Expression of K2P5.1 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Introduction

CD4⁺ T cells express K_2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K_2P5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K_2P5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients.

Methods

Expression levels of K_2P5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K_2P5.1.

Results

K_2P5.1 expression levels in CD4⁺ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K_2P5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K_2P5.1 expression to disease activity parameters during a longitudinal follow-up for six months.

Conclusions

Disease activity in RA patients correlates strongly with K_2P5.1 expression levels in CD4⁺ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K_2P5.1 as a potential biomarker for disease activity and differential diagnosis.     

Prevention of airway inflammation with topical cream containing imiquimod and small interfering RNA for natriuretic peptide receptor

Background

For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants.

Methods

To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34⁺ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls.

Results

Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)⁺ cells; BV173: 9-fold, 37% ± 2% GFP⁺ cells; Lama84: 36-fold, 29% ± 2% GFP⁺ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP⁺ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP⁺ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP⁺ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed.

Conclusion

Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors.     

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

Ecosystem changes in Galápagos highlands by the invasive tree Cinchona pubescens

Background and aims

Salinity is an increasing problem for agricultural production worldwide. Understanding how Na⁺ enters plants is important if reducing Na⁺ influx, a key component of the regulation of Na⁺ accumulation in plants and improving salt tolerance of crop plants, is to be achieved. Our previous work indicated that two distinct low-affinity Na⁺ uptake pathways exist in the halophyte Suaeda maritima. Here, we report the external NaCl concentration at which uptake switches from pathway 1 to pathway 2 and the kinetics of the interaction between external K⁺ concentration and Na⁺ uptake and accumulation in S. maritima in order to determine the roles of K⁺ transporters or channels in low-affinity Na⁺ uptake.

Methods

Na⁺ influx, Na⁺ and K⁺ accumulations in S. maritima exposed to various concentrations of NaCl (0–200 mM) were analyzed in the absence and presence of the inhibitors TEA and Ba⁺ (5 mM TEA or 3 mM Ba²⁺) or KCl (0, 10 or 50 mM).

Results

Our earlier proposal was confirmed and extended that there are two distinct low-affinity Na⁺ uptake pathways in S. maritima: pathway 1 might be mediated by a HKT-type transporter under low salinity conditions and pathway 2 by an AKT1-type channel or a KUP/HAK/KT type transporter under high salinity conditions. The external NaCl concentration at which two distinct low-affinity Na⁺ uptake switches from pathway 1 to pathway 2, the ‘turning point’, is between 90 and 95 mM. Over a short period (12 h) of Na⁺ and K⁺ treatments, a low concentration of K⁺ (10 mM) facilitated Na⁺ uptake by S. maritima under high salinity (100–200 mM NaCl), whether or not the plants had been subjected to a longer (3 d) period of K⁺ starvation. The kinetics suggests that low concentration of K⁺ (10 mM) might activate AKT1-type channels or KUP/HAK/KT-type transporters under high salinity (100–200 mM NaCl).

Conclusions

The turning-point of external NaCl concentrations for the two low-affinity Na⁺ uptake pathways in Suaeda maritima is between 90 and 95 mM. A low concentration of K⁺ (10 mM) might activate AKT1 or KUP/HAK/KT and facilitate Na⁺ uptake under high salinity (100–200 mM NaCl). The kinetics of K⁺ on Na⁺ uptake and accumulation in S maritima are also consistent with there being two low-affinity Na⁺ uptake pathways.     

Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas

Background

Altered levels of erythrocyte Na⁺K⁺-ATPase, atherogenic and anti-atherogenic lipid metabolites have been implicated in diabetic complications but their pattern of interactions remains poorly understood. This study evaluated this relationship in Nigerian patients with Type 1 diabetes mellitus.

Methods

A total of 34 consented Type 1 diabetic patients and age -matched 27 non-diabetic controls were enrolled. Fasting plasma levels of total cholesterol, triglycerides and HDL-cholesterol were determined spectrophotometrically and LDL-cholesterol estimated using Friedewald formula. Total protein content and Na+K+-ATPase activity were also determined spectrophotometrically from ghost erythrocyte membrane prepared by osmotic lysis.

Results

Results indicate significant (P < 0.05) reduction in Na⁺K⁺-ATPase activity in the Type 1 diabetic patients (0.38 ± 0.08 vs. 0.59 ± 0.07 uM Pi/mgprotein/h) compared to the control but with greater reduction in the diabetic subgroup with poor glycemic control (n = 20) and in whom cases of hypercholesterolemia (8.8%), hypertriglyceridemia (2.9%) and elevated LDL-cholesterol (5.9% each) were found. Correlation analyses further revealed significant (P < 0.05) inverse correlations [r = -(0.708-0.797] between all the atherogenic lipid metabolites measured and Na⁺K⁺-ATPase in this subgroup contrary to group with good glycemic control or non-diabetic subjects in which significant (P < 0.05) Na⁺K⁺-ATPase and HDL-C association were found (r = 0.427 - 0.489). The Na⁺K⁺-ATPase from the diabetic patients also exhibited increased sensitivity to digoxin and alterations in kinetic constants Vmax and Km determined by glycemic status of the patients.

Conclusion

It can be concluded that poor glycemic control evokes greater reduction in erythrocyte Na⁺K⁺-ATPase activity and promote enzyme-blood atherogenic lipid relationships in Type 1 diabetic Nigerian patients.     

Effect of sugars,Na+-, and K+-ions on biopterin transport in Crithidia fasciculata

Biopterin uptake by Crithidia fasciculata is pH dependent with optimum at pH 6 and is strongly inhibited by 0.5 mM NAA and DNP,respectively. Both inhibitors also reduce respiration by 40% (NAA) and 97% (DNP). K⁺-ions (1.1%) and K⁺/Na⁺ (0.5% each) stimulate biopterin uptake to the same high extent, but ouabain has no effect, thereby ruling out involvement of Na⁺/K⁺ pump. In absence of these ions, even in 5% glucose solution biopterin uptake is reduced to minimum. Proton excretion seems to be linked to sugar uptake. Both these sugars seem to have the same site of entry, demonstrated by competitive uptake, though D-glucose is taken up much faster by Crithidia than D-galactose. DNP (0.5 mM) causes greater proton excretion in glucose than in galactose medium. With NAA (0.5 mM) proton excretion is inhibited in both glucose and galactose media. D-glucose promotes greater biopterin uptake than D-galactose.     

Salt accumulation and depletion in the root-zone of the halophyte Atriplex nummularia Lindl.: influence of salinity,leaf area and plant water use

Background and aims

Salt is known to accumulate in the root-zone of Na⁺ excluding glycophytes under saline conditions. We examined the effect of soil salinity on Na⁺ and Cl^? depletion or accumulation in the root-zone of the halophyte (Atriplex nummularia Lindl).

Methods

A pot experiment was conducted in soil to examine Na⁺ and Cl^? concentrations adjacent to roots at four initial NaCl treatments (20, 50, 200 or 400 mM NaCl in the soil solution). Plant water use was manipulated by leaving plants with all leaves intact, removing approximately 50 % of leaves, or removing all leaves. Daily evapotranspiration was replaced by watering undrained pots to weight with deionised water. After 35-38 days, samples were taken of the bulk soil and of soil loosely- and closely-adhering to the roots.

Results

In plants with leaves intact grown with 200 and 400 mM NaCl, average Na⁺ and Cl^? concentrations in the closely adhering soil were about twice the concentrations of the bulk soil. Ion accumulation increased with final leaf area and with cumulative transpiration over the duration of the trial. By contrast, in plants grown with the lowest salinity treatment (20 mM NaCl), Na⁺ and Cl^? concentrations decreased in the closely adhering soil with increasing leaf area and increasing cumulative water use.

Conclusions

Our data show that Na⁺ and Cl^? are depleted from the root-zone of A. nummularia at low salinity but accumulate in the root-zone at moderate to high salinity, and that the ions are drawn towards the plant in the transpiration stream.     

Molecular cloning and expression analysis of a gene encoding KUP/HAK/KT-type potassium uptake transporter from Cryptomeria japonica

Key message

The molecular mechanism of potassium ion transport across membranes in conifers is poorly known. We isolated and analyzed a gene encoding a potassium transporter from the conifer Cryptomeria japonica.

Abstract

Potassium ion (K⁺) is an essential and the most abundant intracellular cation in plants. The roles of K⁺ in various aspects of plant life are closely linked to its transport across biological membranes such as the plasma membrane and the tonoplast, which is mediated by membrane-bound transport proteins known as transporters and channels. Information on the molecular basis of K⁺ membrane transport in trees, especially in conifers, is currently limited. In this study, we isolated one complementary DNA, CjKUP1, which is homologous to known plant K⁺ transporters, from Cryptomeria japonica. Complementation tests using an Escherichia coli mutant, which is deficient in K⁺ uptake activity, was conducted to examine the K⁺ uptake function of the protein encoded by CjKUP1. Transformation of the K⁺-uptake-deficient mutant with CjKUP1 complemented the deficiency of this mutant. This result indicates that CjKUP1 has a function of K⁺ uptake. The expression levels of CjKUP1 in male strobili were markedly higher from late September to early October than in other periods. The expression levels in male and female strobili were higher than those in other organs such as needles, inner bark, differentiating xylem, and roots. These results indicate that CjKUP1 is mainly involved in K⁺ membrane transport in the cells of reproductive organs of C. japonica trees, especially in male strobili during pollen differentiation.     

Tolbutamide-sensitive potassium conductance in the basolateral membrane of A6 cells

K⁺ channels sensitive to intracellular ATP (K_ATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K⁺ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of ?83 ± 1 mV and was inward rectifying in spite of the outwardly directed K⁺ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μm and 0.114 ± 0.018 μm for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mm) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K⁺ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP.     

Increased prevalence of tumour infiltrating immune cells in oropharyngeal tumours in comparison to other subsites: relationship to peripheral immunity

Background

The nature of the tumour microenvironment immune response in head and neck cancer patients has an important role in tumour development and metastasis, but it is unknown if this differs between cancer subsites or whether it is related to the peripheral immune response.

Methods

Immune cells (CD4, CD8, Foxp3) in head and neck squamous cell carcinoma tissue (HNSCC; n = 66), detected by immunohistochemistry, have been correlated with tumour subsite and immune cells in the peripheral circulation (CD4⁺CD25^HighFoxp3⁺ Treg and CD4⁺ T cells), identified using flow cytometry.

Results

Oropharyngeal tumours had a greater number of infiltrating immune cells in both tumour and stroma compared with other subsites, but no difference was observed in the circulating levels. Immune cells in the stroma were positively related to those in the tumour with consistently higher levels in stroma. A strong relationship was found between the number of CD4⁺ and Foxp3⁺ cells but not between the number of CD8⁺ and Foxp3⁺ cells in the tumour. The number of Foxp3⁺ cells within the tumour was positively correlated with the percentage of circulating CD4⁺CD25^High cells positive for Foxp3. Late stage laryngeal tumours showed a higher number of Foxp3⁺ lymphocytes compared with early stage malignancies, and oropharyngeal tumours had more CD4⁺ cells in node negative tumours compared with node positive ones.

Conclusion

The level of immune cell infiltration in head and neck squamous cell carcinoma appears to be subsite dependent residing primarily in the stroma and is likely to be dependent on the peripheral immune response.     

Kinetics and mechanisms of cowpea root adaptation to changes in solution calcium

Background and aims

Close regulation of cellular Ca in roots is required in the face of marked changes in soil solution Ca over time and space. This study’s aims were to quantify and gain insights into the ways in which roots respond to changes in solution Ca.

Methods

Root elongation rate (RER) of cowpea (Vigna unguiculata (L.) Walp.) seedlings was determined at 0.05 to 15 mM Ca for up to 24 h both without and with added K, Mg, or Na. Root tip concentrations of Ca, K, Mg, and Na were determined and binding of cations by root tips estimated by subsequent Cu sorption.

Results

Transfer from higher to lower Ca solutions (and with added K at high Ca) resulted in RER?≥?2 mm h^?1 within minutes. This was attributed to greater cell wall relaxation through lower Ca binding aided by a decrease to pH?≤?5.1 in solution. Transfer to higher Ca solutions, which remained at ~pH 5.6, led to an equally rapid decrease in RER to ~0.5 mm h^?1, an effect ascribed to greater cell wall binding of Ca. Thereafter, a gradual increase in RER to ~1.8 mm h^?1 occurred over 24 h, an effect likely due to reduced cell wall Ca binding as shown by decreasing Cu sorption at a rate of 0.027 mmol Cu kg^?1 FM h^?1 over 24 h.

Conclusion

The kinetics of changes in RER and cations in root tips suggest that roots respond to changes in solution Ca through effects on cell wall relaxation of the rhizodermis and outer cortex in the elongation zone.     

cDNA Microarray Analysis of Changes in Gene Expression Associated with MPP+ Toxicity in SH-SY5Y Cells

cDNA microarray analysis of 1-methyl-4-phenyl-pyridinium (MPP⁺) toxicity (1 mM, 72 h) in undifferentiated SH-SY5Y cells identified 48 genes that displayed a signal intensity greater than the mean of all differentially expressed genes and a two-fold or greater difference in normalized expression. RT-PCR analysis of a subset of genes showed that c-Myc and RNA-binding protein 3 (RMB3) expression decreased by 50% after 72 h of exposure to MPP⁺ (1 mM) but did not change after 72 h of exposure to 6-hydroxydopamine (25 M), rotenone (50 nM), and hydrogen peroxide (600 M). Exposure of retinoic acid (RA)-differentiated SH-SY5Y cells to MPP⁺ (1 mM, 72 h) also resulted in a decrease in RMB3 expression and an increase in GADD153 expression. In contrast, c-Myc expression was slightly increased in RA-differentiated cells. Collectively, these data provide new insights into the molecular mechanisms of MPP⁺ toxicity and show that MPP⁺ can elicit distinct patterns of gene expression in undifferentiated and RA-differentiated SH-SY5Y cells.