Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium <Emphasis Type="Italic">Micrococcus luteus</Emphasis> K-3

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.     

Effects of SM-20550, a selective Na+-H+ exchange inhibitor,on the ion transport of myocardial mitochondria

The study investigated the influence of L-carnitine on the formation of malondialdehyde, an indicator of lipid peroxidation, in isolated Langendorff rat hearts. Earlier investigations of hemodynamic parameters and the recovery of ATP and creatine phosphate, carried out by means of ³¹P-NMR spectroscopy, had demonstrated that, depending on the composition of the perfusates (content of glucose, fatty acids, and carnitine), quite strong differences may occur in the reperfusion period after ischemia.In order to determine a possible relationship between these differences and the addition of carnitine, the study investigated whether carnitine penetrated into the tissue during the experiments, and whether it was able to reduce the concentration of detrimental substances. The concentrations of free and total carnitine as well as the malondialdehyde content as an indicator of ischemia/reperfusion damage were determined in different parts of the cardiac tissue as follows: After the Langendorff-experiments the hearts were dissected, homogenized and reconditioned; then carnitine and malondialdehyde were determined. The study included 63 hearts, which were divided into 8 different perfusion groups.Carnitine concentrations in heart tissue perfused with L-carnitine were much higher than those of the controls. Since exogenous L-carnitine and formed esters could be found in the tissue after the experiment, they must have permeated the cellular membrane rapidly. The concentrations of malondialdehyde behaved in an inverted way; as expected they were lower in carnitine-perfused hearts. The favourable effects of L-carnitine, expressed both by improved cardiac dynamics and ATP and CrP recovery in the reperfusion period, are obviously due to the fact that L-carnitine reduces ischemic damage.     

CTNNB1 S45F Mutation Predicts Poor Efficacy of Meloxicam Treatment for Desmoid Tumors: A Pilot Study

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

The Burden and Etiology of Community-Onset Pneumonia in the Aging Japanese Population: A Multicenter Prospective Study

Background

The increasing burden of pneumonia in adults is an emerging health issue in the era of global population aging. This study was conducted to elucidate the burden of community-onset pneumonia (COP) and its etiologic fractions in Japan, the world’s most aged society.

Methods

A multicenter prospective surveillance for COP was conducted from September 2011 to January 2013 in Japan. All pneumonia patients aged ≥15 years, including those with community-acquired pneumonia (CAP) and health care-associated pneumonia (HCAP), were enrolled at four community hospitals on four major islands. The COP burden was estimated based on the surveillance data and national statistics.

Results

A total of 1,772 COP episodes out of 932,080 hospital visits were enrolled during the surveillance. The estimated overall incidence rates of adult COP, hospitalization, and in-hospital death were 16.9 (95% confidence interval, 13.6 to 20.9), 5.3 (4.5 to 6.2), and 0.7 (0.6 to 0.8) per 1,000 person-years (PY), respectively. The incidence rates sharply increased with age; the incidence in people aged ≥85 years was 10-fold higher than that in people aged 15-64 years. The estimated annual number of adult COP cases in the entire Japanese population was 1,880,000, and 69.4% were aged ≥65 years. Aspiration-associated pneumonia (630,000) was the leading etiologic category, followed by Streptococcus pneumoniae-associated pneumonia (530,000), Haemophilus influenzae-associated pneumonia (420,000), and respiratory virus-associated pneumonia (420,000), including influenza-associated pneumonia (30,000).

Conclusions

A substantial portion of the COP burden occurs among elderly members of the Japanese adult population. In addition to the introduction of effective vaccines for S. pneumoniae and influenza, multidimensional approaches are needed to reduce the pneumonia burden in an aging society.     

Chromene-3-carboxamide derivatives discovered from virtual screening as potent inhibitors of the tumour maker,AKR1B10

A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a therapeutic target in the treatment of several types of cancer. In order to identify potential leads for new inhibitors of AKR1B10, we adopted the virtual screening approach using the automated program icm, which resulted in the discovery of several chromene-3-carboxamide derivatives as potent competitive inhibitors. The most potent (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide inhibited the reductase activity of AKR1B10 with a K_i value of 2.7 nM, and the metabolism of farnesal and 4-hydroxynonenal in the AKR1B10-overexpressed cells from 0.1 μM with an IC₅₀ value equal to 0.8 μM.     

Expression and effects of epidermal growth factor on human periodontal ligament cells

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.     

Discovery of a Eukaryotic Pyrroloquinoline Quinone-Dependent Oxidoreductase Belonging to a New Auxiliary Activity Family in the Database of Carbohydrate-Active Enzymes

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.