Effect of angiotensin converting enzyme inhibition in renovascular hypertension.

The unique ability of angiotensin converting enzyme (ACE) inhibitors to inhibit the generation of angiotensin II has made them very useful agents for treating patients with renovascular hypertension. Their efficacy in lowering blood pressure in this type of secondary hypertension is now well established. However, episodes of acute renal failure may occur during ACE inhibition, particularly when renal perfusion is compromised. This is often the case in patients with renal artery stenosis and a single kidney or with bilateral renal artery stenosis. In recent years, investigators have shown concern at the long-term fate of the stenotic kidney in patients with unilateral renal artery stenosis who are treated with ACE inhibitors. Although overall renal function remained stable, a decrease in glomerular filtration was demonstrated in the stenotic kidney under ACE inhibition. The long-term implications of this observation merit further investigations.

Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy.

Autophagy or "self eating" is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to gamma-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific target-siRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells.

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex.

A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency.

Microsatellite instability (MSI) occurs in 10-20% of colorectal tumours and is associated with good prognosis. Here we describe the development and validation of a genomic signature that identifies colorectal cancer patients with MSI caused by DNA mismatch repair deficiency with high accuracy. Microsatellite status for 276 stage II and III colorectal tumours has been determined. Full-genome expression data was used to identify genes that correlate with MSI status. A subset of these samples (n = 73) had sequencing data for 615 genes available. An MSI gene signature of 64 genes was developed and validated in two independent validation sets: the first consisting of frozen samples from 132 stage II patients; and the second consisting of FFPE samples from the PETACC-3 trial (n = 625). The 64-gene MSI signature identified MSI patients in the first validation set with a sensitivity of 90.3% and an overall accuracy of 84.8%, with an AUC of 0.942 (95% CI, 0.888-0.975). In the second validation, the signature also showed excellent performance, with a sensitivity 94.3% and an overall accuracy of 90.6%, with an AUC of 0.965 (95% CI, 0.943-0.988). Besides correct identification of MSI patients, the gene signature identified a group of MSI-like patients that were MSS by standard assessment but MSI by signature assessment. The MSI-signature could be linked to a deficient MMR phenotype, as both MSI and MSI-like patients showed a high mutation frequency (8.2% and 6.4% of 615 genes assayed, respectively) as compared to patients classified as MSS (1.6% mutation frequency). The MSI signature showed prognostic power in stage II patients (n = 215) with a hazard ratio of 0.252 (p = 0.0145). Patients with an MSI-like phenotype had also an improved survival when compared to MSS patients. The MSI signature was translated to a diagnostic microarray and technically and clinically validated in FFPE and frozen samples.

A corneal dystrophy associated with transforming growth factor beta-induced Gly623Asp mutation an amyloidogenic phenotype.

PURPOSE: To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset. DESIGN: Experimental study. PARTICIPANTS: Four affected and 6 nonaffected family members. METHODS: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15. MAIN OUTCOME MEASURES: Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. RESULTS: The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein. CONCLUSIONS: In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions.

The orexigenic neurotransmitter neuropeptide Y (NPY) plays a central role in the hypothalamic control of food intake and energy balance. NPY also exerts an inhibition of the gonadotrope axis that could be important in the response to poor metabolic conditions. In contrast, leptin provides an anorexigenic signal to centrally control the body needs in energy. Moreover, leptin contributes to preserve adequate reproductive functions by stimulating the activity of the gonadotrope axis. It is of interest that hypothalamic NPY represents a primary target of leptin actions. To evaluate the importance of the NPY Y1 and Y5 receptors in the downstream pathways modulated by leptin and controlling energy metabolism as well as the activity of the gonadotrope axis, we studied the effects of leptin administration on food intake and reproductive functions in mice deficient for the expression of either the Y1 or the Y5 receptor. Furthermore, the role of the Y1 receptor in leptin resistance was determined in leptin-deficient ob/ob mice bearing a null mutation in the NPY Y1 locus. Results point to a crucial role for the NPY Y1 receptor in mediating the NPY pathways situated downstream of leptin actions and controlling food intake, the onset of puberty, and the maintenance of reproductive functions.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir.

OBJECTIVES: The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking. METHODS: In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010. RESULTS: The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent. DISCUSSION: Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir.

Prenatal manifestation and management of a mother and child affected by spondyloperipheral dysplasia with a C-propeptide mutation in COL2A1: case report.

It is not unusual for patients with "rare" conditions, such as skeletal dysplasias, to remain undiagnosed until adulthood. In such cases, a pregnancy may unexpectedly reveal hidden problems and special needs. A 28 year old primigravida was referred to us at 17 weeks for counselling with an undiagnosed skeletal dysplasia with specific skeletal anomalies suggesting the collagen 2 disorder, spondyloperipheral dysplasia (SPD; MIM 156550).She was counselled about the probability of dominant inheritance and was offered a prenatal diagnosis by sonography. US examination at 17, 18 and 20 weeks revealed fetal macrocephaly, a narrow thorax, and shortening and bowing of long bones. The parents elected to continue the pregnancy. At birth the baby showed severe respiratory distress for four weeks which then resolved. Mutation analysis of both mother and child revealed a hitherto undescribed heterozygous nonsense mutation in the C-propeptide coding region of COL2A1 confirming the diagnosis of SPD while reinforcing the genotype-phenotype correlations between C-propeptide COL2A1 mutations and the SPD-Torrance spectrum. This case demonstrates the importance of a correct diagnosis even in adulthood, enabling individuals affected by rare conditions to be made aware about recurrence and pregnancy-associated risks, and potential complications in the newborn.

Rôle de l'activation de c-Jun N-terminal kinase (JNK) dans un modèle de glaucome chez le rat et neuroprotection par inhibition de la voie de JNK

RESUME : Dans ce travail effectué chez le rat adulte, l'excitotoxicité rétinienne est élicitée par injection intravitréenne de NMDA. Les lésions en résultant sont localisées dans la rétine interne. Elles prennent la forme de pycnoses dans la couche des cellules ganglionnaires (corps cellulaires des cellules ganglionnaires et amacrines déplacées) et dans la partie interne de la couche nucléaire interne (cellules amacrines). Cette localisation est liée à la présence de récepteurs au glutamate de type NMDA sur ces cellules. L'activation de ces récepteurs entraîne un influx calcique et l'activation de diverses enzymes (phospholipase A, calpaïnes, calmoduline, synthase d'oxyde nitrique). La signalisation se poursuit en aval en partie par les voies des Mitogen Activated Protein Kinase (MAPK) : ERK, p38, ]NK. Dans les expériences présentées, toutes trois sont activées après l'injection de NMDA. Dans les cascades de signalisation de JNK, trois kinases s'ancrent sur une protéine scaffold. Les MAPKKK phosphorylent MKK4 et MKK7, qui phosphorylent JNK. JNK a de nombreuses cibles nucléaires (dont le facteur de transcription c-Jun) et cytoplasmiques. La voie de JNK est bloquée par l'inhibiteur peptidique D-JNKI-1 en empêchant l'interaction de la kinase avec son substrat. L'inhibiteur est formé de 20 acides aminés du domaine de liaison JBD et de 10 acides aminés de la partie TAT du virus HIV. L'injection intravitréenne de D-JNKI-1 permet une diminution des taux de JNK et c-Jun phosphorylés dans les lysats de rétine. L'effet prépondérant est la restriction importante des altérations histologiques des couches internes de la rétine. L'évaluation par électrorétinogramme met en sus en évidence une sauvegarde de la fonction cellulaire. Ce travail a ainsi permis d'établir la protection morphologique et fonctionnelle des cellules de la rétine interne par inhibition spécifique de la voie de JNK lors d'excitotoxicité. SUMMARY Excitotoxicity in the retina associates with several pathologies like retinal ischemia, traumatic optic neuropathy and glaucoma. In this study, excitotoxicity is elicited by intravitreal NMDA injection in adult rats. Lesions localise in the inner retina. They present as pyknotic cells in the ganglion cell layer (ganglion cells and displaced amacrines) and the inner nuclear layer (amacrine cells). These cells express NMDA glutamate receptors. The receptor activation leads to a calcium flow into the cell and hence enzyme activation (phospholipase, calpains, calmodulin, nitric oxide synthase). The subsequent signaling pathways can involve the Mitogen Activated Protein Kinases (MAPK): ERK, p38 end JNK. These were all activated in our experiments. The signaling cascade organises around several scaffold proteins. The various MAPKKK phosphorylate MKK4 and MKK7, which phosphorylate JNK. JNK targets are of nuclear (c-Jun transcription factor) or cytoplasmic localisation. The peptidic inhibitor D-JNKI-1, 20 amino acids from the JNK binding domain JBD coupled to 10 amino acids of the TAT transporter, disrupts the binding of JNK with its substrate. Intravitreal injection of the inhibitor lowers phosphorylated forms of JNK and c-Jun in retinal extracts. It protects strongly against histological lesions in the inner retina and allows functional rescue.

De novo mutation in the KCNQ1 gene causal to Jervell and Lange-Nielsen syndrome.

Jervell and Lange-Nielsen syndrome (JLNS) is an autosomal recessive disorder, clinically characterized by severe cardiac arrhythmias [due to prolonged QTc interval in electrocardiogram (ECG)] and bilateral sensory neural deafness. Molecular defects causal to JLNS are either homozygous or compound heterozygous mutations, predominantly in the KCNQ1 gene and occasionally in the KCNE1 gene. As the molecular defect is bi-allelic, JLNS patients inherit one pathogenic mutation causal to the disorder from each parent. In this report, we show for the first time that such a disorder could also occur due to a spontaneous de novo mutation in the affected individual, not inherited from the parent, which makes this case unique unlike the previously reported JLNS cases.

Angiotensin-converting enzyme inhibition by hydroxamic zinc-binding idrapril in humans.

The new angiotensin-converting enzyme (ACE) inhibitor idrapril acts by binding the catalytically important zinc ion to a hydroxamic group. We investigated its pharmacodynamic and pharmacokinetic properties in 8 healthy men: Increasing doses of 1, 5, and 25 mg idrapril as well as placebo or 5 mg captopril were administered intravenously (i.v.) at 1-week intervals. Six of the subjects received 100 mg idrapril orally (p.o.) last, and two ingested oral placebo as a double-blind control. Blood pressure (BP) and heart rate (HR) remained unchanged. No serious side effects were observed. ACE inhibition in vivo was evaluated by changes in the ratio of specifically measured plasma angiotensin II (AngII) and AngI concentrations determined by high-performance liquid chromatography/radioimmunoassay (HPLC/RIA) techniques. Plasma ACE activity in vitro was estimated by radioenzymatic assay; it was suppressed by > or = 93% at 15 min after injection of 25 mg idrapril or 5 mg captopril and by 96% 2 h after idrapril intake. Mean AngII levels were decreased dose dependently at 15 min after idrapril injections. At the same time, plasma renin activity (PRA) and AngI increased according to the doses. The AngII/AngI ratio was clearly related to plasma idrapril levels (r = -0.88, n = 60). Oral idrapril inhibited ACE maximally at 1-4 h after dosing, when < 7% of initial ACE activity was observed in vitro and in vivo. Idrapril is a safe and efficient ACE inhibitor in human subjects. It is well absorbed orally. Besides having a slightly slower onset of action, idrapril has pharmacodynamic effects comparable to those of captopril.

Symposium swissYPG 2011 "La Pharmacie en mutation"

Comme en 2009, le groupe des jeunes pharma- ciens suisses (swissYPG) organise cette année un nouveau symposium de formation et de ré- flexion à destination des jeunes pharmaciennes et pharmaciens suisses. Ce dernier aura lieu à Interlaken le 30 novembre 2011, dans le cadre du 1er congrès suisse des pharmaciens.