Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines as inhibitors of phenylethanolamine N-methyltransferase

A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the alpha(2)-adrenoceptor. The THTP nucleus was suggested as an isosteric replacement for the 1,2,3,4-tetrahydroisoquinoline (THIQ) ring system on the basis that 3-thienylmethylamine (18) was more potent as an inhibitor of hPNMT and more selective toward the alpha(2)-adrenoceptor than benzylamine (15). Although the isosterism was confirmed, with similar influence of functional groups and chirality in both systems on hPNMT inhibitory potency and selectivity, the THTP compounds proved, in general, to be less potent as inhibitors of hPNMT than their THIQ counterparts, with the drop in potency being primarily attributed to the electronic properties of the thiophene ring. A hypothesis for the reduced hPNMT inhibitory potency of these compounds has been formed on the basis of molecular modeling and docking studies using the X-ray crystal structures of hPNMT co-crystallized with THIQ-type inhibitors and S-adenosyl-L-homocysteine as a template.     

AtCDKA;1 silencing in Arabidopsis thaliana reduces reproduction of sedentary plant-parasitic nematodes

SUMMARY: The activity of the Arabidopsis thaliana cyclin-dependent kinase AtCDKA;1 is important throughout G(1)/S and G(2)/M transitions and guarantees the progression of the cell cycle. Inhibitor studies have shown that activation of the cell cycle is important for the development of nematode feeding sites. The aim of this study was to silence the expression of the AtCDKA;1 gene in nematode feeding sites to interfere with their development. Therefore, sense and antisense constructs were made for the AtCDKA;1 gene and fused to a nematode-inducible promoter which was activated in nematode feeding sites at an earlier time point than AtCDKA;1. Two transgenic A. thaliana lines (S266 and S306) containing inverted repeats of the AtCDKA;1 gene and with reduced AtCDKA;1 expression in seedlings and galls were analysed in more detail. When the lines were infected with the root-knot nematode Meloidogyne incognita, significantly fewer galls and egg masses developed on the roots of the transgenic than wild-type plants. Infection of the AtCDKA;1-silenced lines with Heterodera schachtii resulted in significantly fewer cysts compared with controls. The S266 and S306 lines showed no phenotypic aberrations in root morphology, and analysis at different time points after infection demonstrated that the number of penetrating nematodes was the same, but fewer nematodes developed to maturity in the silenced lines. In conclusion, our results demonstrate that silencing of CDKA;1 can be used as a strategy to produce transgenic plants less susceptible to plant-parasitic nematodes.     

Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate,cosubstrate and products of the aldose reductase reaction

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbital synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold [1]. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent K_m of cellular sorbitol production for glucose was identical in control and diabetic rats ($\text{56 ± 18}\text{vs.}\text{59 ± 14}\text{mmol/l}\text{d}\text{-glucose}$), whereas V_max increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 ± 5 μmol sorbitol/g protein, sorbitol synthesis slightly lower (?15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 ± 4 μmol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 ± 0.3 vs. 5.6 ± 0.4 μmol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 ± 5.1 to 8.6 ± 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.2 ± 0.67 μmol/g protein vs. 7.41 ± 0.5 μmol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent K_m of 7.3 ± 1.9 μmol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with a apparent K_i of 183 ± 31 μmol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K_i = 0.85 · 10^?6 mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/ NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.     

No evidence of altered alveolar macrophage polarization,but reduced expression of TLR2, in bronchoalveolar lavage cells in sarcoidosis

Background

Sarcoidosis is a granulomatous inflammatory disease, possibly of infectious aetiology. We aimed to investigate whether the degree of functional polarization of alveolar macrophages (AMs), or Toll-like receptor (TLR) expression, is associated with sarcoidosis or with distinct clinical manifestations of this disease.

Methods

Total BAL cells (cultured four or 24 h in medium, or stimulated 24 h with LPS) from 14 patients and six healthy subjects, sorted AMs from 22 patients (Löfgren''s syndrome n = 11) and 11 healthy subjects, and sorted CD4+ T cells from 26 patients (Löfgren''s syndrome n = 13) and seven healthy subjects, were included. Using real-time PCR, the relative gene expression of IL-10, IL-12p35, IL-12p40, IL-23p19, CCR2, CCR7, iNOS, CXCL10, CXCL11, CXCL16, CCL18, CCL20, CD80, and CD86, and innate immune receptors TLR2, TLR4, and TLR9, was quantified in sorted AMs, and for selected genes in total BAL cells, while IL-17A was quantified in T cells.

Results

We did not find evidence of a difference with regard to alveolar macrophage M1/M2 polarization between sarcoidosis patients and healthy controls. TLR2 gene expression was significantly lower in sorted AMs from patients, particular in Löfgren''s patients. CCL18 gene expression in AMs was significantly higher in patients compared to controls. Additionally, the IL-17A expression was lower in Löfgren''s patients'' CD4+ T cells.

Conclusions

Overall, there was no evidence for alveolar macrophage polarization in sarcoidosis. However, there was a reduced TLR2 mRNA expression in patients with Löfgren''s syndrome, which may be of relevance for macrophage interactions with a postulated sarcoidosis pathogen, and for the characteristics of the ensuing T cell response.     

Cell recovery in bronchoalveolar lavage fluid in smokers is dependent on cumulative smoking history

Background

Smoking is a risk factor for various lung diseases in which BAL may be used as a part of a clinical investigation. Interpretation of BAL fluid cellularity is however difficult due to high variability, in particular among smokers. In this study we aimed to evaluate the effect of smoking on BAL cellular components in asymptomatic smokers. The effects of smoking cessation, age and gender were also investigated in groups of smokers and exsmokers.

Methods

We performed a retrospective review of BAL findings, to our knowledge the largest single center investigation, in our department from 1999 to 2009. One hundred thirty two current smokers (48 males and 84 females) and 44 ex-smokers (16 males and 28 females) were included. A group of 295 (132 males and 163 females) never-smokers served as reference.

Result

The median [5–95 pctl] total number of cells and cell concentration in current smokers were 63.4 [28.6–132.1]×10⁶ and 382.1 [189.7–864.3]×10⁶/L respectively and correlated positively to the cumulative smoking history. Macrophages were the predominant cell type (96.7% [90.4–99.0]) followed by lymphocytes (2% [0.8–7.7]) and neutrophils (0.6% [0–2.9]). The concentration of all inflammatory cells was increased in smokers compared to never smokers and ex-smokers. BAL fluid recovery was negatively correlated with age (p<0.001). Smoking men had a lower BAL fluid recovery than smoking women.

Conclusion

Smoking has a profound effect on BAL fluid cellularity, which is dependent on smoking history. Our results performed on a large group of current smokers and ex-smokers in a well standardized way, can contribute to better interpretation of BAL fluid cellularity in clinical context.     

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.     

Phenylethanolamine N-methyltransferase kinetics: bovine versus recombinant human enzyme.

Epinephrine (Epi) acts as a neurotransmitter in the brain, but its function therein is not well understood. Phenylethanolamine N-methyltransferase (PNMT) catalyzes the final step in the biosynthesis of Epi and is thus a pharmacological target to investigate the function of Epi in the central nervous system. The kinetic differences between bovine adrenal PNMT and human brain PNMT for a number of substrates and inhibitors are examined and the results reported.