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  • 1995-1999  (10)
  • Immunohistochemistry  (5)
  • Nitrate reductase  (5)
  • Fibronectin  (2)
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    International journal of legal medicine 110 (1997), S. 18-21 
    ISSN: 1437-1596
    Keywords: Key words Proteinase inhibitors ; Fibronectin ; Lysozyme ; Immunohistochemistry ; Autolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Law
    Notes: Abstract The influence of postmortem damage of tissues on the immunohistochemical diagnosis of wound age has not as yet been clarified. We utilized antibodies against the proteinase inhibitors α-1-antichymotrypsin and α-2-macroglobulin, fibronectin and lysozyme to study samples of skin which had been intact intravitally, but were damaged postmortem either by autolysis or compression with a surgical clamp at the time of dissection. Even in the absence of autolysis, antibodies against the proteinase inhibitors and fibronectin exhibited staining of tissue margins. Autolysis caused an increase in false positive results. In contrast, antibodies against lysozyme did not give false positive staining. There were no antigens sensitive to postmortem clamping and false positive results were not observed. Antibodies against proteinase inhibitors are not useful for the diagnosis of wound age because of a high number of false positive reactions in marginal areas. Fibronectin also showed false positive band-shaped staining patterns at the tissue margin. In addition, autolytic processes increase the number of false positives. The antibody against lysozyme is much less sensitive to autolysis and no false positive reactions were observed in our series of tests.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    International journal of legal medicine 110 (1997), S. 18-21 
    ISSN: 1437-1596
    Keywords: Proteinase inhibitors ; Fibronectin ; Lysozyme ; Immunohistochemistry ; Autolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Law
    Notes: Abstract The influence of postmortem damage of tissues on the immunohistochemical diagnosis of wound age has not as yet been clarified. We utilized antibodies against the proteinase inhibitors α-1-antichymotrypsin and α-2-macroglobulin, fibronectin and lysozyme to study samples of skin which had been intact intravitally, but were damaged postmortem either by autolysis or compression with a surgical clamp at the time of dissection. Even in the absence of autolysis, antibodies against the proteinase inhibitors and fibronectin exhibited staining of tissue margins. Autolysis caused an increase in false positive results. In contrast, antibodies against lysozyme did not give false positive staining. There were no antigens sensitive to postmortem clamping and false positive results were not observed. Antibodies against proteinase inhibitors are not useful for the diagnosis of wound age because of a high number of false positive reactions in marginal areas. Fibronectin also showed false positive band-shaped staining patterns at the tissue margin. In addition, autolytic processes increase the number of false positives. The antibody against lysozyme is much less sensitive to autolysis and no false positive reactions were observed in our series of tests.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2307
    Keywords: Lymphoma ; Hodgkin's disease ; Polymerase chain reaction ; Immunohistochemistry ; Histological classification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Ninety-one Hodgkin's lymphomas (HD), 52 non-Hodgkin lymphomas (NHL) and 33 specimens of non-neoplastic lymphatic tissues were investigated by polymerase chain reaction (PCR) for the presence of the bcl-2/JH gene rearrangement. The majority of the HD cases were drawn from the files of the German Hodgkin trial where diagnoses are established by a panel of four independent histopathologists. Using the very sensitive PCR method which detected 1 positive among 10000 negative cells, the bcl-2/JH gene rearrangement was found in 7/52 NHL and 3/16 tonsils with follicular hyperplasia, but in none of the 91 HD. The bcl-2 protein, however, was expressed by malignant cells of B and T cell lymphomas and by the giant tumour cells in 2/13 HD lymphocyte predominant, 11/28 HD nodular sclerosing I, 14/17 HD nodular sclerosing II, 10/27 HD mixed cellularity and 3/3 HD lymphocyte depleted. The bcl-2/JH rearrangement is thus independent of protein over-expression, the latter being found in all types of lymphomas. Our results do not confirm the findings of others who have detected the bcl-2/JH rearrangement in HD. These discrepancies may be explained by differences in choice of material, the gene rearrangement actually occuring in bystander cells but not in Reed-Sternberg or Hodgkin cells, or by contamination.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Planta 196 (1995), S. 1-6 
    ISSN: 1432-2048
    Keywords: Acid-base loading ; Nitrate reductase ; pH regulation (intracellular) ; Protein phosphorylation ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of acid or base-loading of spinach (Spinacia oleracea L.) leaf discs on the activation status of nitrate reductase (NR) in the dark and in the light was investigated. Activity of NR (NRA), measured in crude extracts of leaf discs with removed lower epidermis, which had been floating on Mes-buffer [2-(N-morpholino)ethane sulfonic acid] pH 5.2 in the dark, was at a similar low level as in whole, darkened leaves. By addition of acetate or propionic acid, butyric acid or benzoic acid, NR was activated to or beyond the light level. The pH of crude tissue extracts was decreased by 0.5–1 pH units. Tissue acidification caused an inhibition of photosynthesis and of dark CO2 fixation. The acid-induced activation of NR in vivo was largely prevented by okadaic acid, an inhibitor of Type 1 and Type 2A protein phosphatases. This indicates that acid-induced activation was mediated by protein dephosphorylation. When, on the other hand, leaf discs were illuminated on Ches-buffer (2-[ N-cyclohexylamino]ethane sulfonic acid) pH 9 in the presence of bicarbonate (80 mM), their NR was as active as in intact leaves. Addition of ammonium chloride (up to 6 mM) caused a pH increase of the tissue extract up to 0.9 pH units. At the same time NR was inactivated to the dark level. Methionine sulfoximine did not prevent the ammonium effect. Photosynthesis and dark CO2 fixation were stimulated at pH 9 by ammonium chloride (1–2· mol· m −3) and were only slightly inhibited by up to 6 mol· m−3. The modulation of NR by acid-base treatment in vivo was fully reversible. The response of the NR system to acid or base treatment is consistent with a proposed role of nitrate reduction in the cellular pH-stat. The observation also indicates that cytosolic pH changes may be involved the signal chain triggering the modulation of NR.
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  • 5
    ISSN: 1432-2048
    Keywords: Key words: Activation state (nitrate reductase) ; Anoxia ; Hordeum (roots) ; Nitrate reductase ; Protein phosphory-lation ; Protein turnover
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-2048
    Keywords: Key words: Activation state (nitrate reductase) ; Hordeum (nitrate reductase) ; Nitrate reductase ; Nitrate supply ; Phosphate deficiency ; Signal metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRAmax) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRAmax and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRAmax and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRAmax and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRAmax was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRAmax in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NRmax by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRAmax.
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  • 7
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Knochenmarkhistologie ; Kunststoffeinbettung ; Methylmethacrylat ; Immunhistochemie ; Key words Bone marrow histology ; Plastic embedding ; Methyl-methacrylate ; Immunohistochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary A patented low-temperature polymerization method for methylmethacrylate (MMA) infiltrated bone marrow biopsies is described: it has been developed from our previous MMA technique and is a patented procedure. Differences from the previous method are (1) removal of stabilizer from the MMA monomer before its application, (2) the use of a different starter, (3) avoidance of O2 influence during polymerization by means of vacuum exchange with N2, and (4) polymerisation in a water bath to draw off residual heat. After this procedure, all immunohistochemical reactions are possible provided that the previous fixation is adequate. The effects of different fixatives are reviewed briefly without detailed analysis. Technically, this plastic embedding can be performed at least as rapidly as the classic paraffin embedding after decalcification. The advantages over the latter method are: (1) the cells can be better differentiated because semi-thin sections can be made; (2) the immunoreactions can also be performed on the basis of semi-thin sections, which means they can be interpreted more easily; (3) morphometric analyses yield more reliable results because of the constant thickness of sections; (4) osteological examination of bone trabeculae, especially the search for mineralisation deficiencies, is possible; (5) the plastic embedding procedure is less dependent on individual instabilities in the quality of performance of the staff members involved. Furthermore, it is worth mentioning that the costs for additional equipment necessary remain below DM 100,000 including an excellent microtome.
    Notes: Zusammenfassung Eine patentierte Methode zur Einbettung von Knochenmarkbiopsien durch Kaltpolymerisation von Methylmethacrylat (MMA) wird beschrieben, die als patentiertes Verfahren aus unserer bisherigen MMA-Technik entwickelt worden ist. Die Unterschiede zum bisherigen Verfahren sind die Destabilisierung des Monomers Methylmethacrylat, die Verwendung eines anderer Starters, der Ersatz von Raumluft durch Stickstoff vor der Polymerisation und die Polymerisation unter Restwärmeabführung im Wasserbad. Danach sind alle immunhistochemischen Reaktionen an Zellen und Geweben möglich, vorausgesetzt, daß in geeigneter Form fixiert worden ist. Auf die Unterschiede der Fixierungslösungen wird kurz eingegangen, ohne sie detailliert zu analysieren. Technisch läßt sich diese Kunststoffeinbettung mindestens ebenso schnell wie die Entkalkung und Paraffineinbettung durchführen. Die Vorteile gegenüber dem Entkalkungs-Paraffinierungs-Verfahren sind, daß• die Zellen besser zu differenzieren sind, weil Semidünnschnitte angefertigt werden können, • die Immunreaktionen ebenfalls am Semidünnschnittpräparat durchgeführt und die Reaktionsergebnisse deshalb leichter zugeordnet werden können, • morphometrische Analysen wegen der konstanten Schnittstärke zuverlässigere Werte ergeben und • osteologische Untersuchungen, insbesondere die Beurteilung von Mineralisationsstörungen, möglich sind. Ein fünfter Vorteil ist, daß das Kunststoffverfahren unempfindlicher gegenüber subjektiven Leistungsschwankungen der beteiligten Mitarbeiter ist. Die zusätzlich notwendigen Geräteinvestitionen liegen unter DM 100 000.–, worin ein optimales Mikrotom, z. B. Polycut, eingeschlossen ist.
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  • 8
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Chondroblastom ; Knochentumoren ; Immunhistologie ; Proliferation ; Key words Chondroblastoma ; Bone tumors ; Immunohistochemistry ; Proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Representing only about 1 % of all primary bone tumors, chondroblastoma constitutes a very rare bone tumor entity. 56 cases of chondroblastoma, that had been collected by the Hamburg Bone Tumor Registry from 1972 to 1995, were examined histologically together with the radiological and clinical findings. In addition immunohistochemistry with antibodies against S 100, PGM1, LCA and the proliferationmarker MIB 1 was performed. The mean age was 20.4 years and male patients being the majority with a gender ratio of 2.7 : 1. Predominant localisation was the epiphyses of the long bones, although almost 40 % of the tumors were located at untypical sites. Usually a well-circumscribed lysis could be seen on plain X-Ray examination, however partial cortical destruction could be observed in one third of the cases. Histologically characteristic was a polygonal cell component with a weblike chonroid matrix, sometimes with a plane-like appearance. 5 cases showed a distinct nuclear polymorphism making a distinction from osteosarcoma difficult. Using immunohistochemistry all tumors except for one showed positive reaction for S 100 protein. Although the histogenesis of chondroblastoma is not completely understood, morphological findings as well as the observed reactivity with the S 100 protein indicate the chondroid origin. No reactivity for PGM 1 (CD 68) or LCA could be detected. All chondroblastoma showed a low rate of proliferation, thereby being distinguishable from high malignant bone tumors. In general chondroblastoma show a benign biological behavior. Different behavior was observed in 2 cases. One relapse located in the pelvis revealed local aggressive growth while in another case in the humerus a malignant transformation had taken place.
    Notes: Zusammenfassung 56 Chondroblastome, die im Hamburger Knochentumorregister im Zeitraum von 1972 bis 1995 archiviert wurden, wurden retrospektiv histologisch untersucht, unter Berücksichtigung des radiologischen Befunds sowie der klinischen Angaben. Zusätzlich wurden immunhistologische Färbungen für S 100, PGM 1, LCA und den Proliferationsmarker MIB 1 durchgeführt. Das Durchschnittsalter der Patienten betrug 20,4 Jahre unter Bevorzugung männlicher Patienten mit einem Geschlechtsverhältnis von 2,7 : 1. Bevorzugter Lokalisationsort waren die Epiphysen der langen Röhrenknochen. Radiologisch stellt sich typischerweise eine umschriebene Lyse mit umgebendem Randsaum dar. Charakteristisch ist histologisch eine polygonale Zellkomponente mit einer netzartigen chondroiden Matrix. In 5 Fällen lag eine deutliche Kernpolymorphie vor, die eine Abgrenzung zum Osteosarkom schwierig machte. Immunhistologisch waren mit Ausnahme eines Falle alle Tumoren positiv für S 100. Allen Chondroblastomen war eine niedrige Proliferationsrate gemeinsam, die diese deutlich von hochmalignen Knochentumoren unterschied. Chrondroblastome besitzen üblicherweise ein gutartiges biologisches Verhalten. Zwei Fälle dieser Studie zeigten einen davon abweichenden Verlauf. Bei einem Rezidivtumor im Becken zeigte sich ein lokal aggressives Wachstum, in einem anderen Fall im Humerus war es zu einer malignen Transformation gekommen.
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  • 9
    ISSN: 1432-2048
    Keywords: Inhibitor protein ; Nitrate reductase ; Protein kinase ; Protein phosphatase ; Protein turnover
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (〈3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5′-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with γ-[32P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.
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  • 10
    ISSN: 1432-2048
    Keywords: Inhibitor protein ; Nitrate reductase ; Protein phosphorylation ; Protein kinase ; Spinacia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The function of two proteins (P67 and P100) required for the MgATP-dependent inactivation of nitrate reductase (NR) from spinach leaves (Spinacia oleracea L.) was studied. When NR was incubated with γ-[32P]ATP and P67, NR-protein was phosphorylated, but without a change in NR activity. Protein P100 by itself was neither able to phosphorylate nor to inactivate NR, and when added together with P67 it did not change the extent of NR phosphorylation. However, when NR was first phosphorylated with MgATP and P67, subsequent addition of P100 after removal of unreacted ATP caused an immediate NR inactivation. In presence of both P67 and P100 the time-course of ATP-dependent NR phosphorylation paralleled the time course of inactivation. The extent of NR phosphorylation and of NR inactivation (in the presence of P67 plus P100) was similarly affected by metabolites or high salt concentrations. Magnesium (Mg2+) played a dual role in the inactivation process: the phosphorylation of NR by P67 was strictly Mg2+-dependent. Further, phospho-NR (+P100) was inactive only in the presence of Mg2+, but active in the presence of excess EDTA. Dephospho-NR appeared to be Mg2+-insensitive. The observations suggest that phosphorylation of NR by P67 is obligatory, but not sufficient for inactivation. In addition to protein phosphorylation, inactivation requires “binding” of an inhibitor protein (P100) to phospho-NR.
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