Synthesis and Antibacterial Activity of Spiro[4H-pyran-3,3'-oxindoles] Catalyzed by Tröger's Base Derivative.

OBJECTIVE: Two classes of spiro[4H-pyran-3,3'-oxindole] derivatives were prepared via the one pot reaction of chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins and malononitrile successfully catalyzed by a Tröger's base derivative 1b (5,12-dimethyl-3,10-diphenyl-bis-1H-pyrazol[b,f][4,5]-1,5-diazadicyclo[3.3.1]-2,6-octadiene). The antibacterial activity of products against three wild-type bacteria (B. subtilis, S. aureus, and E. coli) and two resistant strains (Methicillin-resistant S. aureus (18H8) and E. coli carrying the BlaNDM-1 gene (18H5)) was evaluated using the minimum inhibitory concentration (MIC).. METHODS: 1-Phenyl-1,3-butanedione 2 or dibenzoylmethane 2' (0.42 mmol), substituted isatin 3 (0.4 mmol), malononitrile 4 (0.8 mmol), Tröger's base derivative 1b (0.08 mmol), and 10 mL of acetonitrile were added to a 50 mL round bottom flask and refluxed. After the completion (TLC monitoring), water (10 mL) was added to the reaction mixture; pH = 7 was adjusted with saturated NaHCO3 (aq.), and the mixture was extracted with CH2Cl2 (50 mL × 3). Organic layers were combined and dried with anhydrous Na2SO4; the solvent was removed under vacuum, and the residue was purified by column chromatography (VDCM: VMeOH = 80: 1) to afford product 5. The antibacterial activity was tested by the MTT method. RESULTS: Seventeen spiro[4H-pyran-3,3'-oxindole] derivatives were synthesized through the reaction of chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins, and malononitrile in one-pot in medium to high yields. Four compounds showed antibacterial activity, and two of them showed the same activity as the positive control Ceftazidime on S. aureus (MIC = 12.5 µg/mL). CONCLUSION: Two classes of spiro[4H-pyran-3,3'-oxindole] derivatives were prepared, and their antibacterial activity was evaluated. Tröger's base derivative 1b (5,12-dimethyl-3,10-diphenyl-bis-1H-pyrazol[b,f][4,5]- 1,5-diazadicyclo[3,3,1]-2,6-octadiene) was used as an efficient organocatalyst for the reaction of low reactive chain diketones (1-phenyl-1,3-butanedione or dibenzoyl methane), substituted isatins, and malononitrile in one-pot successfully and effectively by providing multiple active sites and alkaline environment. By the theoretical calculation, we explained the possible reaction sequence and mechanism. Due to the superiority and high efficiency of the TB framework as an organocatalyst, the reaction showed many advantages, including mild reaction conditions, low catalyst loading, and a wide substrate range. It expanded the application of Tröger's base to the multicomponent reaction in organocatalysis. Some products were screened due to their high antibacterial activity in vitro, showing their potential in new antibacterial drug development.

Recombinant streptavidin fusion proteins as signal reporters in rapid test of human hepatitis C virus infection.

BACKGROUND: Early diagnosis of hepatitis C virus (HCV) infection is very important for the treatment of the disease. Development of sensitive and specific rapid detection assays is of great significance for the diagnosis. Here, we describe a promising method of using gold-labeled streptavidin fusion proteins as novel signal reporter in a rapid detection assay for HCV infection. METHODS: Recombinant genes encoding streptavidin fused with Escherichia coli maltose-binding protein (MBP) or with a portion of bacterial translational initiation factor 2 were cloned in expression vectors pMAL-5CX and pET28 and transformed in proper Escherichia coli host strains. The genes were induced and streptavidin fusion proteins, named M-STV and IF-STV, respectively, were purified by affinity chromatography to over 90% purity. The biotin-binding activity of M-STV and IF-STV was tested by enzyme-linked immunosorbent assay (ELISA). M-STV was labeled with colloidal gold nanoparticles and used as a signal reporter to develop a lateral flow-based rapid test for detecting anti-HCV antibodies in human blood samples. RESULTS: M-STV showed slightly higher biotin-binding activity and similar binding specificity as compared to commercial streptavidin. The gold-labeled M-STV bound specifically to biotin moieties immobilized on the rapid test strips in a dose-responsive manner and was successfully used in detecting HCV antibodies in serum samples of patients infected with HCV. The rapid test displayed higher detection sensitivity than gold-labeled commercial NeutrAvidin. CONCLUSION: Our results indicate that gold-labeled M-STV is a promising agent in rapid tests of HCV infection and possibly other viral infections.

Characterization of the inflammatory and apoptotic cells in the aortas of patients with ascending thoracic aortic aneurysms and dissections.

OBJECTIVE: The histopathologic abnormality underlying ascending aortic aneurysm and dissection is medial degeneration, a lesion that is described as the noninflammatory loss of smooth muscle cells and elastic fibers. This study sought to determine whether inflammatory cells are present in medial degeneration and assess any possible contribution of these cells to apoptosis of smooth muscle cells. METHODS: Aortic specimens were obtained from patients undergoing prophylactic surgical repair of an ascending aortic aneurysm (n = 9) and type A dissection (n = 7), along with control patients dying of causes unrelated to aortic disease (n = 5). Immunohistochemical staining was performed to evaluate the presence of lymphocytes and macrophages, and markers of apoptosis were assessed in the aortas of patients with ascending aortic aneurysm and dissection. RESULTS: Immunohistochemical study indicated significantly more CD3+ cells in the aortas of patients with aneurysms or dissections than in control aortas (P = .020 and P = .0022, respectively). In addition, aortas of patients with aneurysms or dissections had more CD68+ cells (P = .01 and P = .005, respectively). CD3+ cells were localized in the media and surrounding the vasa vasorum in the adventitia. Cells yielding a positive result on in situ terminal transferase-mediated deoxyuridine triphosphate nick end-labeling were found in increased numbers in the aortas of patients with aneurysms or dissections relative to control aortas (P = .005 and P = .002, respectively). Furthermore, Fas and FasL were increased in the aortic samples from patients with aneurysms and dissections relative to control aortas. CONCLUSION: The coexistence of inflammatory cells with markers of apoptotic vascular cell death in the media of ascending aortas with aneurysms and type A dissections raises the possibility that activated T cells and macrophages may contribute to the elimination of smooth muscle cells and degradation of the matrix associated with thoracic aortic aneurysms and dissections.

Frameshift mutations in the bax gene are not involved in development of ovarian endometrioid carcinoma.

The purpose of this study was to determine whether mutations in the Bax gene play a role in the development of ovarian endometrioid carcinoma with a microsatellite instability phenotype. We analyzed a total of 60 tumor specimens, 49 ovarian endometrioid carcinomas and 11 concurrent endometrial endometrioid carcinomas from 49 patients. Fourteen ovarian endometrioid carcinomas and 6 endometrial endometrioid carcinomas showed a microsatellite instability-high phenotype. Tumor and normal-tissue specimens from eight patients with a microsatellite instability-high phenotype colorectal carcinoma were included in this study as controls. The presence or absence of a mutation in the poly (G) 8 tract of the Bax gene was determined by polymerase chain reaction followed by direct DNA sequence analysis. A 1-base pair deletion at the poly (G) 8 tract and no expression of Bax and Bcl-2 proteins were identified in one microsatellite instability-high endometrial endometrioid carcinoma. Immunohistochemical staining for Bax and Bcl-2 proteins was negative on the tumor specimen that had this 1-base pair deletion. No mutations were found in the synchronous microsatellite instability-high ovarian endometrioid carcinoma from the same patient. In contrast, four (50%) of the eight microsatellite instability-high sporadic colorectal carcinomas had a mutation in the poly (G) 8 tract. Although Bax plays an important role in carcinogenesis of the colorectum with microsatellite instability-high phenotype, Bax may not play a direct role in the genesis of ovarian endometrioid carcinoma, regardless of microsatellite instability status.

Profibrillin-1 maturation by human dermal fibroblasts: proteolytic processing and molecular chaperones.

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.

Activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract and oleandrin from human serum: monitoring the effect by measuring apparent digoxin concentration.

Accidental poisoning from oleander leaf or oleander tea can be life threatening. The authors studied the effectiveness of activated charcoal and equilibrium dialysis in removing oleander leaf extract and commercially available oleandrin as well as oleandrigenin, the active components of oleander plant, from human serum. Oleander leaf extract was prepared in distilled water and drug-free serum was supplemented with the extract. Then serum was treated with activated charcoal at room temperature and an aliquot was removed at 0 minutes, 10 minutes, 20 minutes, and finally 30 minutes to study the presence of oleander extract by measuring the apparent digoxin concentration using the FPIA for digoxin. The authors observed effective removal of oleander extract by activated charcoal. When the authors supplemented other drug-free serum pools with pure oleandrin or oleandrigenin and then subsequently treated them with activated charcoal, the authors observed complete removal of digoxin-like immunoreactivity at the end of 30 minutes' treatment. When drug-free serum pool supplemented with either oleander leaf extract, oleandrin, or oleandrigenin was passed through a small column packed with activated charcoal, the authors observed almost no apparent digoxin concentration following the passage through the column indicating that activated charcoal is very effective in removing oleander from human serum in vitro. In contrast, when serum pools containing either oleander leaf extract or oleandrin were subjected to equilibrium dialysis against phosphate buffer at pH 7.4, the authors observed no significant reduction in apparent digoxin concentration even after 24 hours. The authors conclude that activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract from human serum.

Melanocytes: morphological basis for an exteroceptive sensory system for monitoring ultraviolet radiation.

From the conventional medical perspective, melanocytes have been traditionally viewed as epidermal responders/reactors to ultraviolet radiation. In this paper we have begun an analysis of the functional significance of melanocytes as monitors for ultraviolet radiation with their neuronal, endocrinological and immunological intercalations. This preliminary study was performed using as a human model unilateral sural nerve sensory blockade before and after ultraviolet exposure to both feet while the unblocked foot acted as the control. Full thickness surgical skin biopsies were taken of (I), normal pre-exposure skin, in the center of the two centimeter exposure area of both the sural nerve blocked area (II-III) and control (IV-V) one centimeter outside the exposed area in both the sural nerve blocked foot and the control or non-blocked foot. The objective clinical, blinded morphological and immuno-histochemical data from this experiment support the initial conclusion that neuronal connection is necessary for the normal ultraviolet exposure dermal reaction. Based upon this study, we further propose the existence of an exteroceptive sensory system in which melanocytes, with direct nervous system connection initiate an ultraviolet radiation reactive response that mobilizes both conventional endocrine and immune pathways.