Crystal structures of ADP and AMPPNP-bound propionate kinase (TdcD) from Salmonella typhimurium: comparison with members of acetate and sugar kinase/heat shock cognate 70/actin superfamily

Recently, it has been shown that l-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase (TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2A and 2.3A, respectively, by molecular replacement using Methanosarcina thermophila acetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology betabetabetaalphabetaalphabetaalpha, identical with that of glycerol kinase, hexokinase, heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. This was further confirmed by kinetic studies with the purified enzyme, which showed about ten times lower K(m) for propionate (2.3 mM) than for acetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well-characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure.     

Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle

The smooth muscle of the gut expresses mainly G(s) protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC(2) receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC(2) receptors via the phosphorylation of GRK2 at Ser(685). VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to G betagamma. VIP-induced phosphorylation of VPAC(2) receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC(2) receptor internalization (determined from residual (125)I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC(2) receptor degradation (determined from residual (125)I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC(2) receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC(2) receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC(2) receptor phosphorylation, internalization, desensitization, and degradation.     

Solid phase synthesis of quinolones.

Solid phase syntheses of ethyl 6-carboxyquinol-4(1H-)-one-3-carboxylate (5) and N-substituted 6-carboxyquinol-4(1H)-one-3-carboxamides 7a-d have been described. Antifilarial in vitro activities of 5,7a-d against Brugia malayi have also been delineated.     

Neurobiology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in neurobiology.     

In vitro propagation of the endangered orchid,Geodorum densiflorum (Lam.) Schltr. through rhizome section culture

Micropropagation of the endangered terrestrial orchid, Geodorum densiflorum (Lam.) Schltr. was achieved through rhizome section culture from in vitro seed- derived rhizomes. Rhizome sections were cultured on Murashige and Skoog (MS) and Knudson C(KC) media supplemented with various growth regulators and 0.1% activated charcoal. The rhizome sections responded on MS medium. Naphthaleneacetic acid (NAA) at 2.0 μM stimulated rhizome growth. However, benzyladenine (BA) at 5.0 μM induced multiple shoots within four weeks of culture and inhibited rhizome growth. The regenerated shoots rooted on MS only or with NAA at 1.0 μM. Well-developed plantlets were transferred to community pots and then to a greenhouse where the plants have been acclimatised. This revised version was published online in June 2006 with corrections to the Cover Date.     

Luminescence and dosimetry approach in terbium(III)-activated tungstate double perovskite

A series of tungstate double perovskite Ca₃WO₆ doped with Tb³⁺ was prepared by a combustion process using urea as a flux. The crystal structure identification of Ca₃WO₆:Tb³⁺ phosphors was done using X-ray diffraction patterns, and a monoclinic structure was discovered. The Fourier transform infrared spectrum of Ca₃WO₆:Tb³⁺ displayed characteristic vibrations of tungstate bonds. Under 278 nm excitation, Ca₃WO₆:Tb³⁺ exhibited intense downconversion green emission, which corresponded to the ⁵D₄–⁷F_J (J = 4,5) transitions of Tb³⁺. The phosphor exhibited the highest photoluminescence (PL) intensity when it was doped with 1 mol% of Tb³⁺; later intensity quenching appeared to be due to the multipolar interaction at higher dopant concentrations. Moreover, high-quality thermoluminescence (TL) was detected when phosphors were irradiated using beta rays. The effects of Tb³⁺ concentration and beta dose on TL intensity were the two major aspects studied in detail. The TL intensity demonstrated excellent linear response to the applied range of beta dose. The trap parameters of the studied phosphors were computed by the peak shape approach and glow curve deconvolution. The fading effect on TL intensity was studied by recording the TL glow curves after 1 month of beta irradiation. Obtained results from the PL and TL characterizations showed that the phosphors under study have the potential to be used in lighting displays and in thermoluminescence dosimetry.     

Optimization of mycophenolic acid production in solid state fermentation using response surface methodology

Mycophenolic acid (MPA) can be produced in solid state fermentation. An isolate of Penicillium brevi-compactum ATCC 16024 grown on moist wheat bran produced a titre of 425 mg per kg of wheat bran. Central composite rotatable design and response surface methodology were employed to derive a statistical model for media optimization towards production of mycophenolic acid. Five levels with a five factorial design were adopted. The correlation coefficient was 0.82, ensuring a satisfactory adjustment of the model to the experimental values. This statistical design was very effective in improving the titre of mycophenolic acid up to 3286 mg per kg of wheat bran. Received 24 July 1998/ Accepted in revised form 4 December 1998     

Use of an extended KDIGO definition to diagnose acute kidney injury in patients with COVID-19: A multinational study using the ISARIC–WHO clinical characterisation protocol

BackgroundAcute kidney injury (AKI) is one of the most common and significant problems in patients with Coronavirus Disease 2019 (COVID-19). However, little is known about the incidence and impact of AKI occurring in the community or early in the hospital admission. The traditional Kidney Disease Improving Global Outcomes (KDIGO) definition can fail to identify patients for whom hospitalisation coincides with recovery of AKI as manifested by a decrease in serum creatinine (sCr). We hypothesised that an extended KDIGO (eKDIGO) definition, adapted from the International Society of Nephrology (ISN) 0by25 studies, would identify more cases of AKI in patients with COVID-19 and that these may correspond to community-acquired AKI (CA-AKI) with similarly poor outcomes as previously reported in this population.Methods and findingsAll individuals recruited using the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC)–World Health Organization (WHO) Clinical Characterisation Protocol (CCP) and admitted to 1,609 hospitals in 54 countries with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection from February 15, 2020 to February 1, 2021 were included in the study. Data were collected and analysed for the duration of a patient’s admission. Incidence, staging, and timing of AKI were evaluated using a traditional and eKDIGO definition, which incorporated a commensurate decrease in sCr. Patients within eKDIGO diagnosed with AKI by a decrease in sCr were labelled as deKDIGO. Clinical characteristics and outcomes—intensive care unit (ICU) admission, invasive mechanical ventilation, and in-hospital death—were compared for all 3 groups of patients. The relationship between eKDIGO AKI and in-hospital death was assessed using survival curves and logistic regression, adjusting for disease severity and AKI susceptibility. A total of 75,670 patients were included in the final analysis cohort. Median length of admission was 12 days (interquartile range [IQR] 7, 20). There were twice as many patients with AKI identified by eKDIGO than KDIGO (31.7% versus 16.8%). Those in the eKDIGO group had a greater proportion of stage 1 AKI (58% versus 36% in KDIGO patients). Peak AKI occurred early in the admission more frequently among eKDIGO than KDIGO patients. Compared to those without AKI, patients in the eKDIGO group had worse renal function on admission, more in-hospital complications, higher rates of ICU admission (54% versus 23%) invasive ventilation (45% versus 15%), and increased mortality (38% versus 19%). Patients in the eKDIGO group had a higher risk of in-hospital death than those without AKI (adjusted odds ratio: 1.78, 95% confidence interval: 1.71 to 1.80, p-value < 0.001). Mortality and rate of ICU admission were lower among deKDIGO than KDIGO patients (25% versus 50% death and 35% versus 70% ICU admission) but significantly higher when compared to patients with no AKI (25% versus 19% death and 35% versus 23% ICU admission) (all p-values <5 × 10⁻⁵). Limitations include ad hoc sCr sampling, exclusion of patients with less than two sCr measurements, and limited availability of sCr measurements prior to initiation of acute dialysis.ConclusionsAn extended KDIGO definition of AKI resulted in a significantly higher detection rate in this population. These additional cases of AKI occurred early in the hospital admission and were associated with worse outcomes compared to patients without AKI.

Marina Wainstein and colleagues examine acute kidney injury (AKI) incidence, severity, and outcomes among patients with COVID-19 using both a traditional and extended definition of AKI.     

Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1beta

The pro-inflammatory cytokine IL-1beta contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel diseases. However, the mechanisms are not well understood. The effects of IL-1beta on the signaling targets mediating acetylcholine (ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, myosin light chain (MLC(20)) phosphorylation, and activation of PLC-beta and Rho kinase. Expression levels of the signaling targets were determined by Western blot analysis and real-time RT-PCR. Short interfering RNAs (siRNAs) for regulator of G protein signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1beta treatment of muscle strips inhibited both initial and sustained contraction and MLC(20) phosphorylation in isolated muscle cells. IL-1beta treatment increased RGS4 expression but had no effect on muscarinic receptor binding or Galpha(q) expression. In contrast, IL-1beta decreased the expression and phosphorylation of CPI-17 but had no effect on RhoA expression or ACh-induced Rho kinase activity. Upregulation of RGS4 and downregulation of CPI-17 by IL-1beta in muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1beta on initial contraction and PLC-beta activation, whereas overexpression of RGS4 inhibited PLC-beta activation. These data suggest that IL-1beta upregulates RGS4 expression, resulting in the inhibition of initial contraction and downregulation of CPI-17 expression during sustained contraction in colonic smooth muscle.     

Alterations in Adenosine Metabolism and Signaling in Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Background

Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.

Methodology/Principal Findings

The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A_2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A_2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A_2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A_2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.

Conclusions/Significance

These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A_2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.