Neonatal lethality in knockout mice expressing the kinase-dead form of the gefitinib target GAK is caused by pulmonary dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.     

Structure-based functional identification of a novel heme-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8

The TT1485 gene from Thermus thermophilus HB8 encodes a hypothetical protein of unknown function with about 20 sequence homologs of bacterial or archaeal origin. Together they form a family of uncharacterized proteins, the cluster of orthologous group COG3253. Using a combination of amino acid sequence analysis, three-dimensional structural studies and biochemical assays, we identified TT1485 as a novel heme-binding protein. The crystal structure reveals that this protein is a pentamer and each monomer exhibits a β-barrel fold. TT1485 is structurally similar to muconolactone isomerase, but this provided no functional clues. Amino acid sequence analysis revealed remote homology to a heme enzyme, chlorite dismutase. Strikingly, amino acid residues that are highly conserved in the homologous hypothetical proteins and chlorite dismutase cluster around a deep cavity on the surface of each monomer. Molecular modeling shows that the cavity can accommodate a heme group with a strictly conserved His as a heme ligand. TT1485 reconstituted with iron protoporphyrin IX chloride gave a low chlorite dismutase activity, indicating that TT1485 catalyzes a reaction other than chlorite degradation. The presence of a possible Fe–His–Asp triad in the heme proximal site suggests that TT1485 functions as a novel heme peroxidase to detoxify hydrogen peroxide within the cell.     

Roles of C-terminal processing, and involvement in transacylation reaction of human group IVC phospholipase A2 (cPLA2gamma)

The phospholipase A2s (PLA2s) are a diverse group of enzymes that hydrolyze the sn-2 fatty acid from phospholipids and play a role in a wide range of physiological functions. A 61-kDa calcium-independent PLA2, termed cPLA2gamma, was identified as an ortholog of cPLA2alpha with approximately 30% overall sequence identity. cPLA2gamma contains a potential prenylation motif at its C terminus, and is known to have PLA2 and lysophospholipase activities, but its physiological roles have not been clarified. In the present study, we expressed various forms of recombinant cPLA2gamma, including non-prenylated and non-cleaved forms, in order to investigate the effects of C-terminal processing. We examined the expression of the wild type and non-prenylated (SCLA) forms of cPLA2gamma, and found that the SCLA form was expressed normally and retained almost full activity. Expression of the prenylated and non-cleaved form of cPLA2gamma using yeast mutants lacking prenyl protein proteases AFC1 (a-factor-converting enzyme) and RCE1 (Ras-converting enzyme) revealed decreased expression in the mutant strain compared to that in the wild type yeast, suggesting that complete C-terminal processing is important for the functional expression of cPLA2gamma. In addition, cPLA2gamma was found to have coenzyme A (CoA)-independent transacylation and lysophospholipid (LPL) dismutase (LPLase/transacylase) activities, suggesting that it may be involved in fatty acid remodeling of phospholipids and the clearance of toxic lysophospholipids in cells.     

Synthesis, structure and property of manganese(II) complexes with mixed tetradentate imidazole-containing ligand and benzenedicarboxylate

Three new coordination complexes [Mn(L)(H₂O)₂](1,4-BDC)·2H₂O (1), [Mn(L)_0.5(1,4-BDC)]CH₃OH·H₂O (2) and [Mn(L)(H₂O)₂](1,2-HBDC)₂·2H₂O (3) were synthesized by solvothermal reactions of 1,2,4,5-tetrakis(imidazol-1-ylmethyl)benzene (L) and 1,4-benzenedicarboxylic acid (1,4-H₂BDC) or 1,2-benzenedicarboxylic acid (1,2-H₂BDC) with Mn(II) salt, and characterized by single crystal X-ray diffraction, IR, thermogravimetric and elemental analyses. In complexes 1 and 3, each ligand L links four Mn(II) atoms to form two-dimensional (2D) cationic network with non-coordinated 1,4-BDC²⁻ and 1,2-HBDC⁻ anions lying in the voids between the two adjacent layers, respectively. The 2D layers are further connected together by hydrogen bonds to give three-dimensional (3D) supramolecular structures. However, the 1,4-BDC²⁻ in 2 acts not only as counteranion, but also as bridging ligand leading to the formation of 2-fold interpenetrated 3D framework with pcu (primitive cubic unit) topology. The Mn(II) atoms bridged by carboxylate groups in 2 show antiferromagnetic interactions.     

Colonization and community structure of root-associated microorganisms of Sabina vulgaris with soil depth in a semiarid desert ecosystem with shallow groundwater

Arbuscular mycorrhizal fungi (AMF) have been observed in deep soil layers in arid lands. However, change in AMF community structure with soil depth and vertical distributions of the other root-associated microorganisms are unclear. Here, we examined colonization by AMF and dark septate fungi (DSF), as well as the community structure of AMF and endophytic fungi (EF) and endophytic bacteria (EB) in association with soil depth in a semiarid desert with shallow groundwater. Roots of Sabina vulgaris and soils were collected from surface to groundwater level at 20-cm intervals. Soil chemistry (water content, total N, and available P) and colonization of AMF and DSF were measured. Community structures of AMF, EF, and EB were examined by terminal restriction fragment length polymorphism analysis. AMF colonization decreased with soil depth, although it was mostly higher than 50%. Number of AMF phylotypes decreased with soil depth, but more than five phylotypes were observed at depths up to 100 cm. Number of AMF phylotypes had a significant and positive relationship with soil moisture level within 0-15% of soil water content. DSF colonization was high but limited to soil surface. Number of phylotypes of EF and EB were diverse even in deep soil layers, and the community composition was associated with the colonization and community composition of AMF. This study indicates that AMF species richness in roots decreases but is maintained in deep soil layers in semiarid regions, and change in AMF colonization and community structure associates with community structure of the other root-associated microorganisms.     

Efficacy of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on body weight, eating behavior, and glycemic control, in Japanese obese type 2 diabetes

ABSTRACT: BACKGROUND: We recently reported that short-term treatment with liraglutide (20.0 +/- 6.4 days) reduced body weight and improved some scales of eating behavior in Japanese type 2 diabetes inpatients. However, it remained uncertain whether such liraglutide-induced improvement is maintained after discharge from the hospital. The aim of the present study was to determine the long-term effects of liraglutide on body weight, glycemic control, and eating behavior in Japanese obese type 2 diabetics. METHODS: Patients with obesity (body mass index (BMI) >25 kg/m2) and type 2 diabetes were hospitalized at Osaka University Hospital between November 2010 and December 2011. BMI and glycated hemoglobin (HbA1c) were examined on admission, at discharge and at 1, 3, and 6 months after discharge. For the liraglutide group (BMI; 31.3 +/- 5.3 kg/m2, n = 29), patients were introduced to liraglutide after correction of hyperglycemic by insulin or oral glucose-lowering drugs and maintained on liraglutide after discharge. Eating behavior was assessed in patients treated with liraglutide using The Guideline For Obesity questionnaire issued by the Japan Society for the Study of Obesity, at admission, discharge, 3 and 6 months after discharge. For the insulin group (BMI; 29.1 +/- 3.0 kg/m2, n = 28), each patient was treated with insulin during hospitalization and glycemic control maintained by insulin after discharge. RESULTS: Liraglutide induced significant and persistent weight loss from admission up to 6 months after discharge, while no change in body weight after discharge was noted in the insulin group. Liraglutide produced significant improvements in all major scores of eating behavior questionnaire items and such effect was maintained at 6 months after discharge. Weight loss correlated significantly with the decrease in scores for recognition of weight and constitution, sense of hunger, and eating style. CONCLUSION: Liraglutide produced meaningful long-term weight loss and significantly improved eating behavior in obese Japanese patients with type 2 diabetes.