Screening for primary aldosteronism in a cohort of Brazilian patients with resistant hypertension

The objective was to estimate the prevalence of plasma aldosterone concentration:plasma renin activity ratio >30 ng/dL:ng/mL/h in patients with resistant hypertension and to describe the computed tomography findings of adrenal glands in those with elevated ratios. In a cross-sectional design, 492 patients were enrolled. All patients with plasma aldosterone concentration:plasma renin activity ratio >or=30 ng/dL:ng/mL/h (n=77) underwent abdominal computed tomography. Patients with an adrenal image of possible aldosterone-producing adenoma underwent a saline-loading test. The prevalence of elevated plasma aldosterone concentration:plasma renin activity ratio was 15.7% (95% confidence interval, 12.6-19.2). Twelve patients showed adrenal abnormalities on computed tomography. The level of renin was low in 50% of the sample. Results indicate a low prevalence of aldosterone-producing adenoma. Our evidence points out the importance of confirming the hypothesis that essential hypertension, low-renin hypertension, and idiopathic hyperaldosteronism could be the same disease, but at different neurohormonal stages, and aldosterone-producing adenoma may be yet another disease.     

Successful Treatment of Carbapenemase-Producing Pandrug-Resistant Klebsiella pneumoniae Bacteremia

New antibiotic options are urgently needed for the treatment of carbapenem-resistant Enterobacteriaceae infections. We report a 64-year-old female with prolonged hospitalization following an intestinal transplant who developed refractory bacteremia due to a serine carbapenemase-producing pandrug-resistant isolate of Klebsiella pneumoniae. After failing multiple antimicrobial regimens, the patient was successfully treated.     

A low molecular weight OLED material: 2-(4-((2-hydroxyethyl)(methyl)amino)benzylidene)malononitrile. Synthesis,crystal structure,thin film morphology,spectroscopic characterization and DFT calculations

2-(4-((2-Hydroxyethyl)(methyl)amino)benzylidene)malononitrile (HEMABM) was synthesized from 4-[hydroxymethyl(methyl)amino]benzaldehyde and propanedinitrile to obtain a low molecular weight fluorescent material with an efficient solid-state emission and electroluminescence properties comparable to the well-known poly(2-methoxy-5(2′-ethyl)hexoxyphenylenevinylene) (MEH-PPV). The HEMABM was used to prepare an organic light-emitting diode by a solution process. Despite the title compound being a small molecule, it showed optical properties and notable capacity to form a film with smooth morphology (10.81 nm) closer to that of polymer MEH-PPV (10.63 nm). The preparation of the device was by spin coating, the electrical properties such as threshold voltage were about 1.0 V for both HEMABM and MEH-PPV, and the luminance 1300 cd m⁻² for HEMABM and 2600 cd m⁻² for MEH-PPV. This low molecular weight compound was characterized by SCXRD, IR, NMR, and EI. Besides a quantitative analysis of the intermolecular interactions by PIXEL, density functional theory (DFT) calculations are reported.

A low molecular weight fluorescent malononitrile derivative showed an efficient solid-state emission and electroluminescence properties.     

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.Cell perception of extracellular stimuli is followed by a transient variation in cytosolic calcium concentration. Plants have evolved to produce the specific molecular machinery to interpret this primary information and to transmit this signal to the components that organize the cell response (1–4). The plant family of serine/threonine protein kinases PKS or CIPKs (hereinafter CIPKs) and their activators, the calcium-binding proteins SCaBPs or CBLs (hereinafter CBLs) (5, 6) function together in decoding calcium signals caused by different environmental stimuli. Available data suggest a mechanism in which calcium mediates the formation of stable CIPK–CBL complexes that regulate the phosphorylation state and activity of various ion transporters involved in the maintenance of cell ion homeostasis and abiotic stress responses in plants. Among them, the Arabidopsis thaliana CIPK24/SOS2-CBL4/SOS3 complex activates the Na⁺/H⁺ antiporter SOS1 to maintain intracellular levels of the toxic Na⁺ low under salt stress (7–9), the CIPK11–CBL2 pair regulates the plasma membrane H⁺-ATPase AHA2 to control the transmembrane pH gradient (10), the CIPK23–CBL1/9 (11, 12) regulates the activity of the K⁺ transporter AKT1 to increase the plant K⁺ uptake capability under limiting K⁺ supply conditions (12, 13), and CIPK23–CBL1 mediates nitrate sensing and uptake by phosphorylation of the nitrate transporter CHL1 (14). Together these findings show that understanding the molecular mechanisms underling CIPKs function provides opportunities to increase plant tolerance to abiotic stress and to improve plants for human benefit.CIPKs and CBLs contain discrete structural modules that are involved in the calcium-dependent regulation of the activity of the system and ensure the colocalization of the CIPK–CBL interacting pairs with their substrates at particular sites within the cell (15–17). CIPKs include an N-terminal kinase catalytic domain followed by a characteristic self-inhibitory motif known as FISL or NAF motif (hereinafter NAF, Pfam no. PF03822) (1, 6) and a protein phosphatase 2C binding domain designated as PPI (11, 18, 19). The NAF motif directly interacts with the catalytic domain and inhibits the kinase activity. The calcium-dependent interaction of CBLs with the NAF motif relieves the self-inhibition and activates the CIPKs (5, 6, 19, 20). The calcium binding to CBLs is mediated by four EF hand-like calcium binding motives. In addition, several CBLs are myristoylated and/or palmitoylated. These modifications are essential for recruiting their interacting CIPK partner to the plasma or vacuolar membrane (17, 21–23), and they may also be involved in the interaction of the CIPK–CBL complexes with their substrates (24). In addition, the phosphorylation of a conserved serine residue at the C terminus of CBLs by its interacting CIPK is required for activation of transporter substrates. It has been proposed that this process may stabilize the CIPK–CBL complex and trigger conformational changes to the binary complex that enhance its specificity toward target proteins (13, 25).Like many other kinases, CIPKs are also regulated by the phosphorylation of the activation loop by upstream kinases. This loop undergoes large conformational changes upon phosphorylation, allowing the entrance and the stabilization of substrates at the kinase active site (26). The activation loop of the CIPKs contains three conserved Tyr, Thr, or Ser residues. For some members of the family, the mutation of one of these residues to Asp mimics phosphorylation and produces the activation of the kinase, partly overcoming the effect of the self-inhibitory NAF motif. In fact, these phosphorylation-mimicking mutations and the deletion of the inhibitory domain produce a synergistic effect on the CIPK activity (6, 27–29). Transgenic plants expressing these CIPK24/SOS2 mutant proteins show improved salt tolerance (30).The kinase self-phosphorylation is another regulatory mechanism used by CIPKs. CIPK24/SOS2 is able to self-phosphorylate, and the autophosphorylation is important for its activity (31). Although the default state of CIPKs is inactive, some degree of autophosphorylation activity has been observed even for dephosphorylated and CBL-unbound CIPKs, which suggests that some CIPKs display basal activity (6). Indeed, it has been shown that the general regulatory factor 14-3-3 proteins (32) interact with CIPK24/SOS2 and repress its basal kinase activity when plants are grown in the absence of salt stress (33).The crystal structure of the binary complex of Ca²⁺-CBL4/SOS3 with the C-terminal regulatory moiety of CIPK24/SOS2 revealed the molecular mechanism underlying CBL-mediated activation of the CIPKs. The structure showed that the CIPK24/SOS2 self-inhibitory NAF motif is bound to CBL4/SOS3 and, consequently, it is not accessible to the kinase domain (19, 20). However, whether the CBL-unbound NAF blocks the active site or inhibits the enzyme by an allosteric mechanism is not known. To determine the molecular and structural basis for the CIPKs autoinhibition by the NAF and the activation by upstream kinases, we solved the structures of CIPK23 and CIPK24/SOS2. Our data show that inactivation of the kinases relies on the blockage of the active site by the NAF motif and the activation loop, which constitutes the basis for the conserved NAF-mediated self-inhibition of the CIPKs.