State of transition to Ministry of Health governance of prison healthcare in the Council of Europe region.

OBJECTIVES: This study aimed to examine timebound prison healthcare governance amendments and current structures in Europe two decades after the World Health Organization (WHO) Declaration on Prison Health as part of Public Health adopted in Moscow on 24 October 2003 (Moscow Declaration), which recommended prison health care be closely linked with public health systems to ensure quality prison health care, connected health surveillance, and continuity of care. STUDY DESIGN: We present here a regional evolutionary mapping of the Council of Europe Member State transfer of prison healthcare governance to the auspices of the Ministry of Health. METHODS: The European Committee for the Prevention of Torture database and WHO Regional Office for Europe Health In Prison European Database were scrutinised for Council of Europe (CoE) Member State status regarding the Ministry responsible for prison healthcare governance and if this had changed since the adoption of the Moscow Declaration in 2003. RESULTS: As of October 2023, completed transfer of governance to the Ministry of Health nationally is documented in 13 CoE Member States and in one CoE Member State candidate (Kosovo). Partial transfer is documented in Spain (Catalonia and Basque Autonomous Community) and Switzerland (cantons of Geneva, Valais, Vaud, Neuchatel, and Basel-Stadt). Three CoE Member States operate joint governance of prison health care between Ministries (Malta, Portugal, Türkiye). Transfer is a lengthy process (up to 10 years). CONCLUSIONS: Successful transition requires political commitment, cooperation, needs assessment, resourcing, and evaluation. Monitoring of cost and prison healthcare standards, due process for complaints, and cooperation with independent/Committee against Torture inspections is critical.

Cognitive impairment in early stages of multiple sclerosis is associated with high cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain.

BACKGROUND AND PURPOSE: Chitinase 3-like 1 (CHI3L1) and neurofilament light chain (NF-L) are promising biomarkers of disability in multiple sclerosis (MS). However, their role in cognitive dysfunction remains elusive. Here, we aimed to correlate cerebrospinal fluid (CSF) levels of CHI3L1 and NF-L with cognitive status in MS. METHODS: Fifty one recently diagnosed patients were cognitively evaluated and CSF was collected. Levels of CHI3L1 and NF-L were determined by ELISA. Spearman's partial correlation coefficient was performed. RESULTS: After adjusting cognitive scores by age, anxiety and EDSS, association was detected between CHI3L1 levels and Trail Making Test A (rs = 0.348; p = 0.016) and between NF-L levels and Word List Generation (rs = -0.324; p = 0.025). CONCLUSION: High levels of CSF CHI3L1 and NF-L are associated with cognitive impairment in the early phases of MS.

Dominant isolated renal magnesium loss is caused by misrouting of the Na(+),K(+)-ATPase gamma-subunit.

Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg(2+) wasting, often associated with disturbances in Ca(2+) excretion. We identified a putative dominant-negative mutation in the gene encoding the Na(+), K(+)-ATPase gamma-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.

[Intramammary use of antibiotics in dairy farms in the canton of Ticino before, during and after Staphylococcus aureus genotype B elimination]. / Intramammärer Antibiotikaeinsatz vor, während und nach einer Staphylococcus aureus Genotyp B Sanierung in Milchkuhbetrieben im Kanton Tessin.

INTRODUCTION: The aim of the study was to evaluate the use of intramammary antibiotics before, during and after the elimination of Staphylococcus aureus genotype B (SAGTB). Data on intramammary antibiotic use was collected in 65 dairy farms as part of a pilot project for SAGTB elimination in the canton of Ticino from 2017 to 2019. The investigated farms were divided into 46 affected farms (with at least one SAGTB-positive animal) and 19 control farms (SAGTB-free farms). Data on antibiotic use were requested from veterinarians and treatment incidence, as a measure of antibiotic use, was calculated based on medical records and veterinary prescriptions. In addition, the treatment incidence was calculated for 47 farms during alpine farming period. In 2018 (elimination year), the mean incidence of treatment during lactation in the SAGTB-positive farms was significantly higher than in the control farms (p=0,003). In 2019 no significantly lower antibiotic use during lactation or dry period was detected between 2017 (before elimination) and 2019 (after elimination). Alpine farming places where only S. aureus genotype B-negative animals had access to had a significantly lower antibiotic use during lactation (p=0,004). The new federal database (Antibiotics Information System in Veterinary Medicine, IS ABV) should allow continuous monitoring and to confirm the reduction of antibiotic use in the coming years.

INTRODUCTION: L'objectif de l'étude était d'évaluer la consommation d'antibiotiques intramammaires avant, pendant et après l'assainissement de Staphylococcus aureus génotype B (SAGTB). Dans le cadre d'un projet pilote d'assainissement SAGTB dans le canton du Tessin, l'étude a recueilli des données sur la consommation d'antibiotiques intramammaires de 2017 à 2019 dans 65 exploitations laitières réparties en 46 exploitations test (avec au moins un animal positif au SAGTB) et 19 exploitations témoins (exploitations exemptes de SAGTB). Les données relatives à la consommation d'antibiotiques ont été demandées aux vétérinaires et, grâce aux enregistrements et aux prescriptions des vétérinaires, il a été possible de calculer l'incidence du traitement en tant que mesure de la consommation d'antibiotiques. En outre, l'incidence des traitements pendant l'alpage a également pu être calculée pour 47 exploitations. En 2018 (année d'assainissement), l'incidence des traitements pendant la lactation était en moyenne significativement plus élevée dans les exploitations SAGTB-positives que pour les exploitations de contrôle (p=0,003). En comparant 2017 (avant l'assainissement) et 2019 (après l'assainissement), il n'y a pas eu de baisse significative de la consommation d'antibiotiques pendant la lactation et le tarissement en 2019. Dans les exploitations d'estivage, on a constaté l'année suivant l'assainissement (2019) une consommation d'antibiotiques significativement plus faible pendant la lactation pour les alpages qui n'accueillaient que des animaux négatifs à S. aureus génotype B (p=0,004). Il faut espérer qu'avec l'aide de la nouvelle base de données fédérale (Système d'information sur les antibiotiques en médecine vétérinaire, IS ABV), le monitoring pourra être poursuivi dans les années à venir et que la réduction de la consommation d'antibiotiques sera confirmée dans les années suivantes.

[Economic importance of antibiotic sale for private food animal practices in Switzerland - example veal production]. / Ökonomische Bedeutung des Umsatzes mit Antibiotika für private Nutztierpraxen in der Schweiz ­ Beispiel Kälbermast.

INTRODUCTION: The use of antibiotics in Swiss veal production is considered an established method for controlling bacterial infectious diseases. Although the veterinary profession aims to ensure animal welfare, the veterinary business income needs to be ensured at the same time. Against the background of increasing problems with resistant pathogens in human and veterinary medicine, the use of antibiotics should be significantly reduced and used more selectively. The associated economic consequences for food animal practitioners are unknown. The aim of this study was to determine the economic importance of antibiotic sale volume for private food animal practitioners in veal production. An anonymized questionnaire was sent to 120 mixed veterinary practices in Switzerland, which offered services to veal and beef cattle farmers. Questions involved the pharmaceutical sale volume, details on veterinary invoices from three farms with average, below and above average animal health throughout 2017. Twenty-nine complete questionnaires (response rate: 24.2%) and veterinary invoices of 84 farms were returned. The study is not representative, but it allows a rough assessment of the economic framework in Swiss livestock practice. The majority of the total turnover with livestock farms was generated by the sale of antibiotics (54%). Antibiotic sales per animal were higher as expected in farms with a below-average animal health than in farms with an average or above-average animal health. Consulting services turnover contributed only 0.5% to the total sale volume in veal farming. The results document, that antibiotic reduction measurements in veal and beef production will have economic consequences for veterinary livestock practices. In the medium term, the profitable existence of livestock veterinary practice requires a change to cost based consulting services.

INTRODUCTION: En Suisse, dans le cadre de la pratique de l'engraissement des veaux, l'utilisation d'antibiotiques est principalement considérée comme une méthode éprouvée pour lutter contre les maladies infectieuses bactériennes. L'activité vétérinaire vise à prévenir ou minimiser les souffrances chez les animaux. Mais pour autant, les vétérinaires restent des entrepreneurs qui doivent assurer un revenu adéquat de leur pratique. Dans un contexte de problèmes croissants rencontrés avec les agents pathogènes résistants en médecine humaine et vétérinaire, l'utilisation d'antibiotiques devrait être considérablement réduite et ciblée. Les conséquences économiques associées pour la pratique vétérinaire rurale sont peu connues. Le but de la présente étude était de déterminer l'importance économique des ventes d'antibiotiques pour les pratiques vétérinaires rurales privées en utilisant l'exemple de l'engraissement des veaux. À cet effet, un questionnaire anonymisé a été envoyé à 120 cabinets vétérinaires mixtes en Suisse, qui s'occupent entre autres d'exploitations d'engraissement de veaux, de broutards et de taurillons. Les participants ont été interrogés sur le niveau des ventes de médicaments. En outre, nous les avons invités à envoyer des factures vétérinaires concernant trois entreprises d'engraissement avec un niveau de santé animale inférieur, égal et supérieur à la moyenne tout au long de l'année 2017. Vingt-neuf questionnaires complets (taux de réponse: 24,2%) ainsi que des factures vétérinaires concernant au total 84 exploitations d'engraissement de veaux, de broutards ou de taurillons nous ont été retournés. L'étude n'est certes pas représentative, mais elle permet une évaluation approximative du cadre économique des pratiques rurales en Suisse. La majorité du chiffre d'affaires total sur les exploitations d'engraissement de veaux, de broutards ou taurillons a été générée par la vente d'antibiotiques (54%). Il apparait que les ventes d'antibiotiques étaient plus élevées dans les exploitations où la santé animale était inférieure à la moyenne que dans les établissements où elle était supérieure ou égale à la moyenne. Le chiffre d'affaires des prestations de conseil ne représentait que 0,5% du chiffre d'affaires total dans le domaine des veaux. Ces résultats montrent que les mesures visant à réduire les antibiotiques dans les conditions actuelles de production de veau et de boeuf auront probablement des conséquences économiques non négligeables sur la part des exploitations d'engraissement dans les revenus des pratiques vétérinaire rurales. A moyen terme, afin d'assurer la rentabilité d'une pratique rurale, une nouvelle orientation de l'activité vétérinaire sera nécessaire: la mise en place d'un service de conseils payants pour les exploitations d'engraissement, ayant pour but l'établissement de concepts de prévention.

A novel breast tissue density classification methodology.

It has been shown that the accuracy of mammographic abnormality detection methods is strongly dependent on the breast tissue characteristics, where a dense breast drastically reduces detection sensitivity. In addition, breast tissue density is widely accepted to be an important risk indicator for the development of breast cancer. Here, we describe the development of an automatic breast tissue classification methodology, which can be summarized in a number of distinct steps: 1) the segmentation of the breast area into fatty versus dense mammographic tissue; 2) the extraction of morphological and texture features from the segmented breast areas; and 3) the use of a Bayesian combination of a number of classifiers. The evaluation, based on a large kappa = 0.81 and 0.67 for the two data sets) between automatic and expert-based Breast Imaging Reporting and Data System mammographic density assessment.

[Importance of nocturia and its impact on quality of sleep and quality of life in patient with benign prostatic hyperplasia]. / Importancia de la nocturia y su impacto en la calidad del sueño y en la calidad de vida en el paciente con hiperplasia benigna de próstata.

INTRODUCTION: Nocturia is the main cause of insomnia or sleep interruption in adult men, which has a negative impact on daily activities, quality of life (QoL) and quality of sleep (QoS). The assessment of nocturia and its impact on QoL and QoS in patients suffering from benign prostatic hyperplasia (BPH) has been poor in terms of clinical research, moreover there is a lack of specific methods to assess this impact. OBJECTIVES: The objectives of BPH treatment should include the improvement of patient's QoL by controlling both diurnal and nocturnal symptoms. In order to assess how nocturia affects QoL and also QoS, some specific tools, such as N-QoL questionnaire or the number of Hours of Undisturbed Sleep (HUS), have been recently developed. Therefore, it would be interesting to assess how nocturia reduction due to LUTS/BPH treatment can impact on some objective parameters such as HUS and also how nocturia reduction improves QoS and QoL. This assessment should be developed during the application of pharmacological treatments in clinical practice by means of these specific tools. With the aim of tackling nocturia as an urologic problem in patients with LUTS/BPH, as well as knowing the physiology of sleep and the effect of nocturia on the sleep and QoL, a meeting of expert urologists, that gathered about fifty specialists of all around Spain, was held in Madrid. This article presents the main ideas and concepts exposed in this meeting. CONCLUSIONS: Nocturia is a symptom with a high prevalence in older patients with STUI/BPH. The PreNoc study has showed a Nocturia prevalence in Spain of 83% in patients > or =60 years old diagnosed of BPH. Nocturia is the most bothersome symptom in patients with STUI/BPH.

Hematologic disorders associated with primary mediastinal nonseminomatous germ cell tumors.

BACKGROUND: The association between primary germ cell tumors of the mediastinum (the space between the lung pleura that contains the heart and other chest viscera) and hematologic malignancies has been described by retrospective analysis of patients treated at individual clinical centers. To better characterize the risk of hematologic disorders in patients with extragonadal germ cell tumors and to describe the clinical and biologic features of the disorders, we studied an unselected population in a large, international, multicenter database. METHODS: Six hundred thirty-five patients treated at 11 centers in the United States and Europe from 1975 through 1996 were evaluated retrospectively. RESULTS: A hematologic disorder was observed in 17 patients with germ cell tumors. All cases developed among the 287 patients with primary mediastinal nonseminomatous germ cell tumors, giving an incidence rate in this group of 2.0% (95% confidence interval [CI] = 1.1%-3.1%) per year over a median follow-up time of 3 years. The risk of developing hematologic disorders was statistically significantly increased in patients with primary mediastinal nonseminomatous germ cell tumors in comparison with the age-matched general population (standardized incidence ratio = 250; 95% CI = 140-405). The median time to onset of hematologic neoplasia was 6 months (range, 0-47 months), and the median survival after diagnosis of the hematologic disorder was 5 months (range, 0-16 months) (two-sided P<.0001, comparing survival from the time of diagnosis of the germ cell tumor of patients with and without hematologic disorders). CONCLUSION: In our study, approximately one in 17 patients with primary mediastinal nonseminomatous germ cell tumors was affected by a hematologic disorder, whereas no cases were seen among 334 patients with other extragonadal germ cell tumors. The hematologic disorder had a statistically significant impact on prognosis, with none of the 17 reported patients surviving for more than 2 years.

Incidence of metachronous testicular cancer in patients with extragonadal germ cell tumors.

BACKGROUND: The frequency of subsequent testicular cancer (referred to as metachronous testicular cancer) in men who have had previous testicular cancer is relatively high. The rate of metachronous testicular cancer in men with extragonadal germ cell tumors (EGCTs), however, is largely unknown. We conducted a retrospective study of EGCT patients to determine the incidence, cumulative risk, and specific risk factors for metachronous testicular cancers. METHODS: A standardized questionnaire about patient characteristics, the extent of EGCT disease, any second malignancies, and treatments received was completed for 635 patients with EGCTs identified from the medical records of 11 cancer centers in Europe and the United States from 1975 through 1996. Comparisons with age group-specific data from the Saarland, Germany, population-based cancer registry were used to calculate the standardized incidence ratio (SIR). The Kaplan-Meier method was used to analyze survival data and cumulative risk. All statistical tests were two-sided. RESULTS: Sixteen EGCT patients (4.1%) developed metachronous testicular cancers, with a median time between diagnoses of 60 months (range, 14-102 months). The risk of developing metachronous testicular cancers was statistically significantly increased in patients with EGCTs (observed = 16; expected = 0.26; SIR = 62; 95% confidence interval [CI] = 36 to 99) and in subsets of EGCT patients with mediastinal location (SIR = 31; 95% CI = 8 to 59), retroperitoneal location (SIR = 100; 95% CI = 54 to 172), and nonseminomatous histology (SIR = 75; 95% CI = 43 to 123). The cumulative risk of developing a metachronous testicular cancer 10 years after a diagnosis of EGCT was 10.3% (95% CI = 4.9% to 15.6%) and was higher among patients with nonseminomatous EGCTs (14.3%; 95% CI = 6.7% to 21.9%) and retroperitoneal EGCTs (14.2%; 95% CI = 5.6% to 22.8%) than among patients with seminomatous EGCTs (1.4%; 95% CI = 0.0% to 4.2%) and mediastinal EGCTs (6.2%; 95% CI = 0.1% to 12.2%). CONCLUSIONS: Patients with EGCTs, particularly those with retroperitoneal or nonseminomatous tumors, but also those with primary mediastinal EGCTs, are at an increased risk of metachronous testicular cancer.

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.

Cation sidedness in the phosphorylation step of Na+/K+-ATPase.

Na+/K+ -ATPase, reconstituted into phospholipid vesicles, has been used to study the localisation of binding sites of ligands involved in the phosphorylation reaction. Inside-out oriented Na+/K+ -ATPase molecules are the only population in this system, which can be phosphorylated, as the rightside-out oriented as well as the non-incorporated enzyme molecules are inhibited by ouabain. In addition, the right-side-out oriented Na+/K+ -ATPase molecules have their ATP binding site intravesicularly and are thus not accessible to substrate added to the extravesicular medium. Functional binding sites for the following ligands have been demonstrated: (i) Potassium, acting at the extracellular side with high affinity (stimulating the dephosphorylation rate of the E2P conformation) and low affinity (inducing the non-phosphorylating E2K complex). (ii) Potassium, acting at the cytoplasmic side with both high and low affinity. The latter sites are also responsible for the formation of an E2K complex and complete with Na+ for its binding sites. (iii) Sodium at the cytoplasmic side responsible for stimulation of the phosphorylation reaction. (iv) Sodium (and amine buffers) at the extracellular side enhancing the phosphorylation level of Na+/K+ -ATPase where choline chloride has no effect. (v) Magnesium at the cytoplasmic side, stimulating the phosphorylation reaction and inhibiting it above optimal concentrations.

Ouabain- and potassium-induced stimulation of amylase release in fragments and acini of rabbit pancreas.

Ouabain increases the enzyme secretion from the isolated rabbit pancreas and pancreatic fragments, but not from isolated pancreatic acini. The increase occurs after a delay of 45-60 min and is not accompanied by an increase in lactate dehydrogenase release. The stimulatory effect of ouabain (10(-5) M) is dependent on the presence of extracellular calcium, and is not antagonized by 10(-4) M atropin, 10(-4) M propranolol, 10(-5) M phentolamine, 10(-3) M dibutyryl-cyclic GMP, 10(-6) M tetrodotoxin, 10(-4) M verapamil or 10(-4) M D-600. Elevation of the extracellular potassium concentration to 120 mM in the presence of 10(-4) M atropin also increases the enzyme secretion from rabbit pancreatic fragments. The increase is again dependent on the presence of extracellular calcium and is resistant to adrenergic blockade and to tetrodotoxin, verapamil or D-600. Forskolin also stimulates a Ca2+-dependent release of amylase from pancreatic fragments but not from pancreatic acini. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IMX), ouabain (10(-5) M) and K+ (120 mM) cause an immediate increase in the cyclic AMP content of pancreatic fragments which does not occur in the absence of extracellular calcium. In pancreatic acini, the cAMP production is only slightly increased by ouabain. In the absence of IMX, the cAMP levels in fragments or acini are not detectably altered by ouabain or K+. The results suggest that the stimulation of enzyme secretion by ouabain and high K+ is an indirect effect, mediated by the release of an endogenous transmitter from non-cholinergic, non-adrenergic nerves in the intact preparations. The release and/or the effect of the transmitter appears to be mediated primarily by Ca2+ and secondarily by cyclic AMP.

A monoclonal antibody against pig gastric H+/K(+)-ATPase, which binds to the cytosolic E1.K+ form.

Monoclonal antibodies were raised against a purified membrane fraction from hog gastric mucosa containing H+/K(+)-ATPase. The properties of one of these monoclonal antibodies (5B6) were further evaluated. On immunoblot it recognized the 95 kDa peptide of the H+/K(+)-ATPase-rich membrane fraction. The K(+)-ATPase activity was inhibited by 65% under standard assay conditions (pH 7.0). At pH 6.0 and 8.0 this enzyme activity was inhibited by 40% and 100%, respectively. The maximal inhibition in inside-out vesicles was also 65% at pH 7.0. The inhibition was uncompetitive with respect to K+ and noncompetitive with respect to ATP. Mg2(+)-ATPase activity and K(+)-dependent p-nitrophenylphosphatase activity were not influenced. The monoclonal antibody lowered the steady-state phosphorylation level at pH 6.0, 7.0 and 8.0 by 30%, 40% and 60% respectively. The rate of the K(+)-stimulated dephosphorylation step was not inhibited. These findings demonstrate that 5-B6 recognizes the E1.K+ dephosphoenzyme at the cytosolic side.

Binding of unsaturated fatty acids to Na+, K(+)-ATPase leading to inhibition and inactivation.

The effects of free fatty acids on the mechanism of action of Na+, K(+)-ATPase were studied. Unsaturated free fatty acids (palmitoleic acid, oleic acid, linoleic acid and arachidonic acid) inhibited the Na+, K(+)-ATPase activity within a narrow range, while saturated and methylated fatty acids had little or no effect. The following effects of oleic acid were found: (1) The affinity for K+ on the overall ATPase and the p-nitrophenylphosphatase reaction as well as the maximal activities were decreased. (2) The Na(+)-ATPase activity was also inhibited but the '0'-ATPase activity was hardly changed. (3) The steady-state ATP phosphorylation level in the presence of Na+ was not influenced. (4) The dephosphorylation rate constant of the phosphointermediate was slightly decreased, resulting in elevated phosphorylation levels in the absence of Na+. (5) The inhibitory effect of ATP on the dephosphorylation rate was not affected. (6) The K+ sensitivity of the phosphoenzyme in the presence as well as in the absence of Na+ was decreased. (7) Ouabain binding was inhibited. Both the affinity and the number of binding sites were lowered. In addition it was found that Na+, K(+)-ATPase binds oleic acid linearly with the fatty acid concentration up to more than 100 mol oleic acid per mol alpha beta oligomer of Na+, K(+)-ATPase. Prolonged incubation with oleic acid led to irreversible inactivation of the enzyme. This inactivation was dependent on the reaction conditions: ligands, temperature, enzyme concentration, time and fatty acid concentration. The combined presence of inactivation (long term effects) and the effects on the (K(+)-activated) dephosphorylation (short term effects) explain the mixed type inhibition of free fatty acids as observed in assays for K(+)-activated ATPase, K(+)-activated p-nitrophenylphosphatase and ouabain binding. It also explains the sharp inhibition curve in the Na+, K(+)-ATPase activity test.

Transepithelial permeability in the rabbit pancreas.

1. The transepithelial permeability in the isolated rabbit pancreas has been studied with the aid of radioactive markers added to the bathing medium. 2. After addition of these compounds in 2 mM concentration to the medium, they equilibrate within 30 min to a steady-state concentration in the secreted fluid. The latter concentrations, expressed as percent of those in the bathing medium, are: urea 100%, glycerol 90%, erythritol 95%, mannitol 60%, lactose 5%, sucrose 4% and inulin 3%. 3. Addition of 10(-5) M carbachol to the bathing medium after 60 or 90 min of incubation results in an increase of the concentrations of mannitol, lactose sucrose and inulin in the secreted fluid. Maximal concentrations, reached about 35 min after addition of the stimulant, are: mannitol 65%, lactose 31%, sucrose 23%, inulin 8%. 4. No change in the concentration of urea is observed, while the concentrations of glycerol and erythritol increase always to 100% after addition of 10(-5) M carbachol. 5. For sucrose and lactose the increase in permeability appears to be dependent on the concentration of carbachol. 6. There is no increase in the extracellular space for lactose, sucrose and inulin after incubating fragments of the rabbit pancreas with 10(-5) M carbachol. 7. Addition of atropine 5 min or more after carbachol stimulation has no effect on enzyme secretion, but markedly inhibits the increase in sucrose permeability. 8. These results indicate that: (a) the permeability of the transcellular transport route in the isolated rabbit pancreas is determined by the size of the permeating molecules, (b) this route is probably extracellular, (c) its permeability is increased by a cholinergic agent in dose-dependent fashion, (d) the increase in permeability is not caused by the enzyme secretion as such.

Rat pancreas adenylate cyclase. VI. Role of the enzyme in secretin stimulated enzyme secretion.

1. The responsiveness of adenylate cyclase and enzyme secretin for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, but not in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimulating the adenylate cyclase system.

Transport ratios of reconstituted (H+ + K+)-ATPase.

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol vesicles by means of a freeze-thaw-sonication procedure. The passive and active transport mediated by these vesicles were measured (Skrabanja, A.T.P., Asty, P., Soumarmon, A., De Pont, J.J.H.H.M. and Lewin, M.J.M. (1986) Biochim. Biophys. Acta 860, 131-136). To determine real initial velocities, the proteoliposomes were separated from non-incorporated enzyme, by means of centrifugation on a sucrose gradient. The purified proteoliposomes were used to measure active H+ and Rb+ transport, giving at room-temperature velocities of 46.3 and 42.5 mumol per mg per h, respectively. A transport ratio of two cations per ATP hydrolyzed was also measured. These figures indicate that the enzyme catalyzes an electroneutral H+-Rb+ exchange.

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla studies on (Na+ + K+)-activated ATPase, XXXIX.

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas.

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.