A novel family VII esterase with industrial potential from compost metagenomic library

<p>Abstract</p> <p>Background</p> <p>Among the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of <it>estCS2 </it>was selected for lipolytic properties on a tributyrin-containing medium.</p> <p>Results</p> <p>The <it>estCS2 </it>sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45%) the carboxylesterase from <it>Haliangium ochraceum </it>DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser₂₄₅-Glu₃₆₃-His₄₆₆that is typical of an α/β hydrolase. The Ser₂₄₅residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward <it>p</it>-nitrophenyl caproate (C6), and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v) dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries.</p> <p>Conclusions</p> <p>The high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.</p

Transcortical Alterations in Na+-K+ ATPase and Microtubule-Associated Proteins Immunoreactivity in the Rat Cortical Atrophy Model Induced by Hypoxic Ischemia

To identify the chronological transcortical change in the contralateral hemisphere following ischemic insults, we investigated the changes in microtubule associated protein (MAP) and Na+-K+ ATPase expressions in the peri-infarct zone and contralateral hemisphere, including the hippocampus. Two days after hypoxic ischemia, Na+-K+ ATPase immunoreactivity was significantly enhanced in the contralateral cortex and was maintained up to 7 days after ischemia, whereas Na+-K+ ATPase immunoreactivity in the peri- and infarct zones was unaffected by hypoxic ischemia. In contrast, 2 to 7 days after ischemia, MAP1A and MAP2 immunoreactivity in the ipsi- and contralateral cortex significantly decreased, whereas in layer V, MAP1 immunoreactivity obviously accumulated in the neurons and their processes. In the hippocampus, 2 days after insults both MAP1A and MAP2 immunoreactivity was significantly reduced within the ipsi- and contralateral hippocampus. In the contralateral hippocampus, however, the distribution of MAP2 immunoreactivity recovered to the sham level 7 days after ischemia, whereas MAP1A immunoreactive axons remained 2 months after ischemia. The results suggest that the unilateral elevation of Na+-K+ ATPase immunoreactivity reflects elevated neuronal activity. In addition, this asymmetric hyperexcitability might play an important role in the recovery or the reorganization of the brain, accompanied by transcortical changes in MAPs expression

Comparison of Anti-Inflammatory Effects of Flavonoid-Rich Common and Tartary Buckwheat Sprout Extracts in Lipopolysaccharide-Stimulated RAW 264.7 and Peritoneal Macrophages

Buckwheat sprouts have been widely consumed all around world due to their great abundance of bioactive compounds. In this study, the anti-inflammatory effects of flavonoid-rich common buckwheat sprout (CBS) and tartary buckwheat sprout (TBS) extracts were evaluated in lipopolysaccharide- (LPS-) stimulated RAW 264.7 murine macrophages and primary peritoneal macrophages from male BALB/c mice. Based on the reversed-phase HPLC analysis, the major flavonoids in CBS were determined to be C-glycosylflavones (orientin, isoorientin, vitexin, and isovitexin), quercetin-3-O-robinobioside, and rutin, whereas TBS contained only high amounts of rutin. The TBS extract exhibited higher inhibitory activity as assessed by the production of proinflammatory mediators such as nitric oxide and cytokines including tumor necrosis factor-α, interleukin- (IL-) 6, and IL-12 in LPS-stimulated RAW 264.7 macrophages than CBS extract. In addition, TBS extract suppressed nuclear factor-kappa B activation by preventing inhibitor kappa B-alpha degradation and mitogen-activated protein kinase phosphorylation in LPS-stimulated RAW 264.7 macrophages. Moreover, the TBS extract markedly reduced LPS-induced cytokine production in peritoneal macrophages. Taken together, these findings suggest that TBS extract can be a potential source of anti-inflammatory agents that may influence macrophage-mediated inflammatory disorders

Comparison of acute phase reactant levels of Kawasaki disease patients who visited with less than 5 days duration of fever and with 5 days or longer

Purpose Kawasaki disease (KD) is a common, acute systemic vasculitis in children. Acute phase reactants (APRs) have been used to assist diagnosis, and to predict outcome in children with KD. However, it remains unknown on levels of APRs depending on duration of fever. We aimed to compare APR levels of children with KD who visited with < 5 days duration of fever and with ≥ 5 days. Methods Children (≤ 15 years) with complete KD who visited the emergency department were enrolled from March 2012 through February 2018. The children were divided into the early (fever < 5 days) and late (fever ≥ 5 days) presenters. The baseline characteristics, APR levels, such as platelet count, and outcomes were compared between the 2 groups. Results A total of 145 children with complete KD were enrolled. Median age was 27.0 (interquartile range [IQR], 12.0-46.5) months, and boys accounted for 60.0%. The early presenters (63 [43.4%]) had a younger age (17.0 [IQR, 7.0-45.0] vs. 32.5 [IQR, 14.0-48.0] months; P = 0.006), shorter duration of fever (3.0 [IQR, 2.0-4.0] days vs. 6.0 [IQR, 5.0-7.0] days; P < 0.001), and a lower platelet count (336.7 ± 105.2 [× 103/μL] vs. 381.6 ± 121.8 [× 103/μL], P = 0.02) than the late presenters. The other APR levels, and frequency of resistance to intravenous immunoglobulin and coronary artery abnormalities showed no differences between the 2 groups. Conclusion Children with KD who visited with < 5 days duration of fever had a lower platelet count compared to those with ≥ 5 days. No differences were found in the other APR levels and the outcomes. It may be necessary to consider the differences in APR levels depending on duration of fever when treating children with KD

Comparison of the prevalence and histology between primary benign bladder tumors and recurrent benign lesions after transurethral resection of malignant bladder tumors

Background Benign bladder tumors are rare disease entities, and insufficient studies have assessed their epidemiological characteristics. The authors investigated the prevalence of benign bladder tumors by retrospectively investigating pathology reports of transurethral resection of bladder tumor (TURBT) procedures over the past 20 years. Methods We analyzed 1,674 pathology reports of TURBT conducted in 1,160 patients from January 1, 2000, to April 30, 2022. The prevalence of benign tumors and histological classification according to the presence of primary (group 1) and recurrent (group 2) bladder lesions were retrospectively investigated. Results The mean age of patients was 65.2±11.5 years, and 1,284 cases (79.1%) were in men. Benign bladder tumors comprised 278 cases (248 patients) accounting for about 17.1% of the total TURBT cases (278/1,624). Furthermore, 184 patients (16.0%, 184/1,147) belonged to group 1 and 78 patients (27.4%, 78/285) belonged to group 2. Among all benign lesions that underwent TURBT, cystitis was the most common (41.0%, 114/278), and this rate was higher in group 2 (64/184 [34.8%] vs. 50/94 [53.2%], p<0.001). The prevalence of non-neoplastic lesions was higher in group 1 (44/184 [23.9] vs. 11/94 [11.7%], p<0.001). There was no difference in the prevalence of noninvasive urothelial neoplasms between the two groups (22/184 [12.0%] vs. 8/94 [8.5%], p=0.86). Conclusions The probability of benign lesions in TURBT was 17.1%, among which cystitis was the most common. When TURBT was performed for recurrent lesions, the frequency of benign tumors was higher than that of primary benign bladder tumors