expression of other genes in addition to ER and HER2 characterize the IBC phenotype. The molecular signature of IBC revealed the overexpression of a large number of NF-kappaB target genes, explaining at least part of the aggressive nature of IBC. Successful validation of some of the overexpressed genes by immunohistochemistry or real-time quantitative PCR demonstrated the robustness of the cDNA microarray experiments. The results of our study provide potential targets for the treatment of patients with IBC.

Belgium↗
PubMed1994

Antigen presentation and cytotoxic T lymphocyte killing studied in individual, living cells.

Interactions between individual, living fibroblasts and cytotoxic T lymphocyte (CTL) clones were analyzed by using video-enhanced differential interference contrast and fluorescence microscopy in a multimode configuration. Fibroblasts expressing known major histocompatibility complex I alleles (MC57: H-2b; Balb: H-2d) were sensitized for killing by incubating or microinjecting them with peptide fragments of lymphocytic choriomeningitis virus. Previous determination of the CTL clones' specificity for these peptides and MHC-I alleles enabled us to study CTL killing of fibroblasts, and nonlethal CTL interaction with targets due to "mismatches" of the CTL, target, and/or peptide. During viral peptide-specific MHC-restricted CTL killing, distinct morphological alterations were observed (CTL shape changes, movements of granules in CTL cytoplasm, and target cell contraction and blebbing). When no killing occurred, CTL engaged in prolonged, nonrandom movement on the target cells. Alloreactive and virus-specific CTL displayed the same morphology during killing. To study antigen presentation further within individual, living cells, a LCMV glycoprotein peptide (aa 272-286, LSDSSGVENPGGYCL) was covalently labeled with tetramethylrhodamine. In 51Cr release assays, the labeled peptide specifically induced potent CTL killing, but neither labeled nor unlabeled peptide proved toxic for unsensitized targets. Microinjection of the labeled peptide into the cytoplasm of fibroblast cells led to CTL killing of those cells, yet nearby uninjected cells contacted by CTL were not killed, indicating that killing was due to presentation of microinjected peptide rather than binding of extracellular peptide to cell surface MHC. Peptide-injected target cells were killed only when combined with CTL specific for the peptide and for the MHC allele of the injected cell.

Amino Acid Sequence↗
PubMed2006

Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse.

Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray-based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb-spaced genome-wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse-associated loci included losses at 1q32.1, 2q36.3-2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p(-)/1q(+), a breakpoint was identified at 1p13. Multiple low-level sub-megabase gains along the length of 1q were identified using chromosome 1 tiling-path arrays. One such recurrent region at 1q22-q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT-PCR. These data provide a high-resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours.

Chromosome Aberrations↗
PubMed2004

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.

Base Sequence↗
PubMed2004

Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival.

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, 5-year survival rates have only approached 50-60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In line with the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with an unfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application of chemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copy numbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. The combination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.

Adolescent↗