PINCH proteins regulate cardiac contractility by modulating integrin-linked kinase-protein kinase B signaling

Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.     

NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.     

Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.     

EBV interferes with the sensitivity of Burkitt lymphoma Akata cells to etoposide

Burkitt lymphoma (BL) commonly exhibits Epstein-Barr virus (EBV) positivity associated with latent chronic infection. Models of acute EBV infection have been associated with cellular resistance to apoptosis. However, the effect of latent long-term EBV infection on apoptosis induced by drugs is not well defined. To determine this, we have studied the response of the Akata EBV+ cell line (type I latency) to etoposide, before and after downregulating EBV gene expression. We observed that downregulating EBV nuclear antigen-1 (EBNA-1) expression with siRNAs reverted cellular sensitivity to etoposide. In accordance with this finding, Akata EBV+ cells showed increased sensitivity to etoposide, when compared to the Akata EBV- cells. We also observed that Akata EBV+ cells presented increased apoptosis levels and decreased Bcl-xL mRNA and protein levels, when compared to the Akata EBV- cells. In addition, Akata EBV+ cells contained less endoplasmic reticulum (ER) than EBV- cells. Finally, downregulation of EBV with EBNA-1 siRNAs caused an increase in the expression of Bcl-xL indicating that EBV is responsible for the differences found between the Akata EBV+ and EBV- cell lines.     

How primary health care professionals and residents assess issues related to the oral health of older persons?

doi: 10.1111/j.1741‐2358.2009.00355.x
How primary health care professionals and residents assess issues related to the oral health of older persons? Background and objectives: It is known that older persons need integrated primary health care. However, oral health may not be a frequent concern of multi‐professional teams taking care of older persons. The aim of the present study was to evaluate knowledge and practices related to oral health care, as reported by professionals and residents in a primary health care service. Material and methods: One hundred and seventy‐three health professionals and residents were assessed in this cross‐sectional study by means of a structured questionnaire containing questions pertaining to oral health practices and beliefs. Participants were grouped based on their professions into “primary health care dentists” or “other primary health care professionals” and based on their working status into “permanent team” or “residents”. Results: Permanent team members (other professionals) assessed and recommended dental care more frequently than residents. Permanent team members (other professionals) also reported that they felt they were able to inform older patients in respect to oral health‐related issues more frequently than did residents (68.7% vs. 31.3%, respectively). Conclusion: Oral health‐related knowledge and beliefs were frequent among non‐dentists primary health care workers, suggesting that primary health care which integrates oral health represents an attainable goal.     

One-step enzymatic production of fatty acid ethyl ester from high-acidity waste feedstocks in solvent-free media

This work aims to demonstrate the enzymatic production of fatty acid ethyl ester biodiesel from highly acidic feedstock in a single-step reaction, without co-solvents and avoiding the inhibition of the enzyme by ethanol and glycerol. Additionally, an empirical equation is proposed to predict the kinetics of the production reaction as a function of the used feedstock and catalyst concentration. Biodiesel production from highly acidic feedstock perform via simultaneous esterification of free fatty acids and transesterification of triacylglycerols. Enzymatic catalysis is one of the most promising alternative technologies for the biodiesel production. Increasing of the enzymatic bioactivity is crucial for the success of such process in industrial scale. Currently, stepwise addition of the alcohol or the use of co-solvents have been proposed to avoid enzyme inhibition, such strategies add downstream processes to the production. These results can be applied to the development economical-viable enzymatic production of biodiesel in industrial scale.     

6-Methylnitroarachidonate: a novel esterified nitroalkene that potently inhibits platelet aggregation and exerts cGMP-mediated vascular relaxation

Nitro-fatty acids represent endogenously occurring products of oxidant-induced nitration reactions. We have previously synthesized a mixture of four isomers of nitroarachidonic acid, a novel anti-inflammatory signaling mediator. In this study, we synthesized and chemically and biologically characterized for the first time an esterified nitroalkene derived from the nitration of methylarachidonate (AAMet): 6-methylnitroarachidonate (6-AAMetNO(2)). Synthesis was performed by reacting AAMet with sodium nitrite under acidic conditions. Analysis by mass spectrometry (positive-ion ESI-MS) showed an [M+H](+) ion of m/z 364, characteristic of AAMetNO(2). Fragmentation of this ion yielded a daughter ion at m/z 317, corresponding to the neutral loss of the nitro group ([M+H-HNO(2)](+)). Furthermore, IR signal at 1378 cm(-1) and NMR data confirmed the structure of a 6-nitro-positional isomer. This novel esterified nitroalkene was capable of promoting vascular protective actions including: (a) the induction of vasorelaxation via endothelium-independent mechanisms, associated with an increase in smooth muscle cell cGMP levels, and (b) a potent dose-dependent inhibition of human platelet aggregation. We postulate that 6-AAMetNO(2) could be a potential drug for the prevention of vascular and inflammatory diseases, and the presence of the methyl group may increase its pharmacological potential.     

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G_s proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G_s and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G_s protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G_s and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B.Our data reveal that NDPK B regulates G_s function by two different mechanisms. The complex formation of NDPK B with G_s is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G_s activation.     

Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.     

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli

Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.