Pharmacological investigations with different protein kinase C inhibitors on IgE-dependent and IgE-independent activation of human basophils

In the present study different selective inhibitors of the multifunctional serine/threonine kinase protein kinase C (PKC) were investigated on classical activation pathways of basophils in comparison to the nonselective protein kinase inhibitor staurosporine. The potent inhibitors Ro 31-7549, Ro 31-8220, calphostin C and ilmofosine (BM 41.440), which show selectivity for PKCin vitro, significantly potentiated FcRI-mediated histamine release up to 50% vs. controls at concentrations >10^–7 mol/l but were without any intrinsic histamine releasing activity. Direct activation of cellular PKC by phorbol ester was suppressed by all compounds apart from ilmofosine at the same concentrations. We did not observe statistically significant effects of selective PKC inhibitors on exocytosis induced by the peptide formylmeth-leu-phe (FMLP) or the ionophore A23187, whereas staurosporine potentiated the FMLP-induced histamine release in a dose-dependent fashion: maximum potentiation was 63.5±8.9% vs. control at 1 mol/l (n=4). The findings suggest that PKC exhibits differential functions during biochemical events of stimulus-secretion coupling in human basophils supposedly by its distinct subtypes. With respect to the present data, TPA-induced and IgE-mediated signals are not closely correlated.     

Stimulation of protein kinase C activity may increase microvascular permeability to colloidal carbon viaα-isoenzyme

The vasculature of the isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution in vitro. Various phorbol-related compounds that are known to have different affinities for the protein kinase C (PKC) isoenzymes, and bradykinin (BK), were tested for their ability to cause the microvascular endothelium to become permeable to injected colloidal carbon (CC). Phorbol 12,13-dibutyrate (PDB), 12-deoxyphorbol 13-phenylacetate (DOPPA), thymeleatoxin (TMX), and resiniferatoxin (RFX), each at a concentration of 1M, were found to increase permeability. Pretreatment with the PKC inhibitor Ro 31–8220 (1M) significantly reduced the response to all of these compounds. Indomethacin (1M), on the other hand, reduced only the effect of RFX. 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA) (1M) and BK (10M) did not increase CC leakage. These results suggest that the Ca²⁺-dependent PKC-isoenzyme was involved in the increase in endothelial permeability. BK does not appear to stimulate PKC activity in this experimental situation.     

12-deoxyphorbol-13-O-phenylacetate 20 acetate [an agonist of protein kinase Cβ1 (PKCβ1)] induces DNA synthesis,interleukin-2 (IL-2) production,IL-2 receptor α-chain (CD25) and β-chain (CD122) expression,and translocation of PKCβ isozyme in human peripheral blood lymphocytes: Evidence for a role of PKCβ1 in human T cell activation

To determine a role of protein kinase C (PKC) isozymes in lymphocyte activation, human peripheral blood mononuclear cells were activated with 12-deoxyphorbol-13-O-phenylacetate (dPP; an agonist of both calcium-dependent and calcium-independent PKC isozymes), thymeleatoxin (TX; an activator of calcium-dependent PKC, , and ), and 12-deoxyphorbol-13-O-phenylacetate 20 acetate (dPPA; an activator of PKC1 isozyme) and examined for DNA synthesis, lymphocyte proliferation, interleukin-2 (IL-2) production, expression of IL-2 receptor and chains on CD3+, CD4+, and CD8+ T lymphocytes and CD20+ B lymphocytes, and translocation of PKC isozyme from cytosol to membrane fraction. The results show that dPPA activates lymphocytes by inducing the above changes in a manner analogous to that of dPP, TX, and phorbol myristate acetate. These data suggest that PKC1 is involved in the activation of human peripheral blood T and B lymphocytes.     

Effects of protein kinase C activators on human basophils

In the present study the effects of different activators of protein kinase C (PKC) on histamine release from human basophils were studied. The phorbol esters 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), phorbol-12,13-didecanoate, and the non-phorbol mezerein induced a dose-dependent mediator release, whereas 4-phorbol-12,13-didecanoate and 4-phorbol, which both do not activate isolated PKCin vitro, were inactive. However, PDBu and mezerein were significantly more active than TPA, indicating that histamine releasing capacity was independent of their activity on PKC. We suggest, therefore, that different PKC isotypes with distinct biochemical characteristics are activated by these compounds. Alternatively, additional PKC-independent mechanisms might explain our findings. Moreover, a direct comparison of TPA-induced and IgE-mediated histamine release, using basophils from the same subjects, revealed a poor correlation. This may confirm previous observations on their differing effects on PKC levels.     

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.     

Cognitive mediators of the social influence-exercise adherence relationship: A test of the theory of planned behavior

The purpose of this study was to examine cognitive constructs from the theory of planned behavior (i.e., attitude, perceived behavioral control, and intention) as potential mediators of the relationship between selected social influence constructs (i.e., subjective norm, social support, and cohesion) and adherence to structured exercise classes. Sixty-two participants completed self-administered questionnaires during the fourth week (social influence constructs) and eighth week (cognitive constructs) of a 12-week exercise program. Exercise adherence was monitored during weeks 9 through 12 using perceived intensity and attendance. Pearson correlations indicated that social support correlated with perceived behavioral control, whereas cohesion correlated with attitude. Path analysis supported two distinct paths from social influence to exercise adherence: (a) social support perceived behavioral control intention excersise adherence, and (b) cohesion attitude intention exercise adherence. Discussion focuses on the theoretical importance of these findings, conceptual and measurement issues regarding subjective norm, and suggestions for future research.     

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     

Vergleichende Virulenzuntersuchungen an Shigella sonnei mit Hilfe des Conjunctivaltestes und derα-Ketoglutarsäurebestimmung

Zusammenfassung Es wird über vergleichende Virulenzuntersuchungen an Shigella sonnei mit dem Conjunctivalversuch und der Bestimmung des-Ketoglutarsäurebildungsvermögens berichtet. Aus den durchgeführten Untersuchungen kann entnommen werden, daß die Virulenz im-Ketoglutarsäurebildungsvermögen und im Conjunctivaltest bei der Shigella sonnei ein paralleles Verhalten zeigt. Das-Ketoglutarsäurebildungsvermögen stellt also bei der Shigella sonnei einen Maßstab für deren Virulenz dar.     

Protein kinase C inhibitor calphostin C prevents cytokine-induced angiogenesis in the rat

A rat sponge implant model was used to examine the role of protein kinase C (PKC) in angiogenesis, Neovascular response was determined by measurements of relative sponge blood flow by a¹³³Xe clearance technique and confirmed histologically. Morphometric analysis was used to quantitate the amount of fibrovascular growth in the sponges. Daily doses of recombinant human basic fibroblast growth factor (bFGF, 100 ng), tumor necrosis factor-alpha (TNF-, 50 ng), or interleukin1-alpha (IL-1, 50 ng) caused neovascular responses that were blocked by daily coadministration of the selective PKC inhibitor, calphostin C (4g). To confirm that calphostin C was able to inhibit PKC in vivo, its effect on the angiogenic response elicited by the PKC activator, phorbol 12-myristate 13-acetate (PMA, 30g) was examined. The blood flow and morphometric data clearly showed that the intense neovascularization induced by PMA was totally suppressed by coadministration of calphostin C (4g). Thus, these results suggest that cytokine-induced angiogenesis may be mediated in part through the activation of PKC and that selective inhibition of this enzyme could have therapeutic benefit in angiogenic diseases.     

Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

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.     

Einstellzeit der Pt-Elektrode bei Messungen nicht stationärer O2-Partialdrucke

Zusammenfassung Das Meßsignal bei sprunghaftenpO₂-Änderungen wird anhand des Diffusionsfeldes der Elektrode beschrieben. Es wird das zeitliche Verhalten des Meßsignals von blanken und membranbespannten Elektroden in gasförmigen und nicht gasförmigen Meßmedien betrachtet. Aus dem Verhalten des Meßsignals kann jeweils die Einstellzeit alssystematischer Meßfehler abgeleitet werden. In nicht gasförmigen Medien (z. B. biologisches Gewebe) übersteigt das Meßsignal nach einempO₂-Sprung zu höheren Werten das stationäre Endsignal. Daraus ergibt sich eine besondere Betrachtung der Einstellzeit in solchen Medien.Die Einstellzeit für Pt-Elektroden mit einfacher und doppelter Membran wird explizit angegeben. Schließlich wird für biologische Medien die Einstellzeit mit dem Diffusionsfehler [8] verglichen. Die Forderungen an eine Membran der Pt-Elektrode mit kleiner Einstellzeit und gleichzeitig kleinem Diffusionsfehler sind zusammengestellt.

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Erklärung der Symbole a Verhältnis der Diffusionskoeffizienten zweier Membranen - Bunsenscher Löslichkeitskoeffizient des Mediums - _m Bunsenscher Löslichkeitskoeffizient der Membran - b Verhältnis der Diffusionsleitfähigkeiten von Membran und Medium - C ₁,C ₂ Proportionalitätskonstanten zwischen Meßsignal und O₂-Partialdruck - D Diffusionskoeffizient des Mediums - D _m,D _m Diffusionskoeffizienten der Membranen - Diffusionskoeffizient der effektiven Membran - DF Diffusionsfehler - DGl Differentialgleichung - d _m,d _m Dicke der Membranen - Dicke der effektiven Membran - dimensionsloser Parameter des Diffusionsfehlers - erf Fehlerfunktion - exp Exponentialfunktion - F Faradaykonstante - grad Gradient - I stationäres Meßsignal vor dempO₂-Sprung - I stationäres Meßsignal nach dempO₂-Sprung - I(t), I(),I() instationäres Meßsignal als Funktion der Zeit bzw. zeitabhängiger dimensionsloser Parameter - K Diffusionsleitfähigkeit des Mediums - K _m Diffusionsleitfähigkeit der Membran - Diffusionsleitfähigkeit der effektiven Membran - dimensionsloser Parameter - n Summationsindex - pO₂ O₂-Partialdruck - pO₂ als Funktion von Ort und Zeit bzw. zeitabhängiger dimensionsloser Parameter; Diffusionsfeld der Elektrode - p _c konstanterpO₂ vor dempO₂-Sprung - p _c konstanterpO₂ nach dempO₂-Sprung - p(r ₀+d _m , ) pO₂ an der Grenze Membran/Medium in Abhängigkeit des Zeitparameters - p(r,o) Diffusionsfeld zum Zeitpunkt (t=0) despO₂-Sprunges - p(r₀+d_m, o) pO₂ an der Grenze Membran/Medium zum Zeitpunkt despO₂-Sprunges - R Radius der ebenen, kreisförmigen Elektrode - r ₀ Radius der Elektrode mit halbkugelförmiger Pt-Oberfläche - r Kugelkoordinate - ₁,₂ dimensionslose ortsabhängige Parameter - T ₉₀,T ₉₅ Zeit, bis 90% bzw. 95% des Signalunterschiedes nach dempO₂-Sprung ausgeglichen sind (Einstellzeit) - T ₉₀,T ₉₅ Einstellzeit der Elektrode mit Doppelmembran - T _90*,T _95* Zeit, bis sich das Signal nach Übersteigen des stationären Endwertes diesem auf 10% bzw. 5% angenähert hat - dimensionslose Parameter zu den vorangegangenen Einstellzeiten - t Zeitkoordinate - , dimensionslose zeitabhängige Parameter - t _max, _max Zeit maximaler Signalhöhe nachpO₂-Sprung und zugehöriger dimensionsloser Parameter - V(t) Diffusionsgesamtfluß zur Pt-Oberfläche - Stromdichtevektor der diffundierenden O₂-Moleküle - x, y, z Kartesische Koordinaten - Integrationsvariable - ² Laplace-Operator - partielle Ableitung nach der Zeit - Integral über eine Fläche - gerichtetes Flächenelement     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Chloride channel regulation in the skeletal muscle of normal and myotonic goats

External intercostal muscle biopsies from normal and congenitally myotonic goats were studied in vitro at 30° C using a two-microelectrode square-pulse cable analysis assisted by computer. The resting chloride conductance (G _cl) was estimated from the difference between the mean membrane conductance in chloride-containing and chloride-free bathing media. The protein kinase C (PKC) activator, 4--phorbol-12,13-dibutyrate, (0.1–2.0 M) blocks a maximum of 76% of G _cl in normal goat fibers and induces myotonic hyperexcitability similar to that of congenitally myotonic goat fibers. The G _cl block was partially antagonized by pretreatment with the PKC inhibitor, staurosporine (10 M). The inactive 4-phorbol-12, 13,didecanoate had no effect at 50 M, whereas the active 4- isomer blocked 41% G _cl at 1 M. The nearly absent G _cl of congenitally myotonic goat fibers was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Also, forskolin and cholera toxin, which may increase cyclic adenosine monophosphate (cAMP) levels, or the R(+) clofibric acid enantiomers and taurine, which increase G _cl in normal fibers, were also unable to restore G _cl in myotonic goat fibers. The data suggest that PKC may be a chloride channel regulator in normal goat skeletal muscle fibers, however the molecular defect of congenitally myotonic fibers does not appear to be due to excessive activity of PKC.     

Modulation of human monocyte superoxide production by recombinant interleukin-3

We have examined the generation of superoxide by human monocytes isolated from peripheral blood cultured in the presence of recombinant human interleukin-3 in comparison to tumor necrosis factor- and interferon-. The rate of superoxide production of unstimulated and stimulated monocytes [by formylmethionyl-leucyl-phenylalanine (0.1 M) and by phorbolmyristate-acetate (2 ng/ml and 200 ng/ml)] decreased during the culture period in the absence of interleukin-3, whereas cells treated with interleukin-3 maintained or surpassed their initial superoxide-producing capacity. An increase of phorbolmyristate-acetate- and formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production of monocytes cultured with interleukin-3 compared to control cells was detected first after 24 h of monocyte culture. It was maximal after 96 h of monocyte culture. At this time the stimulated superoxide production of monocytes cultured in the presence of interleukin-3 surpassed that of interferon- and tumor necrosis factor- treated cells. Suboptimal concentrations of the stimulus phorbolmyristate-acetate (2 ng/ml) resulted in higher priming than 200 ng/ml phorbolmyristate-acetate. A dose dependence of the effect of interleukin-3 on the superoxide production was observed.Our results demonstrate that interleukin-3 primes cultured human peripheral blood monocytes for enhanced stimulated respiratory burst activity to a higher extent than interferon- and tumor necrosis factor-.     

Diffusionsfehler und Eigenverbrauch der Pt-elektrode beipO2-Messungen im steady state

Zusammenfassung Im steady state beeinflussen Diffusionsfehler und Eigenverbrauch der Pt-Elektrode als systematische Fehler O₂-Partialdruckmessungen. Sie sind abhängig von den geometrischen Eigenschaften der Elektrode, den Diffusionseigenschaften der Membran sowie den Diffusions- und Konvektionseigenschaften des Meßmediums. Das Diffusionsfeld vor der Pt-Oberfläche und das dadurch bestimmte stationäre Meßsignal werden für gasförmige und nicht gasförmige Medien mit und ohne Konvektion berechnet. Daraus resultieren quantitative Aussagen über die systematischen Fehler. Speziell für Messungen in durchbluteten Geweben (z. B. Hirnrinde und Myokard) wird der Einfluß des Eigenverbrauchs von Pt-Elektroden auf den intracapillärenpO₂-Abfall in Durchblutungsrichtung und das intercapillärepO₂-Feld am Meßort der Elektrode ermittelt. Diese Berechnungen erfolgten mit Hilfe eines Digitalmodells.

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Erklärung der Symbole A O₂-Verbrauch des Gewebes - Bunsenscher Löslichkeitskoeffizient des Mediums - _m Bunsenscher Löslichkeitskoeffizient der Membran - C ₁,C ₁,C ₂,C ₂,C ₃ Konstanten - D Diffusionskoeffizient des Mediums - DF, DF Diffusionsfehler bei einfacher und doppelter Membran - DGl Differentialgleichung - d Capillarabstand - d _h Dicke der hydrodynamischen Grenzschicht - d _m ,d _m Dicke der Membranen - , , , , , dimensionslose Parameter - exp Exponentialfunktion - F Faradaykonstante - grad Gradient - I _o stationäres Meßsignal in Medien ohne Konvektion - I stationäres Meßsignal in Gasen - I _o stationäres Meßsignal in Flüssigkeiten mit Konvektion - J _o nullte Bessel-Funktion - K Diffusionsleitfähigkeit des Mediums - KE, KE Konvektionseffekt bei einfacher und doppelter Membran - K _m ,K _m Diffusionsleitfähigkeit der Membranen - l Capillarlänge - l Capillarabschnitt - Viscosität des Mediums - p, pO₂,p(r), p(r,z) O₂-Partialdruck - p mittlerer Partialdruck - P _a O₂-Partialdruck am arteriellen Capillarende - p _c konstanter Partialdruck - P/r _o +d _m O₂-Partialdruck an der Grenze Membran/Medium - P _v O₂-Partialdruck am venösen Capillarende - P _g relativer O₂-Partialdruckabfall im Gewebe - P _v relativer O₂-Partialdruckabfall am venösen Capillarende - R Radius der ebenen kreisförmigen Elektrode - RB Randbedingung - RDF, RDF restlicher Diffusionsfehler einfacher und doppelter Membranen - r _o Radius der Elektrode mit halbkugelförmiger Pt-Oberfläche - r, z Zylinderkoordinaten - r _K Capillarradius - S Sättigungsabfall im Capillarblut ohne Elektrode - S Sättigungsabfall im Capillarblut mit Elektrode - u O₂-Konzentration - V Diffusionsgesamtfluß - V _K Diffusionsfluß aus einem Capillarabschnitt - v _r ,v _z Komponenten des Stromdichtevektors inr- bzw.z-Richtung (Zylinderkoordinaten) - mittlere Stromdichte - Stromdichtevektor des Flusses der O₂-Moleküle - v _c konstante Geschwindigkeit des bewegten Mediums - x, y, z Kartesische Koordinaten - Integrationsvariable - ² Laplace-Operator - partielle Ableitung nach der Zeit     

TPA-enhanced motility and invasion in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1: selective role of PKC-α and its inhibition by a combination of PDBu-induced PKC downregulation and antisense oligonucleotides treatment

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced invasiveness was associated with augmentation of cell motility but not that of metalloproteinase activity in a highly metastatic variant (L-10) of the human colon adenocarcinoma cell line RCM-1 and that this enhancement was possibly mediated by protein kinase C (PKC). In this study, we first intended to determine the specific isoforms of PKC involved in this TPA-enhanced L-10 cell motility that leads to invasion, and then investigated the way to inhibit the enhanced motility and invasion by using antisense oligodeoxynucleotides (ODN) targeting the isoform. An activator of conventional PKC isoforms (cPKC), thymeleatoxin, enhanced L-10 cell motility and invasion like TPA, and an inhibitor of cPKC, Gö-6976, efficiently inhibited TPA-enhanced motility and invasion. TPA treatment induced a shift of PKC-, but not other isoforms, from the cytosol to the membrane fraction, indicating the activation of the isoform. During the assay period, only activation but not downregulation of PKC- occurred with the low concentration of TPA used in our assays. Antisense ODNs specific for PKC- efficiently reduced its expression at the protein levels and inhibited L-10 cell motility in the absence of TPA. With TPA treatment, however, the remaining PKC- was sufficient for activation leading to enhanced invasion. Only a combination of depletion of PKC by prolonged stimulation with a high concentration of phorbol 12,13 dibutyrate (PDBu) and treatment with antisense ODNs effectively inhibited L-10 cell invasion even in the presence of TPA. These results suggested that downregulation of PKC isoforms by treatment with antisense ODNs alone is insufficient to suppress the isoform-mediated cellular events in the presence of PKC activators, and thus that some additional treatments are necessary for the successful downregulation of them.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Natriumtransport in den proximalen Tubuli und den Sammelrohren bei Variation der Natriumkonzentration im umgebenden Intterstitium

Zusammenfassung An Ratten wurden Mikropunktionsuntersuchungen am proximalen Tubulus und am Sammelrohr bei Durchblutung der peritubulären Capillaren bzw. Vasa recta sowie bei künstlicher Perfusion dieser Blutgefäße durchgeführt. In Abhängigkeit von der Höhe der interstitiellen Natriumkonzentration wurden der Nettonatriumtransport (_{Na iso}) bei gleicher Natriumkonzentration zu beiden Seiten der Tubuluswand und die Gleichgewichtskonzentrationsdifferenz (c _Na) bei fehlendem Nettovolumen- und Nettosubstanzfluß gemessen.Bei Variation der Natriumkonzentration im Gewebe durch Perfusion der peritubulären Capillaren mit 155 bzw. 300 mÄq/l Na änderten sich weder _{Na iso} noch c _Na für den proximalen Tubulus (_{Na iso} 8,4 bzw. 7,9·10^–5 Äq · mm^–2 · sec^–1; c _Na 24 bzw. 24 mÄq/l). Veränderung der Natriumkonzentration im Blut durch Infusion hypertoner NaCl Lösung oder Peritonealdialyse mit isotoner Mannitlösung führten zu prinzipiell gleichen Ergebnissen. Bei Perfusion der Vasa recta mit Lösungen, die 145 und 300 mÄq/l Natrium entheilten, blieben _{Na iso} wie c _Na über die Sammelrohrwand ebenfalls konstant (_{Na iso} 4,1 bzw. 4,1·10^–5 Äq · mm^–2 · sec^–1; c _Na 98 bzw. 104 mÄq/l). Proximale Tubuli und Sammelrohre verhalten sich demnach bei Variation der interstitiellen Natriumkonzentration gleich.Da die Wasserresorption aus den Sammelrohren von dem durch Gegenstrommultiplikation erzeugten Natriumkonzentrationsanstieg im Markinterstitium abhängt, die Natriumresorption aus den Sammelrohren aber wie die vorliegenden Befunde zeigen von eben dieser Natriumkonzentration unabhängig ist, ist dem Warmblüterorganismus die Möglichkeit gegeben, Natrium- und Wasserresorption unabhängig voneinander zu variieren. Die Natrium- und Wasserresorption aus den Sammelrohren werden jedoch beide durch den Gehalt der Sammelrohrflüssigkeit an permeablen Nichtelektrolyten, wie z. B. Harnstoff, beeinflußt.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.