Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE)

Background and Purpose

Hydrogen sulfide (H₂S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H₂S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H₂S biosynthesis towards CSE and CBS.

Experimental Approach

To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method.

Key Results

β-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC₅₀ 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC₅₀ 1.1 ± 0.1 μM); the IC₅₀ for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H₂S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC₅₀ for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE.

Conclusions and Implications

In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.     

Asthma Endophenotypes and Polymorphisms in the Histamine Receptor HRH4 Gene

Background: Histamine as an inflammatory mediator plays an important role in chronic allergic and asthmatic conditions. However, the role of genetic polymorphisms of the histamine receptor HRH4 (histamine receptor H4) gene in asthma susceptibility and endophenotypes has not been studied yet. Our aim was to investigate the possible association between single nucleotide polymorphisms (SNPs) in the HRH4 gene and asthma or some endophenotypes of asthma. Methods: Twenty-one SNPs of the HRH4 gene were genotyped in 313 asthmatic patients and 360 controls using Sequenom? iPLEX? Gold Genotyping Technology. Results: Genotype distribution of three HRH4 SNPs, namely rs17187619 [p = 0.002; odds ratio, OR (95% confidence interval, CI) = 2.4 (4.1-1.4)], rs527790 [p = 0.0002; OR (95% CI) = 3.3 (6.1-1.8)] and rs487202 [p = 0.00007; OR (95% CI) = 3.5 (6.6-1.9)] differed significantly between patients with or without infection-induced asthma. Haplotypes, which included the rs4800573-rs527790 CC allele combination, were found to be associated with infection-induced asthma [p = 0.0009, OR (95% CI) = 0.5 (0.4-0.8)]. The rs487202-rs574913 CA haplotype was more frequent among patients with infection-induced asthma [p = 0.0006, OR (95% CI) = 1.9 (1.3-2.6)]. None of the SNPs contributed directly to the risk of asthma. Conclusions: Our results suggest that genetic variation in the HRH4 gene might influence the pathogenesis of infection-induced asthma.     

First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.     

Adenosine-dependent concealed accessory pathway

Adenosine is routinely used during ventricular pacing to exclude the persistence of retrograde accessory pathways conduction after radiofrequency (RF) ablation procedures by blocking conduction over the atrioventricular node. This is the first report of an adenosine-dependent concealed accessory pathway demonstrating transient conduction only after adenosine administration. Our findings may have potential clinical implications in reducing recurrence after accessory pathway ablation. Furthermore, it may add relevant information regarding the ability of adenosine to elicit dormant conduction after RF ablation, a phenomenon that has acquired considerable interest in the era of pulmonary vein isolation.     

Cerebral vasoreactivity to acetazolamide is not impaired in patients with severe sepsis

Introduction

The pathophysiology of sepsis-associated encephalopathy (SAE) is not entirely clear, but one of the possible underlying mechanisms is the alteration of the cerebral microvascular function. The aim of the present work was to test whether cerebral vasomotor reactivity is impaired in patients with severe sepsis.

Methods

Patients fulfilling the criteria of clinical sepsis and showing at least 2 organ dysfunctions were included (n = 16). Nonseptic healthy persons without previous diseases affecting cerebral vasoreactivity served as controls (n = 16). Transcranial Doppler blood flow velocities were measured at rest and at 5, 10, 15, and 20 minutes after intravenous administration of 15 mg/kg acetazolamide. The time course of the acetazolamide effect on cerebral blood flow velocity (cerebrovascular reactivity [CVR]) and the maximal vasodilatory effect of acetazolemide (cerebrovascular reserve capacity [CRC]) were compared among the groups.

Results

Absolute blood flow velocities after administration of the vasodilator drug did not differ between control and septic patients. Assessment of the time course of the vasomotor reaction showed that patients with sepsis reacted in a similar fashion to the vasodilatory stimulus than control persons. When assessing the maximal vasodilatory ability of the cerebral arterioles to acetazolamide during vasomotor testing, we found that there was no difference in vasodilatory ability between septic and healthy subjects (CRC controls, 54.8% ± 11.1%; CRC sepsis-associated encephalopathy, 61.1% ± 34.4%; P = .49).

Conclusions

We conclude that cerebrovascular reactivity is not impaired in patients with severe sepsis. It is conceivable that cerebral vasoreactivity may be differently involved at different severity stages of the septic process.     

Gene expression patterns of the 11β-hydroxysteroid dehydrogenase 2 enzyme in human placenta from intrauterine growth restriction: the role of impaired feto-maternal glucocorticoid metabolism

Objective

To assess 11-β-hydroxysteroid dehydrogenase 2 (11β-HSD2) gene expression patterns in human placental samples from intrauterine growth restriction (IUGR) pregnancies using normal pregnancy as control.

Study design

We compared 11-β-HSD2 gene expression in placental samples from all IUGR pregnancies treated in our clinic between January 1, 2010 and January 1, 2011 vs. 140 normal pregnancy samples from the same study period. Clinical characteristics were also assessed and compared between the IUGR and normal pregnancy groups.

Results

Mean gestational weight gain in the IUGR group was significantly lower than in the control group. Similarly, change in body mass index (BMI) was lower. Impending intrauterine fetal asphyxia was significantly more common in the IUGR group. The 11β-HSD2 gene was underexpressed compared to controls, but this underexpression was only observed after the 33rd gestational week. Within the IUGR group, in cases of impending intrauterine fetal asphyxia the 11β-HSD2 gene was underexpressed compared to both impending asphyxia in non-IUGR cases, or IUGR without impending asphyxia.

Conclusion

Low gestational weight gain appears to predict IUGR. The 11β-HSD2 gene in IUGR is underexpressed and may result in an impaired placental barrier, decreasing protection against maternal glucocorticoids, which are thought to be prominent in fetal programming. Maternal glucocorticoid exposure resulting from an impaired placental barrier may increase the risk for cardiovascular and metobolic disorders later in adult life. In IUGR, before the 33rd gestational week, the expression of the 11β-HSD2 gene remains physiological. The underexpression of this gene after the 33rd week in impending intrauterine fetal asphyxia in IUGR points to an increased sensitivity to hypoxia when impending asphyxia is present in the late phase of IUGR pregnancies.     

Inflammatory Breast Cancer—Comparing the Effectivity of Preoperative Docetaxel-Epirubicine Protocol to Conventional Antracycline-Containing Chemotherapy to Achieve Clinical Benefit and Complete Pathological Response

Our retrospective analysis compared the effectiveness of conventional antracycline-containing protocols (A+) and docetaxel/epirubicine (TE) as primary systemic chemotherapies (PSCT) for inflammatory breast cancer (IBC). Seventy IBC patients received either A?+?(n?=?48) or TE (n?=?22) as PSCT. The objective clinical response and clinical benefit rate of treated patients were 54.3% (A+: 54,2% vs. TE: 54,5%; p = 0,28) and 92.8% (A+: 91,7% vs. TE: 95,5%; p = 0,57), respectively. The clinical complete response rate (cCR) was 23.2% (A+: 27,1% vs. TE:4,5%; χ (2)?=?4,79; p = 0,03) with 7.14% (A+: 10,4% vs. TE:0%; χ (2)?=?2,47; p?=?0,12) of pathological complete responses (pCR). The median progression free (PFS)/local progression free (LPFS)/overall survival (OS) was 2.0/5.4/4.0 years, respectively. Patients achieving cCR had a tendency for better survival parameters than patients with less than cCR. Response rates or survival data were not statistically different in the two chemotherapy (CT) treatment groups. The survival was not influenced by the number of CT cycles in either protocols. In this set of patients, the clinical efficacy of the two alternative primary systemic chemotherapies (A?+?and TE) is equivalent in the treatment of inflammatory breast cancer (IBC), despite of the significant difference in favour of A?+?noticed in CRs. Six cycles of CT could be enough for patients achieving CR, however sequential pre- and/or postoperative CT with non cross-resistant drugs should be considered for non-responders.