A technique for the isolation of mutants of Escherichia coli affected in degradation of cellular RNA.
(25/1051)
Using a semiautomatic technique for handling large numbers of Escherichiacoli colonies, mutants that fail to digest their cellular RNA were isolated. This was achieved by using multiwell plates where each colony is cloned in an individual well. Cells labeled with a radioactive RNA precursor were starved for a carbon source at a high temperature. In order to assess whether or not degradation of cellular RNA took place, aliquots of each culture were subjected to autoradiography. A number of mutants defective in decay of RNA were isolated. One of them was characterized, and was found to be deficient specifically in the enzyme polynucleotide phosphorylase. Experiments carried out with this strain indicate that this enzyme participates in the degradation of "stable" RNA during carbon starvation. (+info)
Gene profiling approach in the analysis of lymphotoxin and TNF deficiencies.
(26/1051)
Mice with combined lymphotoxin-alpha (LTalpha) and tumor necrosis factor (TNF) deficiencies show defects in the structure of peripheral lymphoid organs such as spleen, lymph nodes, and gut-associated lymphoid tissues. To identify genes associated with this defective phenotype in spleen, we applied a gene profiling approach, including subtractive cloning and gene array hybridizations, to mice with combined TNF/LT deficiency. The differentially expressed genes identified by these techniques was then evaluated by Northern blot analysis for splenic expression in knockout mice with single LTalpha or single TNF deficiency. Most of the genes detected in this analysis are directly or indirectly associated with disrupted LT and not TNF signaling. (+info)
Identification and characterization of genes upregulated in cells transformed by v-Jun.
(27/1051)
The transcription factor Jun (c-Jun) functions as a recipient of extracellular growth signals and converts them into patterns of gene expression. An oncogenic variant of c-Jun was isolated from the acutely transforming retrovirus ASV17. Overexpression of this viral Jun (v-Jun) induces transformation of chicken embryo fibroblasts (CEF) in culture and fibrosarcomas in chickens. v-Jun is a constitutively active form of c-Jun and transforms cells presumably by deregulating the expression of specific target genes. In this report, we describe six genes whose transcripts are upregulated in v-Jun-transformed CEF. Three of these genes show homology to known mammalian genes, to MAP kinase phosphatase 2 (MKP-2), to reversion-induced LIM protein (RIL) and to cytokine-inducible SH2-containing protein (CIS). Northern blot analysis, using CEF infected with various Jun mutants or an estrogen-regulatable Jun chimera, revealed distinct induction patterns of individual targets by v-Jun. The chicken RIL homolog showed an expression pattern tightly correlated with the activity of v-Jun. Its expression is also transformation-dependent, suggesting a role for this gene in v-Jun transformation. The newly identified v-Jun targets can serve as molecular markers in the v-Jun transformation process. Oncogene (2000) 19, 3537 - 3545 (+info)
Isolation and characterization of sixteen novel p53 response genes.
(28/1051)
The EB-1 cell line is a stable transfectant of EB, a p53 null colon carcinoma cell line, with an inducible promoter controlling expression of a wild type p53 cDNA. The induced p53 is transcriptionally active and gives rise to apoptosis in these cells. Using this cellular model for presence or absence of the transcription factor p53 and transactivated genes, the Suppression Subtractive Hybridization (SSH) technique permitted the isolation of 17 mRNA candidates (GIPs-Genes induced by p53), whose expression appears to be p53-dependent. Identity has been established for nine of the 17 isolated candidates. These are HGFL/MSP, Zap-70, APOBEC2, Ponsin/SH3P12/CAP/FLAF2, CDCrel2b/H5/Pnutl2, IgG, lats 2, cytokeratin 15 and PIG-3 (quinone oxidoreductase). The latter gene is the only GIP previously demonstrated to be p53 regulated. Of the eight remaining GIPs, six correspond to Unigene clusters. One candidate, GIP #1, is significantly homologous (72% identity) to a chicken zinc finger protein, CTCF, which binds to insulator elements and thus attenuates enhancer cross-talk between physically adjacent promoters. The p53-dependent expression of GIPs was confirmed by dependence of expression upon induction of wt p53 expression in the EB-1 cellular model and by up-regulation following activation of an endogenous wt p53 by treatment with adriamycin. Oncogene (2000) 19, 3978 - 3987. (+info)
Utility of CT angiography and MR angiography for the follow-up of experimental aneurysms treated with stents or Guglielmi detachable coils.
(29/1051)
BACKGROUND AND PURPOSE: Previous studies have depicted arterial and aneurysmal anatomy with three-dimensional time-of-flight (3D-TOF) MR angiography before and after treatment with Guglielmi detachable coils (GDCs) and with CT angiography before and after treatment with stents and stent-grafts. We investigated the ability of time-resolved contrast-enhanced 3D MR angiography (3D MR digital subtraction angiography [DSA]) to accurately depict the anatomy of experimental lateral aneurysms before and after treatment with GDCs and a variety of stents or stent-grafts, and compared these findings with 3D-TOF MR angiography without and with contrast enhancement and CT angiography. METHODS: Two nitinol stents, two nitinol-polytetrafluoroethylene (PTFE) stent-grafts, and two stainless steel stents were deployed in three dogs with experimental carotid aneurysms. In a fourth animal, one of three aneurysms was completely occluded with GDCs. The other two aneurysms were loosely packed to ensure persistence of some residual aneurysmal lumen. Cut-film angiography, CT angiography, 3D-TOF MR angiography without and with contrast enhancement, and 3D MR DSA were performed in all dogs before and 3 weeks after treatment. RESULTS: 3D MR DSA was superior to conventional 3D-TOF MR angiography without and with contrast enhancement in accurately depicting experimental lateral aneurysms and superior to CT angiography in depicting aneurysms treated by GDCs. 3D MR DSA and CT angiography were comparable in depicting vessels treated with nitinol stents and stent-grafts, whereas CT angiography was superior for showing vessels treated by stainless steel stents. CONCLUSION: We recommend further development and clinical evaluation of 3D MR DSA for imaging cerebral aneurysms before and after treatment with GDCs. 3D MR DSA or CT angiography may be useful for evaluating vessels containing nitinol stents or nitinol-PTFE stent-grafts, whereas CT angiography should be used for follow-up of vessels treated by stainless steel stents. (+info)
MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression.
(30/1051)
The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROalpha, beta or gamma in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO effect on melanocyte transformation requires expression of other genes, differential display was performed. One of the mRNA's identified in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/GRO protein. Western blot examination of Ras expression revealed that K- and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The effects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the effects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/GROalpha-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes. (+info)
Does performing image registration and subtraction in ictal brain SPECT help localize neocortical seizures?
(31/1051)
Ictal brain SPECT (IS) findings in neocortical epilepsy (patients without mesiotemporal sclerosis) can be subtle. This study is aimed at assessing how the seizure focus identification was improved by the inclusion of individual IS and interictal brain SPECT (ITS)-MRI image registration as well as performing IS - ITS image subtraction. METHODS: The study involved the posthoc analysis of 64 IS scans using 99mTc-ethyl cysteinate dimer that were obtained in 38 patients without mesiotemporal sclerosis but with or without other abnormalities on MRI. Radiotracer injection occurred during video-electroencephalographic (EEG) monitoring. Patients were injected 2-80 s (median time, 13 s) after clinical or EEG seizure onset. All patients had sufficient follow-up to correlate findings with the SPECT results. All patients had ITS and MRI, including a coronal volume sequence used for registration. Image registration (IS and ITS to MRI) was performed using automated software. After normalization, IS - ITS subtraction was performed. The IS, ITS, and subtraction studies were read by 2 experienced observers who were unaware of the clinical data and who assessed the presence and localization of an identifiable seizure focus before and after image registration and subtraction. Correlation was made with video-EEG (surface and invasive) and clinical and surgical follow-up. RESULTS: Probable or definite foci were identified in 38 (59%) studies in 33 (87%) patients. In 52% of the studies, the image registration aided localization, and in 58% the subtraction images contributed additional information. In 9%, the subtraction images confused the interpretation. In follow-up after surgery, intracranial EEG or video-EEG monitoring (or both) has confirmed close or reasonable localization in 28 (74%) patients. In 6 (16%) patients, SPECT indicated false seizure localization. CONCLUSION: Image registration and image subtraction improve the localization of neocortical seizure foci using IS, but close correlation with the original images is required. False localizations occur in a minority of patients. (+info)
Isolation and identification of psoralen plus ultraviolet A (PUVA)-induced genes in human dermal fibroblasts by polymerase chain reaction-based subtractive hybridization.
(32/1051)
Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for a variety of skin disorders. Recently, we demonstrated that treatment of human dermal fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in a permanent growth arrest with a switch of mitotic to postmitotic fibroblasts. Furthermore, an upregulation of matrix-degrading metalloproteinases and a high level of de novo expression of the senescence-associated beta-galactosidase was detected in the PUVA-treated postmitotic fibroblasts. The molecular basis for this PUVA-induced change in the functional and morphologic phenotype of fibroblasts resembling or mimicking replicative senescence is, however, unknown. Herein after, we have used a polymerase chain reaction-based subtractive hybridization protocol to identify human genes that are induced by PUVA treatment. Application of polymerase chain reaction-Select resulted in the cloning of four PUVA genes. Sequence analysis and homology searches identified three cDNA clones of known genes related to cell cycle regulation (p21waf1/cip1), stress response (ferritin H) and connective tissue metabolism (tissue inhibitor of metalloproteinases-3), whereas one cDNA clone represented a novel gene (no. 478). Northern blot analyses were performed to confirm a PUVA-dependent increase in specific mRNA levels in human dermal fibroblasts in vitro. This report on the identification of growth arrest related genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these known and novel genes in extrinsic and intrinsic aging processes on a molecular and cellular level. (+info)