Functional and morphological effects of tumour necrosis factor α in an interleukin 6-producing pulmonary large cell carcinoma with sarcomatoid features

We established a clonal cell line, HAT.MC8, derived from a human pulmonary large cell carcinoma with sarcomatoid features. This cell line was successfully maintained in a protein-free medium and exhibited sarcomatoid fibroblastic features in vitro. The cells constitutively produced a large amount of interleukin 6 (IL-6) in vitro. Tumour necrosis factor (TNF-) not only stimulated HAT.MC8 cells to produce IL-6, but also induced a morphological change from sarcomatoid fibroblastic to epithelial features. Although this change was related to actin and zonula adherens, there was no evidence that E-cadherin participated in the change. Interleukin 1 (IL-1) had a stimulatory effect on IL-6 production by HAT.MC8 cells, but no influence on the morphology of the cells.     

Serum creatine kinase activity following forearm flexion isometric exercise

Summary The purpose of this study was to 1) compare serum creatine kinase (CK) activity following two forearm flexion isometric exercise regimens differing in work to rest ratio, and 2) examine the CK response to a repeated bout of isometric exercise. Eleven males were tested on two sessions (bouts) spaced 1 week apart. For bout 1, five subjects (group A) performed a forearm flexion isometric exercise consisting of 40 10-s maximal contractions with 20-s inter-trial rests (1020), while six (group B) performed 40 maximal 10-s contractions with 5-s inter-trial rests (105). The increase in serum CK activity following the 1020 exercise (143%) was significantly greater than that following the 105 exercise (52%). The 1020 exercise was also associated with greater tension generation over trials. One week later, both groups performed a bout of 1020 exercise. A substantial reduction in the serum CK response was found following this second bout. The data suggest that for bout 1 the isometric exercise associated with the greater overall tension levels resulted in the greater CK response. However, when the 1020 exercise was repeated 1 week later, a substantial reduction in the CK response was found which was unrelated to the tension generated.This study was supported by a University Faculty Research Grant No. 2-03021     

NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-B in the presence of CM by studying the degradation rate of individual IB isoforms.Results: We show that, as expected, CM induced NF-B translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF- and IL1 naturally present in the CM. However, both the amplitude of NF-B translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF- and IL-1, either separately or together. Our results show that CM induced a faster degradation of the IB- and IB- isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-B activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF- mediated endothelial activation.Received 5 October 2003; returned for revision 2 December 2003; accepted by A. Falus 19 May 2004     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

An in vitro model to study myocardial ischemic injury

Summary This report describes a method to study ischemic and reperfusion damage in cultured ventricular cardiomyocytes. The cardiomyocytes were made ischemic by oxygen deprivation and volume restriction. The ischemic intervals studied were 60, 90, and 120 min. An oxygen tension of 0 mmHg was maintained throughout ischemia. Reperfusion was simulated by bathing the cells in a large volume of normally oxygenated PBSG following ischemia. Ischemia and reperfusion resulted in increased lactate production, increased membrane damage, and ATP degradation. Ischemia caused a decrease in pH. Although 90 min of ischemia caused cell structural changes and alteration of metabolism, a prolonged ischemic interval of 120 min exacerbated these abnormalities. We have found our in vitro ischemia and reperfusion model to closely mimic the in vivo condition. This model using human ventricular cardiomyocytes is a means to studying the effect and mechanism of ischemia and reperfusion at the cellular level under defined conditions.     

Activated charcoal is as effective as fuller's earth or bentonite in paraquat poisoning

Summary In vitro investigations have shown that the adsorption capacity of activated charcoal (Kohle-Compretten, Ultracarbon, E. Merck, Darmstadt, FRG) is just as high as that of Fuller's earth (Surrey powder, Laporte Industries Ltd., Luton, GB) or Bentonite BP W.B. (Steetley Minerals Ltd., Milton Keynes, GB). Fuller's earth (Fullererde) from another manufacturer has had very poor adsorption properties and is thus not suitable for the treatment of paraquat poisoning. Animal experiments have shown that the curative effect of activated charcoal given 0.5, 1, 2, and 3 h after ingestion of 200 and 300 mg paraquat/kg body weight is equally as good or even better than that of Fuller's earth or Bentonite BP W.B.. Activated charcoal is a substitute of equal value to these mineral soils.     

The length dependency of calcium activated contractions in the femoral artery smooth muscle studied with different methods of skinning

The relationship between the calcium concentration and the isometric tension obtained with different techniques of skinning provides information on the biochemical events of contraction in vascular smooth muscle. Muscle preparations of the rabbit femoral artery were skinned with triton X-100, saponin, -escin and -toxin and the relationship between the calcium concentration and isometric tension was determined at different preparation lengths. We determined the calcium sensitivity as a function of muscle length with different techniques of skinning. At a pCa of 6.0, triton X-100 skinned smooth muscle of the femoral artery generated 50% of the maximal tension. In -toxin skinned preparations, this calcium sensitivity was shifted to a pCa of 5.6. The sensitivity of the saponin and -escin skinned preparations were in between those of the triton X-100 and the -toxin skinned preparations. The cooperativity of the regulation of contraction varied among the differently skinned preparations between 3 (-toxin) and 6 (triton X-100). The relationships between the calcium concentration and the isometric tension of the differently skinned preparations up to the optimal length for tension generation did not exhibit any length dependency. The length tension relationship, obtained from the maximal response at the highest calcium concentration is in line with that from other studies. The presence of intracellular proteins and membranes affects the regulation of contraction in smooth muscle of the femoral artery.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

The contractile behaviour of EGTA- and detergent-treated heart muscle

Summary Tension responses of rat ventricular trabeculae subjected to successive treatment with EGTA and Triton X-100 are described in order to investigate the effects of chemical skinning techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the EGTA-treatment fails to render cells hyperpermeable, i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the EGTA-treated tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of hyperpermeability. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after EGTA-treatment alone. This applies to contractures evoked direct from Ca²⁺ concentration 10^–9 m to the test Ca²⁺ concentration at constant total buffer concentration.EGTA-treated fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24h produce preparations showing similar behaviour: whatever the explanation for the EGTA-skinning phenomenon it is not dependent upon low bathing Ca²⁺ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after EGTA-treatment: this treatment does not produce a model of a selectively skinned cardiac cell.     

Stereochemical aspects of the interaction of myosin and actomyosin with nucleotides

Summary The five possible analogues of ATP and the three possible analogues of ADP which contain single non-bridging sulphur atoms instead of oxygen in the polyphosphate structure have been used as probes of the interaction of nucleotides with myosin and actomyosin. Evidence is presented for the requirement of an , , -tridentate complex of magnesium and ATP as the substrate for myosin. Of the four possible tridentate MgATP diastereomers, the exo isomer (nomenclature of Cornelius & Cleland, 1978) appears to be the actual substrate.Abbreviations ATP Adenosine-5-O-triphosphate - ATP(-S) Adenosine-5-O-(1-thiotriphosphate) - ATP(-S) Adenosine-5-O-(2-thiotriphosphate) - ATP(-S) Adenosine-5-O-(3-thiotriphosphate) - ADP Adenosine-5-O-diphosphate - ADP(-S) Adenosine-5-O-(1-thiodiphosphate) - ADP(-S) Adenosine-5-O-(2-thiodiphosphate) - Enzyme Myosin ATPase (EC 3.6.1.3)     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Biochemical characteristics,phage patterns,and O1 factor analysis ofEscherichia coli O1∶K1∶H7∶F11 and O1∶K1∶H−∶F9 strains isolated from patients with urinary tract infections

Biochemical characteristics, O1 antigen factors and phage patterns were examined in 35 urinary O1K1 isolates ofEscherichia coli different in H and F antigen. Fermentation of dulcitol, decarboxylation of ornithine, requirement for nicotinamide, and determination of O1 factor d allowed maximum differentiation. On the basis of these tests the strains could be divided into two major groups which are obviously of different clonal origin. Members of clone 1 represented by serotypes O1K1H7F11 (12 strains) and O1K1H^–F11 (5 strains) were characterized by positive biochemical reactions and absence of O1 antigen factor d. Negative biochemical tests and presence of O1 antigen factor d were shown by strains of clone 2 which were of serotypes O1K1H^–F9 (14 strains) and O1K1H^–F^– (3 strains). Phage patterns are less well correlated with clonal assignment. However, strains of clone 2 were not susceptible to K1-specific phage D and were non-typable with another set of 13 phages.     

Functional mapping of the activity of the R region in the human T-cell leukemia virus type I long terminal repeat to increase gene expression

We previously demonstrated the activity of the R fragment in the long terminal repeat of human T-cell leukemia virus type I for elevation of the level of gene expression. In this study, the fragment was deleted with BAL31 nuclease to determine its functional domain. Series of the shortened R fragments were linked to the simian virus 40 promoter unit, which regulated expression of a reporter gene. Examination with the R fragments deleted from the 5 and 3 ends showed that borders of the functional domain were mapped within nucleotide positions 458 to 473 for the 5 end and nucleotide positions 559 to 594 for the 3 end, respectively. Thus we conclude that a 136-base-pair fragment corresponding to the second half of the R region was sufficient to allow elevation of the level of gene expression.     

Cyclische Nucleotide als intracelluläre Überträgerstoffe bei Hormonwirkungen

Zusammenfassung Hormone dienen als extracelluläre Informationsüberträger zwischen ihrem Bildungsort, einer endokrinen Drüse, und den Zellen, deren Funktion sie regulieren. Durch die Reaktion des Hormons mit den an der Zellmembran gelegenen Receptoren wird die Aktivität der mit diesen eng verknüpften Adenyl-Cyclase beeinflußt. Die meisten Hormone erhöhen in ihrem Zielorgan die Aktivität dieses Enzyms und führen hierdurch zu einem raschen Anstieg der intracellulären Konzentration von Adenosin-3:5-monophosphat (Ado-3:5-P). Dieses cyclische Nucleotid wird durch eine spezifische Phosphodiesterase zu Adenosin-5-monophosphat abgebaut. Auch die Aktivität dieses Enzyms bestimmt die intracelluläre Ado-3:5-P-Konzentration, die im Vergleich zu der anderer Nucleotide sehr gering ist.Ado-3:5-P beeinflußt als zweiter, intracellulärer Überträgerstoff die Aktivität zahlreicher Schlüsselenzyme. Die Ado-3:5-P-Konzentration bestimmt hierdurch das Gleichgewicht verschiedener Stoffwechselwege zueinander und damit die Reaktion einer Zelle auf eine hormonale Stimulierung. An einer Reihe von Enzymen wird die durch Ado-3:5-P bedingte Aktivitäts-Änderung durch einen gleichartigen Mechanismus bewirkt. Das cyclische Nucleotid stimuliert Proteinkinasen, die eine Phosphatgruppe des ATP auf verschiedene Proteine übertragen und hierdurch deren Eigenschaften verändern können. So steigt bei Phosphorylierung durch eine Ado-3:5-P-stimulierbare Proteinkinase die Aktivität der Triglyceridlipase und der Glykogen-Phosphorylase-b-kinase an, dagegen nimmt die Aktivität der Glykogen-Synthetase ab; durch Phosphorylierung von Histonen kann deren Repressorcigenschaft vermindert und die Synthese von Enzymen gesteigert werden.In manchen tierischen Geweben wurde auch eine spezifisch durch Guanosin-3:5-monophosphat (Guo-3:5-P) stimulierbare Proteinkinase nachgewiesen. Dieses cyclische Nucleotid kommt wie Ado-3:5-P in allen Säugerorganen vor. Die Bildung von Guo-3:5-P aus GTP wird durch die Guanyl-Cyclase katalysiert, ein Ferment, das im Gegensatz zur Adenyl-Cyclase zum großen Teil nicht an die Zellmembranen gebunden ist. Die Konzentration von Guo-3:5-P in verschiedenen Geweben, im Blutplasma und im Urin wird durch Hormone beeinflußt. Es ist noch nicht bekannt, welche hormonalen Regulationen durch Guo-3:5-P vermittelt werden; dagegen ist bei vielen, rasch einsetzenden Hormonwirkungen die Beteiligung von Ado-3:5-P nachgewiesen worden.

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Abkürzungen Ado-3:5-P Adenosin-3:5-monophosphat - dAdo-3:5-P Desoxy-adenosin-3:5-monophosphat - Guo-3:5-P Guanosin-3 : 5-monophosphate - Nuc-3:5-P Nucleosid-3:5-monophosphat - NTP Nucleosidtriphosphat - NMP Nuclcosid-5-monophosphat - dATP Desoxyadenosintriphosphat - P_i anorganisches Phosphat - PP_i anorganisches Pyrophosphat - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure - r-RNS ribosomale RNS - m-RNS Boten-RNS - Glykogen-Synthetase UDP-Glucose--1,4-glucan--4-glucosyltransferase - ICSH interstitial-cell-stimulating hormone     

Nephritogenicity and α-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane

Nephritogenicity (anti-GBM-nephritis-inducing activity) and -chain composition of globular-domain (NC1) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different chains of type IV collagen. A purified nephritogenic fraction from renal BM contained 1–6(IV)NC1, whereas a non-nephritogenic fraction contained only 1–2(IV)NC1. Renal and pulmonary NC1 had strong nephritogenic activity; placental NC1 had weak activity. The renal and pulmonary fractions contained 1–6(IV)NC1, and the placental fraction had a large amount of 1–2(IV)NC1 and a very small amount of 3–6(IV)NC1. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained 1–5(IV) chains, but not the 6(IV) chain. The absence of 6(IV) chain in glomerular BM in bovine and in humans indicates that 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of 3–5(IV)NC1.