Role of pirenoxine in the effects of catalin on in vitro ultraviolet-induced lens protein turbidity and selenite-induced cataractogenesis in vivo

Purpose: In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model. Methods: In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was applied to analyze the integrity of crystallin samples. Results: PRX at 1,000 μM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 μM. Results were further confirmed by SDS–PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 μM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 μM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 μM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 μM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 μM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 μM PRX. Conclusions: PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted

Evaluation of Antioxidant and Free Radical Scavenging Capacities of Polyphenolics from Pods of Caesalpinia pulcherrima

Thirteen polyphenolics were isolated from fresh pods of Caesalpinia pulcherrima using various methods of column chromatography. The structures of these polyphenolics were elucidated as gallic acid (1), methyl gallate (2), 6-O-galloyl-d-glucoside (3), methyl 6-O-galloyl-β-d-glucoside (4), methyl 3,6-di-O-galloyl-α-d-glucopyranoside (5), gentisic acid 5-O-α-d-(6′-O-galloyl)glucopyranoside (6), guaiacylglycerol 4-O-β-d-(6′-O-galloyl)glucopyranoside (7), 3-methoxy-4-hydroxyphenol 1-O-β-d-(6′-O-galloyl) glucopyranoside (8), (+)-gallocatechin (9), (+)-catechin (10), (+)-gallocatechin 3-O-gallate (11), myricetin 3-rhamnoside (12), and ampelopsin (13). All isolated compounds were tested for their antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxynitrite radicals scavenging assays. Among those compounds, 11, 12, and 2 exhibited the best DPPH-, hydroxyl-, and peroxynitrite radical-scavenging activities, respectively. Compound 7 is a new compound, and possesses better scavenging activities towards DPPH but has equivalent hydroxyl radical scavenging activity when compared to BHT. The paper is the first report on free radical scavenging properties of components of the fresh pods of Caesalpinia pulcherrima. The results obtained from the current study indicate that the free radical scavenging property of fresh pods of Caesalpinia pulcherrima may be one of the mechanisms by which this herbal medicine is effective in several free radical mediated diseases

Recombinant Zebrafish ␥-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme

ABSTRACT: A cDNA encoding for zebrafish ␥-glutamyl hydrolase (␥GH) was cloned and inserted into a pET43.1a vector via SmaI and EcoRI sites and expressed in Rosetta (DE3) cells as a Nus-His-tag fusion enzyme (NH-z␥GH). After induction with isopropyl thiogalactoside, the enzyme was purified with a Ni-Sepharose column, and approximately 8 mg of pure enzyme was obtained per liter of culture. The primary sequence of the recombinant z␥GH was similar to mammalian ␥GH. Folate is an essential B vitamin and participates in the biosynthesis and metabolism of nucleic acids, proteins, several amino acids, methyl groups, many neurotransmitters, and some vitamins. Mammalian cells are unable to synthesize folates de novo and therefore depend on their food for the supply of folates. Naturally occurring folates are synthesized as poly-␥-glutamate forms (folylpolyglutamate) but are absorbed and transported most efficiently as folylmonoglutamates. The conversion of folylpolyglutamates in dietary food to folylmonoglutamates is catalyzed by carboxypeptidase II (EC 3.4.22.12) in mammals. In a recent study, ␥-glutamyl hydrolase (␥GH, EC 3.4.19.9), a lysosomal cysteine peptidase, was reported to be the enzyme responsible for hydrolyzing dietary folate in rat small intestine Consistent with this notion, the activity of ␥GH to hydrolyze the ␥-glutamyl peptide bonds of folylpolyglutamates has rendered this enzyme a potential target of antifolate chemotherapy and, at the same time, a primary component in regulating the intracellular levels of some antifolate drugs. Antifolate drugs, such as methotrexate, owe much of their effectiveness to being substrates for both folylpoly-␥-glutamate synthetase and ␥GH. Removal of ␥-linked glutamate residues decreases the retention and activity of these drugs. A polymorphism resulting in reduced catalytic activity of ␥GH was observed to be associated with greater accumulation of long-chain methotrexate polyglutamate forms The determination of individual folate derivatives in serum of patients receiving antifolate chemotherapy and in foods is an important current protocol. The first step in these determinations is converting folylpolyglutamates to folylmonoglutamates by ␥GH. Cur- The amino acid numbering used for z␥GH in this study is numbered starting from the first methionine in the full-length peptide with the signal peptide. Article, publication date, and citation information can be found a

Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

[[abstract]]BACKGROUND: Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10(4)/cm(2) ("high" seeding density) or 2x10(3)/cm(2) ("low" density). However, at high seeding density, Y-27632-treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632-treated CCE cells seeded at 2x10(3) or 6.6x10(3) cells/cm(2) did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2x10(3)/cm(2) did not change renewal factors. CONCLUSIONS/SIGNIFICANCE: We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells

Mixed Infections of Helicobacter pylori

Background. Persistent Helicobacter pylori infection may induce several upper gastrointestinal diseases. Two major virulence factors of H. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution of vacA and cag-pathogenicity island (cag-PAI) alleles varies in H. pylori isolated from patients in different geographic regions. Aim. To assess the association between mixed infection of H. pylori clinical isolates from Taiwanese patients and the severity of gastrointestinal diseases. Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes. Results. The prevalence of mixed infections of all H. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P<0.05). Conclusions. H. pylori mixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development

Polycythemia vera as a presentation of renal angiomyolipoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>Angiomyolipoma is a common benign renal tumor composed of thick-walled blood vessels, smooth muscle, and adipose tissue. It may be found incidentally during workup for suspected renal disease. Although angiomyolipoma may present as a palpable, tender renal mass with flank pain and gross or microscopic hematuria, many patients are asymptomatic. Erythrocytosis is an unusual presentation, and malignant transformation may be suspected. This report describes a rare case of a woman diagnosed with renal angiomyolipoma and polycythemia vera. The report discusses the differential diagnosis using erythropoietin, erythropoietin-receptor and Janus kinase 2.</p> <p>Case presentation</p> <p>A 79-year-old Chinese woman was diagnosed with erythrocytosis according to World Health Organization criteria. An upper left renal pole angiomyolipoma was successfully ablated after multiple phlebotomy treatments. Red cell count immediately returned to normal, but gradually increased after 4 months. Polycythemia vera was finally diagnosed by positive mutation of Janus kinase 2 and negative erythropoietin protein expression. Her clinical symptoms improved with regular phlebotomy and hydroxyurea treatment.</p> <p>Conclusion</p> <p>Concurrent occurence of angiomyolipoma and polycythemia vera is rare. Polycythemia vera can be easily missed. Polycythemia vera can be confirmed with high specificity and sensitivity by the acquired somatic mutation. Surgical intervention for this renal tumor should be avoided unless malignancy or renal cell carcinoma is suspected or to prevent spontaneous rupture of larger tumors.</p

Case report: Heterogenous SMARCA4-deficient thoracic non-small cell lung carcinoma with various responses to nivolumab

SMARCA4-deficient non-small cell carcinoma is an aggressive neoplasm with poor outcome. Several studies have highlighted its immunochemistry, pathophysiology, and underlying mechanisms, but studies of its definite treatment are few. Here, we report on a 69-year-old male with heterogenous pathological presentations of SMARCA4-deficient non-small cell carcinoma. He initially presented with neck lymphadenopathies. Immunohistochemistry staining and genomic profiling confirmed the diagnosis of SMARCA4-deficient non-small cell carcinoma. The patient responded well to immune checkpoint inhibitors with nivolumab. However, new lesions with various pathological presentations and various responses to nivolumab appeared during the treatment course. The patient survived more than 3 years from the initial diagnosis. This case shows the efficacy of nivolumab to treat SMARCA4-deficient non-small cell lung carcinoma