Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

Background: In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis.
Results: Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress.
Conclusions: Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.

TAZ Expression as a Prognostic Indicator in Colorectal Cancer

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

The variability in nutrient composition of Anaerobic Digestate granules produced from high shear granulation

This study investigates the production of organic fertilizer using Anaerobic Digestate (as a nutrient source) and limestone powder as the raw materials. A two-level factorial experimental design was used to determine the influence of process variables on the nutrient homogeneity within the granules. Increasing the liquid-to-solid ratio during granulation resulted in increased granule nutrient homogeneity. Increasing the processing time and the impeller speed were also found to increase the nutrient homogeneity. In terms of nutrients release into deionized water, the granules effectively released both potassium and phosphate into solution. © 2012 Elsevier Ltd.

Profiling the ionome of rice and its use in discriminating geographical origins at the regional scale, China

Element profile was investigated for their use to trace the geographical origin of rice (Oryza sativa L.) samples. The concentrations of 13 elements (calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), boron (B), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), arsenic (As), selenium (Se), molybdenum (Mo), and cadmium (Cd)) were determined in the rice samples by inductively coupled plasma optical emission and mass spectrometry. Most of the essential elements for human health in rice were within normal ranges except for Mo and Se. Mo concentrations were twice as high as those in rice from Vietnam and Spain. Meanwhile, Se concentrations were three times lower in the whole province compared to the Chinese average level of 0.088 mg/kg. About 12% of the rice samples failed the Chinese national food safety standard of 0.2 mg/kg for Cd. Combined with the multi-elemental profile in rice, the principal component analysis (PCA), discriminant function analysis (DFA) and Fibonacci index analysis (FIA) were applied to discriminate geographical origins of the samples. Results indicated that the FIA method could achieve a more effective geographical origin classification compared with PCA and DFA, due to its efficiency in making the grouping even when the elemental variability was so high that PCA and DFA showed little discriminatory power. Furthermore, some elements were identified as the most powerful indicators of geographical origin: Ca, Ni, Fe and Cd. This suggests that the newly established methodology of FIA based on the ionome profile can be applied to determine the geographical origin of rice.

Detection and partial characterization of human B cell colony stimulating activity in synovial fluids of patients with rheumatoid arthritis

The joint fluids of 37 patients with rheumatoid arthritis, eight patients with traumatic injuries to their joints, two patients with Reiter's syndrome and three patients with psoriatic arthritis were tested for the presence of B cell colony stimulating activity (B cell CSA). B cell CSA was found in all of the joint fluids from the patients with rheumatoid arthritis but in none of the joint fluids from patients with traumatic injuries to their joints or in the joint fluids from the patients with Reiter's syndrome. A trace of B cell CSA was found in the joint fluid of one of the three patients with psoriatic arthritis. There was a positive correlation (r = 0.796) between the amount of rheumatoid factor present in the joint fluids and the titre of B cell CSA. This correlation was highly significant (P less than 0.001). The B cell CSA was localized to component(s) with molecular weight ranges 115-129 kD and 64-72 kD and an isoelectric point of 6.8. Its activity was sensitive to reduction with 2-mercaptoethanol and to the oxidising action of potassium periodate.

Biochemical and physiochemical characteristics of a pre-B cell stimulating factor produced by the plasmacytoma cell line LICR LON HMY2

We have characterized the pre-B cell colony stimulating activity (pre-B cell CSA) from LICR LON HMY2 conditioned medium (CM) by a variety of biochemical techniques. Pre-B cell CSA was found to be associated with a heat stable glycoprotein which has an isoelectric point of 8.3 and a mol. wt, as determined by polyacrylamide gel electrophoresis, of 28-32 kD. The relationship of this activity to previously described factors acting on cells of the B cell lineage is discussed.

Management of hyperkalaemia

Hyperkalaemia, an elevated extracellular fluid potassium concentration, is a common electrolyte disorder and is present in 1-10% of hospitalised patients. Elevated serum potassium concentrations are usually asymptomatic but may be associated with electrocardiogram (ECG) changes. Hyperkalaemia occasionally leads to life-threatening cardiac arrhythmias. Prompt recognition of this disorder, patient risk management and administration of appropriate treatment can prevent serious cardiac complications of hyperkalaemia. Further assessment of the underlying basis for hyperkalaemia usually reveals a problem with renal potassium excretion (rather than transcellular shift of potassium or excess potassium intake). Reduced potassium excretion is typically associated with decreased potassium secretion in the aldosterone-sensitive distal nephron of the kidney. Common causes for hyperkalaemia include kidney failure, limited delivery of sodium and water to the distal nephron and drugs that inhibit the renin-angiotensin-aldosterone system. Treatment of life-threatening hyperkalaemia (particularly those patients with ECG changes) involves administration of intravenous calcium salts to stabilise the resting cardiac membrane potential. The potassium concentration can be lowered by administration of intravenous insulin combined with an infusion of glucose to stimulate intracellular uptake of potassium. Nebulised β-2 adrenoceptor agonists can augment the effects of intravenous insulin and glucose pending more definitive management of the recurrent hyperkalaemia risk. Additional management steps include stopping further potassium intake and careful review of prescribed drugs that may be adversely affecting potassium homeostasis. Changes to prescribing systems and an agreed institutional protocol for management of hyperkalaemia can improve patient safety for this frequently encountered electrolyte disorder.