Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

A Phase 1b Randomized,Controlled, Double-Blinded Dosage-Escalation Trial to Evaluate the Safety,Reactogenicity and Immunogenicity of an Adenovirus Type 35 Based Circumsporozoite Malaria Vaccine in Burkinabe Healthy Adults 18 to 45 Years of Age

Background

Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa.

Methods

A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10⁹, 10¹⁰, 5X10¹⁰, 10¹¹ vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140.

Results

Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35.

Conclusion

Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies.

Trial Registration

ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459     

Mode of Death on Chagas Heart Disease: Comparison with Other Etiologies. A Subanalysis of the REMADHE Prospective Trial

Background

Sudden death has been considered the main cause of death in patients with Chagas heart disease. Nevertheless, this information comes from a period before the introduction of drugs that changed the natural history of heart failure. We sought to study the mode of death of patients with heart failure caused by Chagas heart disease, comparing with non-Chagas cardiomyopathy.

Methods and results

We examined the REMADHE trial and grouped patients according to etiology (Chagas vs non-Chagas) and mode of death. The primary end-point was all-cause, heart failure and sudden death mortality; 342 patients were analyzed and 185 (54.1%) died. Death occurred in 56.4% Chagas patients and 53.7% non-Chagas patients. The cumulative incidence of all-cause mortality and heart failure mortality was significantly higher in Chagas patients compared to non-Chagas. There was no difference in the cumulative incidence of sudden death mortality between the two groups. In the Cox regression model, Chagas etiology (HR 2.76; CI 1.34–5.69; p = 0.006), LVEDD (left ventricular end diastolic diameter) (HR 1.07; CI 1.04–1.10; p<0.001), creatinine clearance (HR 0.98; CI 0.97–0.99; p = 0.006) and use of amiodarone (HR 3.05; CI 1.47–6.34; p = 0.003) were independently associated with heart failure mortality. LVEDD (HR 1.04; CI 1.01–1.07; p = 0.005) and use of beta-blocker (HR 0.52; CI 0.34–0.94; p = 0.014) were independently associated with sudden death mortality.

Conclusions

In severe Chagas heart disease, progressive heart failure is the most important mode of death. These data challenge the current understanding of Chagas heart disease and may have implications in the selection of treatment choices, considering the mode of death.

Trial Registration

ClinicalTrails.gov {"type":"clinical-trial","attrs":{"text":"NCT00505050","term_id":"NCT00505050"}}NCT00505050 (REMADHE)     

Cold storage effects on flesh quality of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) fed diets containing different vegetable oils

The intention of this experiment was to assess the effects of different sources of dietary lipid on the fatty acid composition of the fillet and liver and the flesh quality traits of rainbow trout (Oncorhynchus mykiss) after a 70‐day feeding period. Four iso‐nitrogenous (approx. 51% crude protein) and iso‐lipidic (approx. 14% crude lipid) experimental diets were formulated. The control diet contained only fish oil (FO) as the primary lipid source. In the other three dietary treatments, fish oil was replaced by 100% (LO30/SO35/SFO35) and 70% (FO30/LO35/SO35 or FO30/SO35/SFO35) sesame oil (SO), linseed oil (LO), or sunflower oil (SFO). Triplicate groups of 40 rainbow trout (~46 g) held under similar culture conditions were hand‐fed daily to apparent satiation for 70 days. At the end of the feeding trials, no difference in growth performance among experimental groups was noted (P > 0.05). There were some differences in the proximate composition of fish fillets (P < 0.05): the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels were highest in fish fed the control diet (P < 0.05); and EPA and DHA levels in fish fed the FO30/LO35/SO35 diet were closest to the control diet (P < 0.05). In contrast, fish fed the diet containing 100% plant oils (LO30/SO35/SFO35) had the highest level of total n‐6 fatty acids in the fillet and liver. In a 12‐day refrigerated storage at 1°C the thiobarbituric acid (TBA), trimethylamin nitrogen (TMA‐N) and pH values gradually increased in all dietary groups (P < 0.05). The chemical indicators of spoilage, TBA, TMA‐N, and pH values were within the limit of acceptability for human consumption.     

Exome sequencing discloses <Emphasis Type="Italic">KALRN</Emphasis> homozygous variant as likely cause of intellectual disability and short stature in a consanguineous pedigree

Background

The recent availability of whole-exome sequencing has opened new possibilities for the evaluation of individuals with genetically undiagnosed intellectual disability.

Results

We report two affected siblings, offspring of first-cousin parents, with intellectual disability, hypotonia, short stature, growth hormone deficiency, and delayed bone age. All members of the nuclear family were genotyped, and exome sequencing was performed in one of the affected individuals. We used an in-house algorithm (CATCH v1.1) that combines homozygosity mapping with exome sequencing results and provides a list of candidate variants. One identified novel homozygous missense variant in KALRN (NM_003947.4:c.3644C>A: p.(Thr1215Lys)) was predicted to be pathogenic by all pathogenicity prediction software used (SIFT, PolyPhen, Mutation Taster). KALRN encodes the protein kalirin, which is a GTP-exchange factor protein with a reported role in cytoskeletal remodeling and dendritic spine formation in neurons. It is known that mice with ablation of Kalrn exhibit age-dependent functional deficits and behavioral phenotypes.

Conclusion

Exome sequencing provided initial evidence linking KALRN to monogenic intellectual disability in man, and we propose that KALRN is the causative gene for the autosomal recessive phenotype in this family.