Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

Acute autonomic effects of vitamins and fats in male smokers.

BACKGROUND/OBJECTIVES: Vitamins can help improve cardiovascular control. In contrast, smoking works in the opposite fashion, reducing the baroreflex control of heart rate (HR) possibly via oxidative stress. High-fat challenges also impair cardiovascular regulation. Whether vitamins have acute beneficial effects on the baroreflex control of HR in smokers is unclear. SUBJECTS/METHODS: A randomized, placebo-controlled crossover study in 30 male smokers (34.2+/-6.9 years). Interventions were: (1) moderate (vitamin C (300 mg) and E (75 IU) and folic acid (1 mg)); (2) high doses of vitamins (vitamin C (2 g) and E (800 IU), and folic acid (5 mg)); or, (3) placebo. Vitamins were ingested with cream (a high-fat challenge) or milk (low-fat control). Four hours later, blood was withdrawn and radial pulse wave forms recorded via tonometry. Spontaneous beat-to-beat variations in HR and systolic blood pressure (SBP) were analysed by spectral analysis techniques and sympathovagal control of HR and baroreflex sensitivity (BRS) were assessed. RESULTS: High doses of vitamins increased plasma vitamin C, E and folic acid levels (P<0.05) with no change in SBP, HR or BRS (P>0.05, analysis of variance). Plasma vitamin levels did not correlate with any cardiovascular parameters. Moderate vitamins increased the vagal control of HR (+23%; P<0.05) and cream led to small increases (P<0.05) in SBP (+2 mm Hg) and HR (+2 beats min(-1)) with no change in BRS. CONCLUSIONS: In male smokers, circulating antioxidants had no effect on BRS and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion.

Arterial stiffness, endothelial function and microcirculatory reactivity in healthy young males.

Large (C1) and small (C2) arterial stiffness has been suggested to parallel endothelial reactivity and has led researchers to suggest parameters of arterial stiffness may be alternative measures to brachial sonographic assessments of flow-mediated dilatation (FMD). However, past studies comparing these measures can be criticized. In addition to %FMD responses, we recorded concurrent hyperaemic responses of the microcirculation and both were compared with C1 and C2. Twenty-nine subjects 18-30 years of age were investigated. Radial blood pressure was recorded with a tonometer. Pulse waveform analysis was performed to calculate C1 and C2. These were compared with %FMD responses and responses of finger flux measured by laser Doppler fluxmetry (LDF); pulsatile finger volume measured by photoplethysmography (PPG); and palm skin temperature measured by infrared thermography (Tpalm) (i.e. microcirculatory responses). Responses were determined as % changes from control. We only found weak relationships between C1 and %FMD (r=0.4, P=0.04); C2 and %PPG (r=0.38, P=0.07); and C2 and %LDFdorsal (r=-0.38; P=0.04). Responses of %FMD weakly parallel those of C1. Neither C2 nor C1 are viable indicators of endothelial or microcirculatory reactivity (i.e. hyperaemic or venous constriction) in healthy, resting young males. These findings refute the claims that C1 and C2 are substitute measures to sonographic assessments of brachial FMD.

[Entrapped motorists and air rescue services: analysis of tactical rescue approach, rescue techniques, and emergency medical services illustrated by a helicopter emergency medical service]. / Eingeklemmte Fahrzeuginsassen in der Luftrettung: Einsatztaktik, rettungstechnische und notfallmedizinische Analyse am Beispiel einer Rettungshubschrauberstation.

BACKGROUND: Analysis of the results and presentation of a treatment concept of a helicopter emergency medical service (HEMS) in prehospital acute care of entrapped motorists. METHODS: Consecutive patient data collection from primary rescue missions of a helicopter emergency medical service (HEMS) from the years 2000-2004. Evaluation based on data collected regarding emergency medical care, rescue techniques, and tactical rescue approach. RESULTS: A total of 359 cases of entrapped motorists were documented: 237 patients were male, 122 were female, and the average age was 37 (range: 2-82 years). The motor vehicle accidents (MVA) occurred in 21% of the cases on the highway, in 29% on a main road, in 43% on a rural road, and in 7% in city/urban areas. Concerning the vehicle types, 86% were automobiles, 5% vans, and 9% trucks. Drivers accounted for 86% of the patients, front seat passengers for 10.1%, and back seat passengers for 3.9%. The average length of motorist entrapment amounted to 17 min with an average on-scene time of 27 min for the HEMS. The total rescue time averaged 56 min. A Glasgow Coma Scale (GCS) score between 3 and 8 was recorded in 33.7% of the patients; in 24% of the cases the shock index was <1; a respiratory rate of <10/min or >20/min was documented in 25.2% of the patients. An NACA score between I and III was recorded in 34.2% of the cases, NACA IV in 18.9%, and NACA >/=V in 46.8%; 11.9% of the patients died before hospital admission. CONCLUSION: For both the emergency control center personnel and the emergency medical technicians (EMT), a case of motorist entrapment must be considered as a trigger mechanism of injury, activating a sophisticated and time-sensitive prehospital acute care and transportation service. In the German emergency medical service this involves primarily the HEMS. Even in cases of potentially critically injured entrapped motorists, the prehospital adherence to"the golden hour of shock" is made possible, despite the resulting higher personnel and equipment expenses.

Herbal medicines as diuretics: a review of the scientific evidence.

There is increasing interest in the health and wellness benefits of herbs and botanicals. This is with good reason as they might offer a natural safeguard against the development of certain conditions and be a putative treatment for some diseases. One such area may be the lowering of blood pressure in those where it is elevated (i.e., hypertension). One class of clinical medicines used to lower blood pressure are known as diuretics and work by increasing the excretion of urine from the body as well as the amount of sodium in urine. There are a growing number of studies purporting diuretic effects with traditional medicines. The aim of this article was to review these studies and identify which extracts promote diuresis (which we assessed on terms of urine excreted and urinary sodium excretion) and also to identify the research needs in this area. We identified a number of species and genuses reporting diuretic effects. Of these, the most promising, at the present time, are the species Foeniculum vulgare, Fraxinus excelsior, Hibiscus sabdariffa, Petroselinum sativum and Spergularia purpurea, and species from the genuses Cucumis (Cucumis melo and Cucumis trigonus), Equisetum (Equisetum bogotense, Equisetum fluviatile, Equisetum giganteum, Equisetum hiemale var. affine and Equisetum myriochaetum), Lepidium (Lepidium latifolium and Lepidium sativum), Phyllanthus (Phyllanthus amarus, Phyllanthus corcovadensis and Phyllanthus sellowianus) and Sambucus (Sambucus mexicana and Sambucus nigra). However, there the number of studies is limited and we recommend that further studies be conducted to confirm reported effects. Such evidence is needed to provide scientific credence to the folklore use of traditional medicines and even be helpful in the development of future medicines, treatments and treatment guidelines.

The relation of arterial stiffness to endothelial function in healthy subjects.

Local wall stiffness affects endothelial responsiveness but how global measures affect responsiveness is unanswered. We assessed this by comparing reactive hyperaemic responses of brachial diameter (RHRBD) with central (heart-to-brachial artery pulse wave velocity (PWV); large (C1)) and peripheral (C2) arterial stiffness. Twelve healthy subjects were investigated. RHRBD was induced via an upper- or forearm occluding cuff. Arterial diameter changes were measured using echo ultrasound. Arterial stiffness and RHRBD were compared using a Pearson correlation coefficient (r) and Bland-Altman analysis of Z-scores (indicated as 95% confidence intervals (CI) and expressed in units of standard deviation (SD) from the mean). Weak relations were found between upper-arm RHRBD responses and C2 (r = 0.56, P = 0.06; 95% CI +/- 1.84 SDs) and C1 (r = 0.55, P = 0.06; 95% CI +/- 1.86 SDs). An inverse relation was found between upper-arm RHRBD responses and PWV (r = -0.55, P = 0.06), but Bland-Altman plots revealed no agreement between these parameters (P > 0.05; 95% CI +/- 3.46 SDs). Forearm RHRBD were not related to PWV, C1 or C2 (P > 0.05; 95% CI > 2 SDs). The weak relation between upper-arm endothelial responses and C2 and C1 seems to suggest that C2, and also C1, is not a good and reliable method for assessments of endothelial health. Furthermore, if anything, upper-arm mediated RHRBD responses are more affected by arterial stiffness than forearm responses.

Non-invasive methods and stimuli for evaluating the skin's microcirculation.

Vessels in the skin are arranged into superficial and deep horizontal plexuses and they are involved in thermoregulation, oxygen and nutritional support. The skin has a large number of functions and broad appeal spanning basic mechanistic and clinical research. Indeed, the skin can be used as a marker of normal and impaired vascular control and, owing to its accessibility and frequent involvement, is easy to investigate non-invasively. A large number of non-invasive methods are available for investigating the skin, ranging from those that permit the visualisation of microvessels, to those that monitor blood flow or one of its derivatives (e.g., skin temperature and transcutaneous oxygen). Such methods can be combined with non-invasive, dynamic stimuli (e.g., the use of cold or warm stimuli, activation of the peripheral nervous system or local neuronal systems, and the topical application of vasoactive drugs) and this potentially enables the differentiation of underlying disorders (e.g., primary from secondary Raynaud's phenomenon) and also to quantify changes over time or following intervention. The present article outlines the non-invasive methods and dynamic tests that can be used to investigate the microcirculation of the skin.

Cytoplasmic regions of the beta3 subunit of integrin alphaIIbbeta3 involved in platelet adhesion on fibrinogen under flow conditions.

Platelet adhesion to surface-bound fibrinogen depends on integrin alphaIIbbeta3. In the present study, we investigated the role of the regions 749EATSTFT756N and 755TNITYRG762T of the beta3 cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E-N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T-T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S-->P substitution in the E-N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E-N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to alphaIIbbeta3 in platelet suspensions. Under flow, cytochalasin D (1 micro mol L-1) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E-N) and completely (T-T) restored platelet spreading. Thus, the 749EATSTFT756N and 755TNITYRG762T regions of beta3 contribute to the regulation of alphaIIbbeta3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.

Antenatal day care units versus hospital admission for women with complicated pregnancy.

BACKGROUND: The use of antenatal day care units is widely recognized as an alternative for inpatient care for women with complicated pregnancy. OBJECTIVES: The objective of this review was to assess the clinical safety, plus maternal, perinatal and psychosocial consequences for the women and cost effectiveness of this type of care. SEARCH STRATEGY: The Group's Specialised Register of Controlled Trials was searched in May 2001 as well as the Cochrane Controlled Trials Register (CENTRAL/CCTR), CINAHL (1982-04 to 1998-10) and Current Contents (Life Sciences/Clinical Medicine 1995-05 to 1999). Conference proceedings of PSANZ (Perinatal Society of Australia and New Zealand) and FIGO (Fédération Internationale de Gynécologie et d'Obstétrie) (1997) were searched. SELECTION CRITERIA: Randomized trial comparing day care with inpatient care for women with complicated pregnancy. DATA COLLECTION AND ANALYSIS: Trial quality was assessed by two reviewers independently. The author of the study was contacted for additional information. Data were extracted by the same two reviewers independently. MAIN RESULTS: One trial involving 54 women was included. This trial was of average quality. It was found that day care assessment for non-proteinuric hypertension can reduce inpatient stay (difference in mean stay: 4.0 days; 95% confidence interval (CI): 2.1 to 5.9 days). Also a significant increase in the rate of induction of labour in the control group was found (4.9 times more likely: 95% CI: 1.6 to 13.8). The other clinical outcomes did not show a statistically significant difference between the control and intervention group. No other significant differences were observed. REVIEWER'S CONCLUSIONS: Admission to day care for non-proteinuric hypertension reduces the amount of time spent in the hospital and proportion of women induced for labour. However, one trial of 54 women is not sufficient to draw sound conclusions. Additional studies are needed to give more solid evidence to confirm the advantages of antenatal day care units.

Cyclic adenosine monophosphate signaling in the rat vomeronasal organ: role of an adenylyl cyclase type VI.

The present study indicates that male rat urinary components in female rat vomeronasal organ microvillar preparations not only induce a rapid and transient IP(3) signal, but in addition, the level of cAMP decreases with a delayed and sustained time course. This decrease seems to be a consequence of the preceding activation of the phosphoinositol pathway rather than the result of an enhanced phosphodiesterase activity or an inhibition of adenylyl cyclase (AC) via Galpha(i) or Galpha(o). This notion is supported by the finding that activation of the endogenous protein kinase C suppresses basal as well as forskolin-induced cAMP formation. Furthermore, it was observed that elevated levels of calcium inhibit cAMP formation in rat VNO microvillar preparations. These properties of cAMP signaling in the VNO of rats may be mediated by a calcium- and protein kinase C-inhibited AC VI subtype, which is localized in microvillar preparations of the VNO.

Dual regulation of platelet protein kinase B.

Protein kinase B (PKB) is a serine/threonine kinase that is activated by growth hormones and implicated in prevention of apoptosis, glycogen metabolism, and glucose uptake. A key enzyme in PKB activation is phosphatidylinositide 3-kinase (PI-3K), which triggers the dual phosphorylation of PKB by phosphatidylinositol-dependent kinases (PDKs). Here we report that the major PKB subtype in platelets is PKBalpha, which is activated by phosphorylation of Thr(308) and Ser(473) and has a constitutively phosphorylated Thr(450) that does not contribute to PKB activation. alpha-Thrombin and thrombopoietin activate PKBalpha via PI-3K and trigger the concurrent phosphorylation of Thr(308) (via PDK1) and Ser(473) (via a not yet identified PDK2). In addition, alpha-thrombin activates a PI-3K-independent pathway involving phospholipase Cbeta and calcium-dependent protein kinase C subtypes (PKCalpha/beta). This route is specific for phosphorylation of Ser(473) and can be initiated by direct PKC activation with phorbol ester or purified active PKC catalytic fragment in platelet lysate. Different degrees of Ser(473) and Thr(308) phosphorylation correlate with different degrees of enzyme activity. These data reveal a PI-3K-independent PKB activation in which PKCalpha/beta regulates the phosphorylation of Ser(473) in PKBalpha. The independent control of the two phosphorylation sites may contribute to fine regulation of PKBalpha activity.

Identification of a phospholipase C beta subtype in rat taste cells.

From rat circumvallate papillae a novel phospholipase C (PLC) subtype has been cloned and identified as most closely related to human PLC beta2. The corresponding mRNA was only detected in sensory lingual tissue but not in non-taste lingual tissue or any other tissues examined by Northern blot analysis. In situ hybridization revealed that a subset of taste receptor cells of circumvallate papillae was specifically labeled. A functional involvement of this PLC beta subtype in taste signal transduction emerged from biochemical analysis monitoring the second messenger response in circumvallate preparations induced by denatonium benzoate. This bitter agent elicited a rapid and transient increase of the inositol 1,4,5-trisphosphate level; this response was blocked by U73122, a potent inhibitor of PLC, and by PLC beta2-specific antibodies. These data indicate that a phospholipase C beta2 isoform mediates a denatonium benzoate-induced second messenger response of taste sensory cells in the circumvallate papillae.

Odorants selectively activate distinct G protein subtypes in olfactory cilia.

Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.

Pheromone-induced second messenger signaling in the hamster vomeronasal organ.

Compounds present in estrous hamster vaginal discharge modulate male attraction and mounting behavior. These compounds are differentially processed by chemosensory neurons in the main olfactory epithelium (MOE) and vomeronasal organ (VNO). The transduction cascade responsible for this processing is unclear in the VNO, although studies of the MOE suggest that the second messengers cAMP or IP3 may be involved. Here we demonstrate that purified aphrodisin, a hamster mounting pheromone, modulates IP3 production in male VNO membranes without altering cAMP production. Aphrodisin does not alter the concentration of either second messenger in membranes from the MOE. These results confirm the specificity of the VNO in the processing of mounting pheromones and establishes the importance of IP3 cascades in mammalian reproductive behavior.

Phosphatase 2A regulates the responsiveness of olfactory cilia.

The odorant-induced second messenger response in olfactory cilia was monitored in the presence of phosphatase modulators. Okadaic acid, a phosphatase inhibitor, attenuated the odorant-induced cAMP-response in a dose-dependent manner, half maximal inhibition was obtained at 1.5 nM okadaic acid indicating that phosphatase 2A may be involved. Protamine, a selective activator of phosphatase 2A, led to significantly stronger cAMP-responses. Western blot and immunohistochemical analysis employing specific antibodies revealed that phosphatase 2A is present in olfactory tissues in particular in olfactory cilia. The results suggest that phosphatase 2A may play a regulatory role in governing the responsiveness of olfactory neurons.

Regulation of olfactory signalling via cGMP-dependent protein kinase.

Strong odor stimuli elicit a slow and sustained increase of the cGMP concentration in isolated rat olfactory cilia. Elevated cGMP levels appear to attenuate the primary response to odorant stimulation. Incubating cilia with membrane-permeable cGMP derivates caused a significantly reduced cAMP signal in response to odorant stimulation. This inhibitory effect was mimicked by 8-(4-chlorophenlythio)-cGMP, a selective activator of cGMP-activated protein kinases; in contrast, a selective inhibitor, [8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate] of cGMP kinases enhanced the reactivity to odorant stimulation. The data suggest that the responsiveness of olfactory sensory cells is governed by a cGMP-dependent protein kinase. Western-blot analysis using subtype-specific antibodies indicated that cytosolic type-I cGMP kinase, but not the membrane-associated type-II cGMP kinase, is expressed in olfactory sensory neurons.

Calcium controls second-messenger signalling in olfactory cilia.

The increase in intracellular calcium concentration elicited by odorant stimulation seems to be involved in down-regulating the responsiveness of olfactory neurons to subsequent stimuli. The present study suggests that this regulatory effect may be due to a calcium-dependent attenuation of the olfactory signalling cascade; the odor-induced cyclic adenosine monophosphate (cAMP) response in olfactory cilia is diminished by calcium in a dose-dependent manner. This reduced cAMP signal is not due to an activation of phosphodiesterases by elevated calcium levels, but rather seems to be mediated by the inhibition of adenylate cyclase by calcium ions.