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Histochemical Staining (histochemical + staining)
Selected AbstractsMolecular detection and , -glucuronidase expression of gus -marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlingsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2003E. Tsomlexoglou Abstract Aim: To detect L-form bacteria in developing Chinese cabbage seedlings. Methods and Results: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding , -glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of , -glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. , -Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. Conclusions: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. Significance and Impact of the Study: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association. [source] Natural genetic resources of Arabidopsis thaliana reveal a high prevalence and unexpected phenotypic plasticity of RPW8- mediated powdery mildew resistanceNEW PHYTOLOGIST, Issue 3 2008Katharina Göllner Summary ,,Here, an approach based on natural genetic variation was adopted to analyse powdery mildew resistance in Arabidopsis thaliana. ,,Accessions resistant to multiple powdery mildew species were crossed with the susceptible Col-0 ecotype and inheritance of resistance was analysed. Histochemical staining was used to visualize archetypal plant defence responses such as callose deposition, hydrogen peroxide accumulation and host cell death in a subset of these ecotypes. ,,In six accessions, resistance was likely of polygenic origin while 10 accessions exhibited evidence for a single recessively or semi-dominantly inherited resistance locus. Resistance in the latter accessions was mainly manifested at the terminal stage of the fungal life cycle by a failure of abundant conidiophore production. The resistance locus of several of these ecotypes was mapped to a genomic region containing the previously analysed atypical RPW8 powdery mildew resistance genes. Gene silencing revealed that members of the RPW8 locus were responsible for resistance to Golovinomyces orontii in seven accessions. ,,These results suggest that broad-spectrum powdery mildew resistance in A. thaliana is predominantly of polygenic origin or based on RPW8 function. The findings shed new light on the natural variation of inheritance, phenotypic expression and pathogen range of RPW8 -conditioned powdery mildew resistance. [source] Isolation of a novel mouse gene, mSVS-1/SUSD2, reversing tumorigenic phenotypes of cancer cells in vitroCANCER SCIENCE, Issue 6 2007Tetsuo Sugahara We report isolation of a novel tumor-reversing gene, tentatively named SVS-1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein. The gene was found among those downregulated in the activated oncogene-v-K-ras-transformed NIH3T3 cells, Ki3T3, with tumorigenic phenotype. SVS-1 protein harbors several functional domains inherent to adhesion molecules. Histochemical staining of mouse tissues using antibody raised against the protein showed the expression of the protein in restricted regions and cells, for example, strongly positive in apical membranes of epithelial cells in renal tubules and bronchial tubes. The protein inducibly expressed in human fibrosarcoma HT1080 cells and cervical carcinoma HeLa cells was found to be localized primarily on the plasma membrane, as stained with antibodies against FLAG tag in the N -terminus and against the C -terminal peptide of the protein. Expression of the protein in cells induced a variety of biological effects on cancer cells: detachment from the substratum and aggregation of cells and growth inhibition in HeLa cells, but no inhibition in non-tumorigenic mouse NIH3T3 cells. Inhibition of clonogenicity, anchorage-independent growth, migration and invasion through Matrigel was also observed. Taken together these results suggest that the SVS-1 gene is a possible tumor-reversing gene. (Cancer Sci 2007; 98: 900,908) [source] Ocular Melanoma Metastatic to Skin: The Value of HMB-45 StainingDERMATOLOGIC SURGERY, Issue 6 2004Robert A. Schwartz MD Background: Cutaneous metastatic disease is an important finding that may represent the first sign of systemic cancer, or, if already known, that may change tumor staging and thus dramatically altered therapeutic plans. Although cutaneous metastases are relatively frequent in patients with cutaneous melanoma, they are less so from ocular melanoma. Objective: To demonstrate the value of HMB-45, staining in the detection of ocular melanoma metastatic to skin. Methods: The immunohistochemical stain HMB-45 a monoclonal antibody directed against intact human melanoma cells, was employed on a skin biopsy specimen from a cutaneous tumor. Results: HMB-45 staining was positive in the atypical hyperchromatic cells of the deep dermis. Conclusion: HMB-45 may be of value in the detection of ocular melanoma metastatic to skin. Cutaneous metastatic disease is a somewhat common and extremely important diagnosis. Although cutaneous metastases from cutaneous melanoma are relatively frequent, those from ocular melanomas are less so. Use of histochemical staining, especially the HMB-45 stain, allows confirmation of the diagnosis. [source] Transgenic strains of the nematode Caenorhabditis elegans as biomonitors of metal contaminationENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2000L. K. Cioci Abstract Transition metal contamination poses a serious environmental and human health threat. The bioavailability of transition metals in environmental samples can best be assessed with living organisms. A transgenic strain of the free-living soil nematode Caenorhabditis elegans has been engineered for monitoring the bioavailability of metals. A reporter transgene consisting of a fragment of the promoter from the C. elegans metallothionein-2 gene (mtl-2) that controls the transcription of a ,-galactosidase reporter (lacZ) has been integrated into the genome of this organism. By using these transgenic C. elegans, the toxicological response to metals in samples can be quickly measured with a simple histochemical staining assay. The C. elegans that contain the mtl-2:lacZ transgene provide a more sensitive assay of exposure to cadmium, mercury, zinc, and nickel than 24-h LC50 assays or those using nematodes with heat-shock protein,based reporter transgenes. This study demonstrates that C. elegans that contain mtl-2:lacZ transgenes can function as sensitive toxicological indicators of metals. [source] Post-conditioning with cyclosporine A fails to reduce the infarct size in an in vivo porcine modelACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 7 2010R. H. LIE Background: Cyclosporine A has generated intense interest in the field of cardioprotection due to its ability to protect the mitochondria at reperfusion by blocking the opening of the mitochondrial permeability transition pore. The aim of our study was to examine the cardioprotective effect of Sandimmun®, a clinically available formulation of cyclosporine A, in an in vivo large mammal model. Methods: Forty-eight pigs were randomly allocated to one of three groups: (i) Control group (Con, n=19), (ii) Cyclosporine group, (Cyclo, n=19) Sandimmun® 10 mg/kg i.v. bolus 5 min before reperfusion and (iii) Pre-conditioning group (Precon, n=10) two cycles of 10 min ischemia interspersed with 30-min reperfusion. The study was further sub-divided into a metabolic protocol, evaluating myocardial metabolism by measuring changes in the interstitial lactate concentration, and a coronary flow protocol. All animals were subjected to 40 min of left anterior descending coronary artery occlusion, followed by 180 min of reperfusion before histochemical staining and assessment of infarct size by planimetry. Results: Infarct sizes were measured as: Con 51.4 ± 16.5%, Cyclo 47.3 ± 15.7% and Precon 2.4 ± 3.6%, with no significant difference between the Con and Cyclo groups but a highly significant difference between the Precon and Cyclo and Con groups (P<0.0001 for both comparisons). In the Cyclo group, the interstitial lactate concentration was significantly increased compared with the Con group at 6-min reperfusion, although significantly lower at 14 min presumably due to accelerated washout. Conclusion: In this large animal model, a 10 mg/kg bolus administration of Sandimmun® 5 min before reperfusion did not reduce the infarct size. [source] Pagetoid dyskeratosis of the prepuce.JOURNAL OF CUTANEOUS PATHOLOGY, Issue 8 2000An incidental histologic finding resembling extramammary Paget's disease Background: Pale cells resembling those of paget's disease have been seen as an incidental finding within the epidermis in a variety of benign papules most commonly located in intertriginous areas. This lesion, called pagetoid dyskeratosis, is considered a reactive process in which a small part of the normal population of keratinocytes is induced to proliferate. Among the inductors friction is suspected. As far as we know, these cells have not been reported in the penis. Methods: Here we describe the location of the lesion in the foreskin and the incidence of this lesion in a group of 281 unselected patiets surgically treated for phimosis. In selected cases histochemical staining and immunohistochemical studies were performed. Results: Pagetoid dyskeratosis was found in 105 cases (37.4%) but only in 5 cases (1.8%) the lesion was conspicuous. The cells of pagetoid dyskeratosis show an immunohistochemical profile different from the surrounding keratinocytes characterized by premature keratinization. Pagetoid dyskeratosis cells must be distinguished from the artefactual clear cells of the epidermis, from reactive melanocytes, and from pale-cell acanthosis. In cases in which pagetoid dyskeratosis shows a florid expression there is a hazard of overdiagnosis to the patient. The main differential diagnosis includes extramammary Paget's disease, pagetoid squamous cell carcinoma in situ, epidermotropic metastasis, superficial spreading malignant melanoma, clear cell papulosis, and penile koilocytoses. Conclusions: The pathologist should be familiar with the histologic features of pagetoid dyskeratosis in the foreskin in order to avoid misdiagnosis and unnecessary treatment. Routine histologic study is usually sufficient to identify the lesion. [source] Insulin-like growth factor-I ameliorates demyelination induced by tumor necrosis factor-, in transgenic miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007Ping Ye Abstract Our groups have reported that tumor necrosis factor-, (TNF-,) causes myelin damage and apoptosis of oligodendrocytes and their precursors in vitro and in vivo. We also have reported that insulin-like growth factor-I (IGF-I) can protect cultured oligodendrocytes and their precursors from TNF-,-induced damage. In this study, we investigated whether IGF-I can protect oligodendrocytes and myelination from TNF-,-induced damage in vivo by cross-breeding TNF-, transgenic (Tg) mice with IGF-I Tg mice that overexpress IGF-I exclusively in brain. At 8 weeks of age, compared with those of wild-type (WT) mice, the brain weights of TNF-, Tg mice were decreased by ,20%, and those of IGF-I Tg mice were increased by ,20%. The brain weights of mice that carry both TNF-, and IGF-I transgenes (TNF-,/IGF-I Tg mice) did not differ from those of WT mice. As judged by histochemical staining and immunostaining, myelin content in the cerebellum of TNF-,/IGF-I Tg mice was similar to that in WT mice and much more than that in TNF-, Tg mice. Consistently, Western immunoblot analysis showed that myelin basic protein (MBP) abundance in the cerebellum of TNF-,/IGF-I Tg mice was double that in TNF-, Tg mice. In comparison with WT mice, the number of oligodendrocytes was decreased by ,36% in TNF-, Tg mice, whereas it was increased in IGF-I Tg mice by ,40%. Oligodendrocyte number in TNF-,/IGF-I Tg mice was almost twice that in TNF-, Tg mice. Furthermore, IGF-I overexpression significantly reduced TNF-,-induced increases in apoptotic cell number, active caspase-3 abundance, and degradaion of MBP. Our results indicate that IGF-I is capable of protecting myelin and oligodendrocytes from TNF-,-induced damage in vivo. © 2007 Wiley-Liss, Inc. [source] Norovirus binds to blood group A-like antigens in oyster gastrointestinal cellsLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2006P. Tian Abstract Aims:, To determine if histo-blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells. Methods and Results:, HBGA-specific monoclonal antibodies (MAbs) were used to determine the presence of the corresponding HBGA in oyster GI cells. All oyster samples tested contained type A-like HBGA in GI tissue as measured by ELISA. Recombinant Norwalk virus viral like particles (rNVLP) were bound to plates coated with oyster GI homogenate. The binding was inhibited when rNVLPs were pre-incubated with MAbs specific for type A HBGA, or samples of human saliva from type A individuals. Co-localization of rNVLP and type A-like HBGA, but not type B-like or type H-like HBGA, on GI epithelial cells was observed by immunofluorescent histochemical staining and three-channel confocal scanning laser microscopy. Conclusion:, Type A-like HBGA is present in oyster GI cells and responsible for binding of rNVLP. Significance and Impact of the Study:, This is the first report of the presence of type A-like HBGA in oyster GI cells and the specific binding of rNVLP to type A-like HBGA on oyster GI cells. The results of this study suggest that human NoV concentrate in oyster GI cells by specific binding to concentrated type A-like HBGA rather than by a nonmolecular entrapment within the tissues. [source] Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallidaPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2004Catherine J. Lilley Summary The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcI,D86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 ± 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 ± 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence. [source] Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in ratsPSYCHIATRY AND CLINICAL NEUROSCIENCES, Issue 6 2001Mariko Osawa MD Abstract Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 ± 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus. [source] Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal miceTHE JOURNAL OF GENE MEDICINE, Issue 4 2006Elena Fedorova Abstract Background Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal. Methods Four weeks after intrathecal injection into the spinal canal of newborn mice, the expression of the LacZ gene was assessed by histochemical staining and by in situ polymer chain reaction (PCR). Following the spinal infusion of a lentivector carrying the SEAP gene, levels of enzymatically active SEAP were measured in the CSF, blood serum, and in brain extracts. Results Intrathecal spinal canal delivery of lentivectors to newborn mice resulted in patchy, widely scattered areas of ,-gal expression mostly in the meninges. The transduction of the meningeal cells was confirmed by in situ PCR. Following the spinal infusion of a lentivector carrying the SEAP gene, sustained presence of the reporter protein was detected in the CSF, as well as in blood serum, and brain extracts. Conclusions These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier. Copyright © 2006 John Wiley & Sons, Ltd. [source] N -acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entryTHE JOURNAL OF GENE MEDICINE, Issue 1 2002L. Jornot Abstract Background It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N -acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. Methods HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of ,-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24,h and 48,h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Results Pretreatment of cells with NAC prior to Ad infection enhanced ,-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in ,-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, ,-Gal activity was further enhanced, by 15-fold. Augmentation of ,-Gal activity was paralleled by an increase in ,-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Conclusion Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright © 2001 John Wiley & Sons, Ltd. [source] Human liver autofluorescence: An intrinsic tissue parameter discriminating normal and diseased conditions,,LASERS IN SURGERY AND MEDICINE, Issue 5 2010Anna C. Croce PhD Abstract Background and Objective Autofluorescence (AF) emission is an intrinsic parameter that can provide real-time information on morpho-functional properties of biological tissue, being strictly related with their biochemical composition and structural organization. The diagnostic potentials of AF-based techniques have been investigated on normal, fibrotic, and steatotic liver tissues, in reference to histological features as evidenced by specific histochemical stainings. Materials and Methods AF emission under excitation at 366,nm has been examined on cryostatic tissue sections obtained from biopsies collected during surgical operation, by means of fluorescence imaging and microspectrofluorometric techniques. Results NAD(P)H, collagen, and vitamin A were found to be the endogenous fluorophores characterizing normal, fibrotic, and steatotic liver tissue AF, respectively. The differences of their photo-physical properties, in terms of emission amplitude, spectral shape, and response to irradiation, give rise to modifications of overall AF signal collected from tissues that allow the liver conditions to be distinguished. Conclusion The study provides a valid premise for a development of AF-based optical biopsy techniques for a real-time discrimination of liver anatomo-pathological patterns. Lasers Surg. Med. 42:371-378, 2010. © 2010 Wiley-Liss, Inc. [source] Metastatic sclerosing mesothelioma in a cowAUSTRALIAN VETERINARY JOURNAL, Issue 7 2002E BEYTUT Metastatic sclerosing mesothelioma in a crossbred cow is described. Accumulation of fluid in the abdominal cavity and solitary or coalesced nodules on the peritoneum, hepatic capsule and visceral pleurae, were observed after slaughter. Histological examination of the nodules revealed that they were composed of tubular structures supported by massive connective tissue. The lumina of the tubules were lined by solitary neoplastic mesothelial cells, or occasionally small groups of such cells were observed in the lumen. Identification of the mesothelial character of the tumours was dependent upon the histopathological and cytological characteristics of the nodules and histochemical stainings. [source] | ||||||||||||||||||||||