Molekulare Charakterisierung des Gens des Sporenwandproteins von Encephalitozoon hellem durch inverse und verankerte Polymerasekettenreaktion

Der labordiagnostische Nachweis von Mikrosporidieninfektionen bereitet noch immer Probleme. Aufgrund ihrer geringen Größe und ihrer unspezifischen Färbeeigenschaften ist der lichtmikroskopische Direktnachweis schwierig. Der elektronenmikroskopische Nachweis ist aufwendig und insbesondere aus Stuhl wenig sensitiv. Nicht alle Mikrosporidien können in Zellkulturen vermehrt werden. Die beim Menschen häufigste Spezies, Enterocytozoon bieneusi, ist nicht kultivierbar. Molekularbiologische Nachweisverfahren wie die PCR sind ebenfalls aufwendig, noch nicht standardisiert und derzeit noch Speziallaboratorien vorbehalten. Zuverlässige immundiagnostische Tests mit ausreichender Sensitivität und Spezifität fehlen insbesondere für E. bieneusi, da aufgrund der fehlenden Kultivierbarkeit die für eine Testentwicklung nötige Menge und Reinheit des Antigens kaum zu erzielen ist. Gene, durch deren rekombinante Expression dieses Problem gelöst werden könnte, sind bisher noch nicht bekannt. Dabei wäre zum Beispiel die Entwicklung eines Koproantigen-ELISA zum Nachweis von E. bieneusi aus Stuhl von großem praktischem Nutzen. Ziel der hier vorgelegten Arbeit war es, die Voraussetzungen hierfür zu verbessern. Als aussichtsreichstes Antigen wurde das so genannte „Sporenwandprotein“ (SWP) gewählt, der Hauptbestandteil der Sporenwand von Mikrosporidien. In eigenen Vorarbeiten konnte gezeigt werden, dass es allerdings wenig aussichtsreich ist, alleine anhand der bis dahin ausschließlich für Encephalitozoon cuniculi und E. intestinalis bekannten SWP-Gensequenzen PCR-Primer zur Amplifikation des homologen Gens aus E. bieneusi zu konstruieren. Deshalb wurde in der vorliegenden Arbeit zunächst der bis dahin unbekannte, vollständige kodierende Bereich des SWP-Gens einer weiteren Encephalitozoon-Spezies, E. hellem, einschließlich benachbarter Genabschnitte auf Nukleinsäureebene charakterisiert. Durch die Vergleichsmöglichkeit mit dem SWP-Gen einer dritten Mikrosporidienart wurden die Voraussetzungen zur Konstruktion „universeller“ Primer, die homologe Abschnitte des wahrscheinlich auch in E. bieneusi vorhandenen SWP-Gens amplifizieren können, verbessert. Ein weiteres Ziel, das in dieser Arbeit erreicht wurde, war bei dieser Charakterisierung ganz auf das Anlegen von Banken zu verzichten. Im Hinblick auf die fehlende Kultivierbarkeit von E. bieneusi und die damit verbundene Schwierigkeit, das Ausgangsmaterial für genomische oder cDNA-Banken (genomische DNA bzw. mRNA) in ausreichender Menge und Reinheit zu gewinnen, sollte nämlich bewiesen werden, dass es möglich ist, das SWP-Gen einer Mikrosporidienart alleine durch die Anwendung von PCR-Techniken bestimmen zu können. Als Ausgangspunkt für die Charakterisierung des SWP-Gens aus E. hellem war die Amplifizierung eines ersten Genfragments. Dies konnte durch die Konstruktion so genannter „degenerierter“ Primer (Primermischungen) erreicht werden. Nach Kenntnis dieser ersten E. hellem-spezifischen DNA-Sequenz konnten anschließend Primer konstruiert werden, die vollständig zum SWP-Gen von E. hellem passten. In einer „klassischen“ PCR können jedoch nur DNA-Abschnitte zwischen bekannten Bereichen amplifiziert werden, da diese zur Konstruktion der beiden PCR-Primer bekannt sein müssen. Um dennoch die DNA-Sequenz aus dem ersten Genfragment nach beiden Seiten („upstream“ und „downstream“) verlängern zu können, wurden „inverse“ und „verankerte“ PCR-Reaktion als Spezialtechniken eingesetzt und durch Kombination mit bekannten Techniken („geschachtelte“, „halb-geschachtelte“ und „Touch Down“-PCR) optimiert. Schließlich wurden in der vorliegenden Arbeit aus der Primärstruktur (Nukleotidabfolge) ableitbare Eigenschaften des SWP-Gens von E. hellem diskutiert. Durch Sequenzvergleich mit den bereits bekannten SWP-Genen aus E. cuniculi und E. intestinalis wurden zwei konservierte Loci identifiziert, die zur Konstruktion „universeller“ SWP-Primer vorgeschlagen werden. In der vorliegenden Arbeit wurde somit (a) das bis dahin unbekannte Gen des Sporenwandproteins aus E. hellem erstmals auf Nukleotidebene charakterisiert, (b) bewiesen, dass die Charakterisierung eines unbekannten SWP-Gens durch ausschließliche Anwendung von PCR-Spezialtechniken und ohne die Herstellung von genomischen oder cDNA-Banken möglich ist und (c) Methoden der „inversen“ und der „verankerten“ PCR in der Arbeitsgruppe etabliert und optimiert

Pharmacological Modulation of Mucosa-Related Impairment of β-Adrenoceptor-Mediated Relaxation in Human Detrusor

Objectives: The mucosa of human detrusor strips impairs catecholamine-induced relaxation. In order to elucidate which signal transduction pathways are involved in this cross talk between the mucosa and detrusor, we have studied the effects of several pharmacological agonists and antagonists on noradrenaline-mediated relaxation in intact and mucosa-denuded detrusor strips. Patients and Methods: Strips of detrusor tissue were obtained from patients who had undergone cystectomy for bladder cancer and were set up for force measurement. KCl- or carbachol-precontracted strips were relaxed with increasing concentrations of noradrenaline in the absence and in the presence of nitric oxide synthase inhibitor, L-NAME; P2X-receptor antagonist, PPADS; ET A -receptor antagonist, BQ-123; ET B -receptor antagonist, BQ-788; cyclooxygenase inhibitor, diclofenac; AT 1 -receptor antagonist, candesartan; and NK 1 -receptor antagonist, L-703,606. Results: In intact strips, KCl-stimulated force was enhanced by all blockers; carbachol-stimulated force increased with L-703,606. In denuded strips, only L-NAME augmented the KCl-stimulated contraction. Noradrenaline relaxed the precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. L-NAME, PPADS and BQ-123/BQ-788 had little effect on noradrenaline-induced relaxation, whereas diclofenac, candesartan and L-703,606 sensitized intact carbachol-stimulated detrusor strips to noradrenaline-induced relaxation. Conclusion: Inhibition of the noradrenaline-induced relaxation of precontracted human detrusor strips by the mucosa is attenuated by diclofenac, candesartan and L-703,606 suggesting the involvement of prostanoids, angiotensin and neurokinin pathways. Further experiments are required to unravel the exact mechanisms

Is the Post-Radical Prostatectomy Gleason Score a Valid Predictor of Mortality after Neoadjuvant Hormonal Treatment?

Purpose: To evaluate the validity of the Gleason score after neoadjuvant hormonal treatment as predictor of diseasespecific mortality after radical prostatectomy. Patients and Methods: A total of 2,880 patients with a complete data set and a mean follow-up of 10.3 years were studied; 425 of them (15%) had a history of hormonal treatment prior to surgery. The cumulative incidence of deaths from prostate cancer was determined by univariate and multivariate competing risk analysis. Cox proportional hazard models for competing risks were used to study combined effects of the variables on prostate cancer-specific mortality. Results: A higher portion of specimens with a history of neoadjuvant hormonal treatment were assigned Gleason scores of 8–10 (28 vs. 17%, p < 0.0001). The mortality curves in the Gleason score strata <8 vs. 8–10 were at large congruent in patients with and without neoadjuvant hormonal treatment. In patients with neoadjuvant hormonal treatment, a Gleason score of 8–10 was an independent predictor of prostate cancer-specific mortality; the hazard ratio was, however, somewhat lower than in patients without neoadjuvant hormonal treatment. Conclusion: This study suggests that the prognostic value of the post-radical prostatectomy Gleason score is not meaningfully jeopardized by heterogeneous neoadjuvant hormonal treatment in a routine clinical setting

The muscarinic receptor antagonist propiverine exhibits α1-adrenoceptor antagonism in human prostate and porcine trigonum

Combination therapy of male lower urinary tract symptoms with α(1)-adrenoceptor and muscarinic receptor antagonists attracts increasing interest. Propiverine is a muscarinic receptor antagonist possessing additional properties, i.e., block of L-type Ca(2+) channels. Here, we have investigated whether propiverine and its metabolites can additionally antagonize α(1)-adrenoceptors. Human prostate and porcine trigone muscle strips were used to explore inhibition of α(1)-adrenoceptor-mediated contractile responses. Chinese hamster ovary (CHO) cells expressing cloned human α(1)-adrenoceptors were used to determine direct interactions with the receptor in radioligand binding and intracellular Ca(2+) elevation assays. Propiverine concentration-dependently reversed contraction of human prostate pre-contracted with 10 μM phenylephrine (-log IC(50) [M] 4.43 ± 0.08). Similar inhibition was observed in porcine trigone (-log IC(50) 5.01 ± 0.05), and in additional experiments consisted mainly of reduced maximum phenylephrine responses. At concentrations ≥1 μM, the propiverine metabolite M-14 also relaxed phenylephrine pre-contracted trigone strips, whereas metabolites M-5 and M-6 were ineffective. In radioligand binding experiments, propiverine and M-14 exhibited similar affinity for the three α(1)-adrenoceptor subtypes with -log K (i) [M] values ranging from 4.72 to 4.94, whereas the M-5 and M-6 did not affect [(3)H]-prazosin binding. In CHO cells, propiverine inhibited α(1)-adrenoceptor-mediated Ca(2+) elevations with similar potency as radioligand binding, again mainly by reducing maximum responses. In contrast to other muscarinic receptor antagonists, propiverine exerts additional L-type Ca(2+)-channel blocking and α(1)-adrenoceptor antagonist effects. It remains to be determined clinically, how these additional properties contribute to the clinical effects of propiverine, particularly in male voiding dysfunctio

On the problem of supersonic gas flow in two-dimensional channel with the oscillating upper wall

In the present paper we solve the problem of supersonic gas flow in two-dimensional channel with the moving upper wall making oscillations according to the harmonic law. In order to get a numerical solution for gas dynamics equations we have implemented a difference scheme with space and time approximation of the first order and one with space approximation of the second order. Depending on a type of harmonic law and initial gas inflow conditions, the peculiarities of angle-shock wave propagation in moving curvilinear domains have been investigated. It has been determined that the increase of oscillation amplitude causes the increase of shock wave intensity. It has been shown that under particular oscillation amplitude the moving wall has practically no effect on the flow within the domain

β-Adrenoceptor-Mediated Relaxation of Carbachol-Pre-Contracted Mouse Detrusor

Aims: To study the β-adrenoceptor subtypes involved in the relaxation responses to (-)-isoprenaline in carbachol-pre-contracted (CCh) mouse detrusor muscle with intact and denuded mucosa. Methods: Isolated muscle strips from the urinary bladder of male C57BL6 mice or β2-adrenoceptor knockout mice were pre-contracted with CCh, 1 µM and relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin. For estimating the β-AR subtypes involved, subtype-selective receptor blockers were used, that is, CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Unlike in KCl-pre-contracted muscle, the mucosa did not affect the sensitivity of the relaxation response to (-)-isoprenaline in CCh-pre-contracted murine detrusor strips. Increasing concentrations of (-)-isoprenaline produced a biphasic concentration-relaxation response without any difference both during the presence and absence of mucosa. The relaxation fraction produced by low (-)-isoprenaline concentrations was mediated by β2-AR as evidenced by a shift of the concentration-response curve to higher concentrations with ICI 118,551, but not with CGP 20712A and L748,337, and by the absence of this fraction in β2-AR-KO mice. The relaxation response with low sensitivity to (-)-isoprenaline was not affected by any of the β-AR subtype-selective blockers and was the only response detected in detrusor strips from β2-AR-KO mice. Conclusions: In CCh-pre-contracted mouse detrusor, β2-ARs are responsible for the relaxation component with high sensitivity to (-)-isoprenaline as indicated by the conversion of a biphasic into a monophasic CRC with ICI 118,551 or by its absence in β2-AR KO mice. The mucosa does not impair relaxation under these conditions.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

beta-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in beta 2-Adrenoceptor Knockout Mice

Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation

β-adrenoceptor-mediated relaxation of urinary bladder muscle in β2-adrenoceptor knockout mice

Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation