Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia

(1) The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. (2) A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinteic parameters of S-adenosylhomocysteine hydrolase. (3) During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 μM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 μM) caused an increased of 0.37 and 4.17 nmol SAdony/soc per g wet weight during normaxia and ischemia, respectively. (4) The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 115 nmol/g; P < 0.05). Purine release was increased 4-fold during ischemia. (5) The K_m for hydrolysis of SAdoHcy was about 12 μM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 μM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. (6) From the combined in vitro and perfusion studies, we comclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.     

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis

Abstract We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti- Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.     

Verbal Memory Impairment in Polydrug Ecstasy Users: A Clinical Perspective

Background

Ecstasy use has been associated with short-term and long-term memory deficits on a standard Word Learning Task (WLT). The clinical relevance of this has been debated and is currently unknown. The present study aimed at evaluating the clinical relevance of verbal memory impairment in Ecstasy users. To that end, clinical memory impairment was defined as decrement in memory performance that exceeded the cut-off value of 1.5 times the standard deviation of the average score in the healthy control sample. The primary question was whether being an Ecstasy user (E-user) was predictive of having clinically deficient memory performance compared to a healthy control group.

Methods

WLT data were pooled from four experimental MDMA studies that compared memory performance during placebo and MDMA intoxication. Control data were taken from healthy volunteers with no drug use history who completed the WLT as part of a placebo-controlled clinical trial. This resulted in a sample size of 65 E-users and 65 age- and gender-matched healthy drug-naïve controls. All participants were recruited by similar means and were tested at the same testing facilities using identical standard operating procedures. Data were analyzed using linear mixed-effects models, Bayes factor, and logistic regressions.

Results

Findings were that verbal memory performance of placebo-treated E-users did not differ from that of controls, and there was substantial evidence in favor of the null hypothesis. History of use was not predictive of memory impairment. During MDMA intoxication of E-users, verbal memory was impaired.

Conclusion

The combination of the acute and long-term findings demonstrates that, while clinically relevant memory impairment is present during intoxication, it is absent during abstinence. This suggests that use of Ecstasy/MDMA does not lead to clinically deficient memory performance in the long term. Additionally, it has to be investigated whether the current findings apply to more complex cognitive measures in diverse ‘user categories’ using a combination of genetics, imaging techniques and neuropsychological assessments.     

Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three hetero-oligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented.     

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.     

Season of sampling and season of birth influence serotonin metabolite levels in human cerebrospinal fluid

Background

Animal studies have revealed seasonal patterns in cerebrospinal fluid (CSF) monoamine (MA) turnover. In humans, no study had systematically assessed seasonal patterns in CSF MA turnover in a large set of healthy adults.

Methodology/Principal Findings

Standardized amounts of CSF were prospectively collected from 223 healthy individuals undergoing spinal anesthesia for minor surgical procedures. The metabolites of serotonin (5-hydroxyindoleacetic acid, 5-HIAA), dopamine (homovanillic acid, HVA) and norepinephrine (3-methoxy-4-hydroxyphenylglycol, MPHG) were measured using high performance liquid chromatography (HPLC). Concentration measurements by sampling and birth dates were modeled using a non-linear quantile cosine function and locally weighted scatterplot smoothing (LOESS, span = 0.75). The cosine model showed a unimodal season of sampling 5-HIAA zenith in April and a nadir in October (p-value of the amplitude of the cosine = 0.00050), with predicted maximum (PC_max) and minimum (PC_min) concentrations of 173 and 108 nmol/L, respectively, implying a 60% increase from trough to peak. Season of birth showed a unimodal 5-HIAA zenith in May and a nadir in November (p = 0.00339; PC_max = 172 and PC_min = 126). The non-parametric LOESS showed a similar pattern to the cosine in both season of sampling and season of birth models, validating the cosine model. A final model including both sampling and birth months demonstrated that both sampling and birth seasons were independent predictors of 5-HIAA concentrations.

Conclusion

In subjects without mental illness, 5-HT turnover shows circannual variation by season of sampling as well as season of birth, with peaks in spring and troughs in fall.     

Nasal gene expression differentiates COPD from controls and overlaps bronchial gene expression

Background

Nasal gene expression profiling is a promising method to characterize COPD non-invasively. We aimed to identify a nasal gene expression profile to distinguish COPD patients from healthy controls. We investigated whether this COPD-associated gene expression profile in nasal epithelium is comparable with the profile observed in bronchial epithelium.

Methods

Genome wide gene expression analysis was performed on nasal epithelial brushes of 31 severe COPD patients and 22 controls, all current smokers, using Affymetrix Human Gene 1.0 ST Arrays. We repeated the gene expression analysis on bronchial epithelial brushes in 2 independent cohorts of mild-to-moderate COPD patients and controls.

Results

In nasal epithelium, 135 genes were significantly differentially expressed between severe COPD patients and controls, 21 being up- and 114 downregulated in COPD (false discovery rate?<?0.01). Gene Set Enrichment Analysis (GSEA) showed significant concordant enrichment of COPD-associated nasal and bronchial gene expression in both independent cohorts (FDR_GSEA <?0.001).

Conclusion

We identified a nasal gene expression profile that differentiates severe COPD patients from controls. Of interest, part of the nasal gene expression changes in COPD mimics differentially expressed genes in the bronchus. These findings indicate that nasal gene expression profiling is potentially useful as a non-invasive biomarker in COPD.

Trial registration

ClinicalTrials.gov registration number NCT01351792 (registration date May 10, 2011), ClinicalTrials.gov registration number NCT00848406 (registration date February 19, 2009), ClinicalTrials.gov registration number NCT00807469 (registration date December 11, 2008).

     

Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using (14)N- or (15)N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R(Met.Lab.) = 0.98R(DiGE) with r(2) = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.