Adrenal venous sampling in primary hyperaldosteronism: correlation of hormone indices and collimated C-arm CT findings.

PURPOSE: To evaluate the feasibility and effect of an approach to adrenal venous sampling (AVS) analysis by combining established selective cortisol and aldosterone indices with the acquisition of a collimated C-arm CT(CACTColl). METHODS: Overall, 107 consecutive patients (45f,62 m; 54 ± 10 years) undergoing 111 AVS procedures without hormonal stimulation from 7/13 to 2/20 in a single institution were retrospectively analysed. Hormone levels were measured in sequential samples of the suspected adrenal veins and right iliac vein, and selectivity indices (SI) computed. Stand-alone SICortisol and/or SIAldosterone ≥ 2.0 as well as SICortisol and/or SIAldosterone ≥ 1.1 combined with positive right-sided CACTColl of the adrenals (n = 80; opacified right adrenal vein) were defined as a successful AVS procedure. Radiation exposure of CACT was measured via dose area product (DAP) and weighed against an age-/weight-matched cohort (n = 66). RESULTS: Preliminary success rates (SICortisol and/or SIAldosterone ≥ 2.0) were 99.1% (left) and 72.1% (right). These could be significantly increased to a 90.1% success rate on the right, by combining an adjusted SI of 1.1 with a positive CACTColl proving the correct sampling position. Sensitivity for stand-alone collimated CACT (CACTColl) was 0.93, with 74/80 acquired CACTColl confirming selective cannulation by adrenal vein enhancement. Mean DAPColl_CACT measured 2414 ± 958 µGyxm2, while mean DAPFull-FOV_CACT in the matched cohort measured 8766 ± 1956 µGyxm2 (p < 0.001). CONCLUSION: Collimated CACT in AVS procedures is feasible and leads to a significant increase in success rates of (right-sided) selective cannulation and may in combination with adapted hormone indices, offer a successful alternative to previously published AVS analysis algorithms with lower radiation exposure compared to a full-FOV CACT.

[Piritramide : A critical review]. / Piritramid : Eine kritische Übersicht.

BACKGROUND: In many European countries and particularly in Germany, piritramide is the first choice opioid analgesic for the management of postoperative and posttraumatic pain. OBJECTIVE: The aim of this study was to review the pharmacological properties of piritramide and to evaluate whether these result in any clinical advantages with respect to effectiveness, safety and side effect profile compared to other strong opioids. MATERIAL AND METHODS: A systematic literature search was conducted in PubMed and Google Scholar and 27 articles published between 1961 and 2015 were retrieved and included in this review. RESULTS: Piritramide is a strong opioid that can only be administered parenterally. After intravenous injection it is effective after 17 min with pain relief lasting for up to 6 h. It is metabolized in the liver to inactive compounds, which is advantageous compared to morphine where active metabolites can accumulate in patients with renal failure. Piritramide is highly lipophilic resulting in a long context-sensitive half-life, making it unsuitable for continuous infusions. Studies further suggest that the side effect profile of piritramide is comparable to morphine. CONCLUSION: So far there is little evidence to support the widespread use of piritramide as first-line opioid analgesic for postoperative pain management in Germany. Especially lacking are in-depth studies about its mechanisms of action, receptor pharmacology, dose-response relationships and clinical dosing regimens. It is therefore questionable why piritramide is given priority.

Ric-8: different cellular roles for a heterotrimeric G-protein GEF.

Signaling via heterotrimeric G-proteins is evoked by agonist-mediated stimulation of seven transmembrane spanning receptors (GPCRs). During the last decade it has become apparent that Gα subunits can be activated by receptor-independent mechanisms. Ric-8 belongs to a highly conserved protein family that regulates heterotrimeric G-protein function, acting as a non-canonical guanine nucleotide exchange factors (GEF) over a subset of Gα subunits. In this review we discuss the roles of Ric-8 in the regulation of diverse cell functions, emphasizing the contribution of its multiple domain protein structure in these diverse functions.

Cloning and spatiotemporal expression of RIC-8 in Xenopus embryogenesis.

RIC-8 is a highly conserved protein that promotes G protein signaling as it acts as a Guanine nucleotide Exchanging Factor (GEF) over a subset of Gα subunits. In invertebrates, RIC-8 plays crucial roles in synaptic transmission as well as in asymmetric cell division. As a first step to address further studies on RIC-8 function in vertebrates, here we have cloned a ric-8 gene from Xenopus tropicalis (xtric-8) and determined its spatiotemporal expression pattern throughout embryogenesis. The xtric-8 transcript is expressed maternally and zygotically and, as development proceeds, it shows a dynamic expression pattern. At early developmental stages, xtric-8 is expressed in the animal hemisphere, whereas its expression is later restricted to neural tissues, such as the neural tube and the brain, as well as in the eye and neural crest-derived structures, including those of the craniofacial region. Together, our findings suggest that RIC-8 functions are related to the development of the nervous system.

G(alpha)s levels regulate Xenopus laevis oocyte maturation.

Progesterone, produced by follicular cells, induces Xenopus laevis oocyte maturation through a very early event that inhibits the activity of the adenylyl cyclase effector system. The participation of a G-protein has been implicated, based on the fact that the inhibitory effect of the steroid is GTP-dependent, and it has been proposed that progesterone acts interfering with G(alpha)s function at the plasma membrane. Here we investigate whether the change in oocyte G(alpha)s levels affects the maturation process induced by progesterone. Overexpression of X. laevis wild type (wt) G(alpha)s and the constitutive activated G(alpha)s(QL) mutant, both blocked progesterone-induced maturation, G(alpha)s(QL) being much more effective than the wt protein. On the other hand, depletion of G(alpha)s, by the use of antisense oligonucleotides, caused spontaneous maturation measured as MAPK activation, indicating clearly that the presence of G(alpha)s is necessary to keep oocytes arrested. Overexpression of three different G-protein coupled receptors (GPCR), the beta2-adrenergic receptor and the m4 and m5 muscarinic receptors, all caused inhibition of MAPK activation induced by progesterone. These receptors, upon their activation with the respective ligands, might be inducing the release of G(beta)gamma from their respective G(alpha), which together with endogenous G(alpha)s-GTP, activate adenylyl cyclase. Our results indicate that G(alpha)s plays an important role in the maturation process and support previous findings of G(beta)gamma participation, suggesting the presence of a mechanism where a constitutively activated G(alpha)s subunit, together with the G(beta)gamma heterodimer, both maintain high levels of intracellular cAMP levels, blocking the G2/M transition.

Clinical performance of a specific algorithm to reconfirm self-terminating ventricular arrhythmias in current implantable cardioverter-defibrillators.

Inappropriate shock therapy is a frequent problem in patients with implantable cardioverter-defibrillators (ICDs), caused mostly by supraventricular rhythms. Self-terminating ventricular arrhythmias (STVAs), however, may also lead to inappropriate shock discharges even in ICDs with abortive shock capabilities. The aim of this study was to evaluate the clinical performance of a specific ventricular tachycardia/ventricular fibrillation (VT/VF) reconfirmation algorithm implemented in current ICD devices from Medtronic to prevent inappropriate shock discharges due to STVAs. A total of 161 STVA episodes were documented in 59 of 150 patients (39%) within a mean follow-up of 30 +/- 20 months and resulted in 25 inappropriate shock discharges in 15 of 150 patients (10%) despite activation of the reconfirmation algorithm. The first synchronization interval of the algorithm was met in 92% of STVA episodes with and even 38% of STVA episodes without shock delivery. A reduced incidence of inappropriate shocks due to STVAs was found with tachycardia/fibrillation detection intervals (TDI/FDI) programmed to shorter cycle lengths < or =280 ms or the use of the first 2 cycles after the end of charging to be considered for reconfirmation only. Thus, inappropriate shocks due to STVAs still occur in 10% of patients with ICDs despite activation of a specific VT/VF reconfirmation algorithm, and are mainly caused by meeting the first synchronization interval that therefore should be shortened in cycle length. Moreover, to reduce the likelihood of inappropriate shocks, the VF reconfirmation algorithm should be optimized by basing the synchronization intervals exclusively on the FDI with short cycle lengths or using the first 2 cycles for reconfirmation only.

Genetic imbalances in precursor lesions of endometrial cancer detected by comparative genomic hybridization.

Endometrial hyperplasia is regarded as a precursor lesion of endometrioid adenocarcinomas of the endometrium. The genetic events involved in the multistep process from normal endometrial glandular tissue to invasive endometrial carcinomas are primarily unknown. We chose endometrial hyperplasia as a model for identifying chromosomal aberrations occurring during carcinogenesis. Comparative genomic hybridization (CGH) was performed on 47 formalin-fixed, paraffin-embedded specimens of endometrial hyperplasia using the microdissection technique to increase the number of tumor cells in the samples and reduce contamination from normal cells. CGH analysis revealed that 24 out of 47 (51%) samples had detectable chromosomal imbalances, whereas 23 (49%) were in a genetically balanced state. The incidence of aberrant CGH profiles tended to parallel dysplasia grade, ranging from 22% aberrant profiles in simple hyperplasia to 67% in complex hyperplasia with atypia. The most frequent imbalances were 1p, 16p, and 20q underrepresentations and 4q overrepresentations. Copy number changes in 1p were more frequent in atypical complex hyperplasia than in complex lesions without atypical cells or simple lesions (42% versus 20% and 0%). Our results show that endometrial hyperplasia reveals recurrent chromosomal imbalances which tend to increase with the presence of atypical cells. The most frequent aberrations in endometrial cancer, 1q and 8q overrepresentations, are not present or are rare in its precursor lesions. This analysis provides evidence that tumorigenesis proceeds through the accumulation of a series of genetic alterations and suggests a stepwise mode of tumorigenesis.

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

Functional characterization of an extremely thermophilic ATPase in membranes of the crenarchaeon Acidianus ambivalens.

A plasma membrane-bound adenosine triphosphatase with specific activities up to 0.2 micromol min(-1) (mg protein)(-1) at 80 degrees C was detected in the thermoacidophilic crenarchaeon Acidianus ambivalens (DSM 3772). The enzymatic activity exhibited a broad pH-optimum in the neutral range with two suboptima at pH 5.5 and 7.0, respectively. Sulfite activation resulted in only one pH optimum at 6.25. In the presence of the divalent cations Mg2+ and Mn2+ the ATPase activity was maximal. Remarkably, the hydrolytic rates of GTP and ITP were substantially higher than for ATP. ADP and pyrophosphate were only hydrolyzed with small rates, whereas AMP was not hydrolyzed at all. Both activities could be weakly inhibited by the classical F-type ATPase inhibitor N,N'-dicyclohexylcarbodiimide, whereas azide had no influence at all. The classical inhibitor of V-type ATPases, nitrate, also exerted a small inhibitory effect. The strongly specific V-type ATPase inhibitor concanamycin A, however, showed no effect at all. The P-type ATPase inhibitor vanadate had no inhibitory effect on the ATPase activity at pH 7.0, whereas a remarkable inhibition at high concentrations could be observed for the activity at pH 5.5. Arrhenius plots for both membrane bound ATPase activities were linear up to 95 degrees C, reflecting the enormous thermostability of the enzyme.

Detection of myocardial injury during transvenous implantation of automatic cardioverter-defibrillators.

OBJECTIVES: The present study was designed to assess the extent of myocardial injury in patients undergoing transvenous implantation of an automatic implantable cardioverter-defibrillator (ICD) using cardiac troponin I (cTNI), which is a highly specific marker of structural cardiac injury. BACKGROUND: During ICD implantation, repetitive induction and termination of ventricular fibrillation (VF) via endocardial direct current shocks is required to demonstrate the correct function of the device. Transthoracic electrical shocks can cause myocardial cell injury. METHODS: Measurements of total creatine kinase (CK), CK-MB, myoglobin, cardiac troponin T (cTNT) and cTNI were obtained before and after ICD implantation in 49 consecutive patients. Blood samples were drawn before and 2, 4, 8, and 24 h after implantation. RESULTS: Elevations of CK, CK-MB, myoglobin, cTNT and cTNI above cut-off level were found in 25%, 6%, 76%, 37% and 14% of patients, respectively, with peak cTNI concentrations ranging from 1.7 to 5.5 ng/ml. Cumulative defibrillation energy (DFE), mean DFE, cumulative VF time, number of shocks as well as prior myocardial infarction (MI) were found to be significantly related to a rise of cTNI. Mean DFE > or = 18 J and a recent MI were identified as strong risk factors for cTNI rise. CONCLUSIONS: During transvenous ICD implantation myocardial injury as assessed by cTNI rise occurs in about 14% of the patients. Peak cTNI concentrations are only minimally elevated reflecting subtle myocardial cell damage. Patients with a recent MI and a mean DFE > or = 18 J seem to be prone to cTNI rise.

The C2 cytosolic loop of adenylyl cyclase interacts with the activated form of G alpha s.

Using the yeast two-hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC). Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed. Interaction between C2 and human G alpha s (hG alpha s) was also detected and was dependent on G alpha s activation. In contrast X. laevis G alpha s (xlG alpha s), which is 92% identical to hG alpha s, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation. Through the construction of chimeras, we demonstrated that the amino-terminal half of xlG alpha s was responsible for the lack of interaction with AC. Chimeras between mouse G alpha i2 and G alpha s (N-mG alpha i2/C-G alpha s), that have previously shown to activate AC to a higher extent than wild-type G alpha s, also interacted with the C2 cytosolic domain and with a higher affinity. Interestingly, N-mG alpha i2/C-xlG alpha s chimera was not only able to interact with C2 but also with the C1 cytosolic domain.

Dual transduction signaling by a Xenopus muscarinic receptor: adenylyl cyclase inhibition and MAP kinase activation.

Using transient transfection of COS-7 and human embryonic kidney 293 cells, we studied the functional properties of a previously cloned muscarinic Xenopus receptor [Herrera et al. (1994): FEBS Lett 352:175-179] and its coupling to adenylyl cyclase (AC) and mitogen-activated protein kinase (MAPK) pathways. Expression of the Xenopus muscarinic receptor results in the inhibition of AC activity and activation of the MAPK pathway through a mechanism that involves a Pertussis-toxin-sensitive G-protein and the G beta gamma subunits. The signal transduction properties of this receptor are similar to the mammalian m2 and m4 muscarinic receptors. These results strongly support the idea that inhibition of AC and MAPK activation, signaled out from the muscarinic oocyte receptor, are involved in the oocyte maturation process.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes.

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).

[The results of strontium-90 contact therapy to prevent the recurrence of pterygium]. / Ergebnisse der Strontium-90-Kontakttherapie zur Rezidivprophylaxe des Pterygiums.

AIM: Aim of the study was the evaluation of the role of adjuvant radiation therapy in the prevention of recurrence after excision. PATIENTS AND METHODS: Between July 1, 1985 and April 1, 1993, 64 patients (43 male, 21 female) were referred to radiation therapy after excision of a nasal pterygium. Radiation therapy was done with a strontium-90 eye applicator and a total dose of 30 Gy, fractionated in 6 fractions of 5 Gy each, 3 times a week. Forty-nine patients were treated primarily, 15 patients underwent radiation therapy for the first time in case of recurrent pterygium after multiple re-excisions. All patients had a following of 1 to 9 years with a median of 5.5 years. RESULTS: In 8 of 64 irradiated patients recurrent pterygium was detected (12.5%). Differentiated into the 2 groups 4 of the primarily treated patients had recurrent pterygium (8.16%), the other 4 were in the group with multiple former re-excisions (26.7%). With regard to the initiation of the irradiation after surgery pterygium did not recur in any of the primarily treated patients who were irradiated in between 3 days after surgery. In contrary 3 of 7 primarily treated patients (42.9%) who started radiation therapy between 7 and 10 days after surgery had recurrent pterygium. For the patients with primarily recurrent pterygium no dependence of the initiation of radiation therapy after surgery could be detected. CONCLUSIONS: Adjuvant radiation therapy after excision of pterygium lowers the rate of recurrence from about 40% to 12.5%, in a primarily adjuvant situation to 8.16%. In these patients radiation therapy should be initiated within 3 days after surgery. Patients with primarily recurrent pterygium have an elevated risk of recurrence independently of the initiation of radiation therapy.

Results of adjuvant radiation therapy after surgical excision of pterygium.

The aim of the present study was the evaluation of the role of adjuvant radiation therapy in the prevention of recurrence after excision. Between July 1, 1985, and April 1, 1993, 64 patients (43 men, 21 women) were referred for radiation therapy after excision of a nasal pterygium. All patients were followed for 1-9 years (median 5.5 years). Radiation therapy was done with a strontium 90 eye applicator at a total dose of 30 Gy fractionated into six fractions of 5 Gy each. In all, 49 patients were treated after their first excision and 15 patients had undergone multiple prior excisions. In 8 of 64 irradiated patients, recurrent pterygium was detected (12.5%); 4 recurrences developed after first excision and adjuvant radiation therapy (8.16%) and the other 4, following multiple former reexcisions and radiotherapy (26.7%). Pterygium did not recur in any of the primarily treated patients who were irradiated within 3 days of surgery. In contrast, 3 of 7 accordingly treated patients (42.9%) who started radiation therapy at between 7 and 10 days after surgery developed recurrent pterygium. Adjuvant radiation therapy after excision of pterygium lowers the overall rate of recurrence to 12.5%. In an adjuvant situation after first excision, radiation therapy should be initiated within 3 days of surgery, the result being freedom from recurrence.

Site-directed mutants of the beta subunit of protein kinase CK2 demonstrate the important role of Pro-58.

The following amino acids of the Xenopus laevis beta subunit of protein kinase CK2 (casein kinase 2) were changed to alanine: Pro-58 (beta P-->A); Asp-59 and Glu-60 and Glu-61 (beta DEE-->AAA); His-151-153 (beta HHH-->AAA). The last 37 amino acids of the carboxyl end were deleted (beta delta 179-215). Stimulation of CK2 alpha catalytic subunit activity was measured with casein as substrate and the following relative activities were observed: beta P-->A > beta DEE-->AAA >>> beta WT > beta HHH-->AAA >>> beta delta 179-215. The beta DEE-->AAA and beta P-->A were similar to beta WT in reducing CD2 alpha binding to DNA but beta delta 179-215 was less active. The results indicate that both Pro-58 and the surrounding acidic cluster play roles in dampening the activation of CK2 alpha and that the carboxyl end of beta is involved in the interaction with CK2 alpha.

Activity of recombinant alpha and beta subunits of casein kinase II from Xenopus laevis.

Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metal ions on the activity of casein kinase II from Xenopus laevis.

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

The cDNAs coding for the alpha- and beta-subunits of Xenopus laevis casein kinase II.

Using a lambda gt10 cDNA library obtained from Xenopus laevis oocytes and probes derived from the known sequences of the human and Drosophila genes, a cDNA coding for the alpha-subunit of the X. laevis casein kinase II was isolated. The coding sequence of this clone determines a polypeptide of 350 amino acids. The X. laevis sequence is 98% identical to the human and rat proteins in the first 323 amino acids. Using the polymerase chain reaction to generate a 370-nucleotide-long probe, it was possible to clone and sequence a cDNA of 900 nucleotides that coded for the X. laevis beta-subunit of casein kinase II. The derived protein sequence is 215 amino acids long and again shows an extraordinary degree of conservation with other species.