A Supramolecular Complex between Proteinases and β-Cyclodextrin that Preserves Enzymatic Activity

BACKGROUND: Cyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and wound-healing properties. METHODS: In this study, we analyzed the association between beta-cyclodextrin (betaCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity. RESULTS AND DISCUSSION: The physicochemical analyses suggest the formation of a complex between P1G10 and betaCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10 : betaCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and betaCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar betaCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29. CONCLUSION: The reported results underscore the potential for betaCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.     

PVA Hydrogels Containing β-Cyclodextrin for Enhanced Loading and Sustained Release of Ocular Therapeutics

The purpose of this work is to develop poly(vinyl alcohol) (PVA) hydrogels incorporating large amounts of β-cyclodextrin (β-CD) in order to improve ocular drug loading and to sustain drug release. First, the mono-methacrylated-β-CD monomer (MA-β-CD) and the methacrylated-PVA macromer (PVAMA), with a substitution degree of 7%, are synthesized and characterized. Then, the poly(methacrylated-PVA-co-mono-methacrylated-β-cyclodextrin) (pPVA-β-CD) hydrogels are prepared by UV-induced polymerization of MA-β-CD and PVAMA. The highest amount of β-CD incorporated into the hydrogels is 30 wt%. The hydrogels are further characterized by transmittance, FT-IR, equilibrium swelling ratio (ESR), tensile tests and protein deposition. The results show that pPVA-β-CD hydrogels possess good transmittance, while the incorporation of β-CD in the hydrogels improves the tensile strength and decreases the ESR and protein deposition. Finally, puerarin and acetazolamide are used as models to evaluate the drug loading and in vitro release behavior of the pPVA-β-CD hydrogels. The results indicate that the amount of drug loaded into the pPVA-β-CD hydrogels progressively increases, while the release rate decreases with increasing β-CD content. In particular, incorporation of β-CD efficiently decreases the initial burst release of acetazolamide, while the release, which is almost linear, is sustained for 15 days. The pPVA-β-CD hydrogels have potential applications as biomedical devices for sustained release of ocular drugs.     

Activity of doripenem against extended-spectrum β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates

The in vitro activity of doripenem was evaluated against a recent collection of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (201 ESBL-producing Enterobacteriaceae [153 Escherichia coli and 48 Klebsiella pneumoniae] and 201 P. aeruginosa). Comparator agents included amikacin, tobramycin, ciprofloxacin, cefepime, cefotaxime, ceftazidime piperacillin-tazobactam, imipenem, and meropenem. Both doripenem and meropenem inhibited 100% of the ESBL-producing Enterobacteriaceae at ≤0.5 μg/mL. For these isolates, the MIC₉₀ of doripenem (0.12 μg/mL) was 4-fold lower than that of imipenem (0.5 μg/mL). Against P. aeruginosa, the MIC₉₀ of doripenem and meropenem was 2 μg/mL, 4-fold lower than that of imipenem (8 μg/mL). At an MIC of ≤2 μg/mL, doripenem, meropenem, and imipenem inhibited 90.5%, 89.6%, and 82.1% of P. aeruginosa isolates, respectively. Doripenem maintained activity against imipenem-nonsusceptible isolates of P. aeruginosa; at an MIC of ≤4 μg/mL, it inhibited 15 of the 25 isolates with MICs for imipenem of >4 μg/mL. Doripenem is active against ESBL-producing Enterobacteriaceae and P. aeruginosa isolates. Its activity is similar to that of meropenem and slightly better than that of imipenem. The results of this study suggest that doripenem could be an alternative therapeutic agent for infections caused by these organisms.     

Antioxidant Properties of a Nanostructured Clathrate Complex Selenopyran with β-Cyclodextrin in Model Systems of Oxidative Stress

Bulletin of Experimental Biology and Medicine - We studied the effect of nanostructured clathrate complex 9-phenyl-symm-octahydoselenoxanthene (selenopyran) with β-cyclodextrin on the...     

Effect of β-Adrenoceptor Blockade on Cardiac Activity in Rats during Postnatal Ontogeny

Electrical stimulation of the right stellate ganglion produces a positive chronotropic effect in 21-, 56-, and 100-day-old rats. The response of the heart rate to suprathreshold stimulation increased from the 21st to 100th day. By contrast, during beta-adrenoceptor blockade the heart rate response decreased. The data suggest that the role of beta-adrenoceptor in the regulation of cardiac chronotropic function increased with age.     

Exploring Pleiotropic Functions of Canine β-Defensin 103: Nasal Cavity Expression,Antimicrobial Activity,and Melanocortin Receptor Activity

Canine β-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a β-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.     

Biological Effect of Magnetic Field in Mice

With a piece of magnet embeded in mouse body tomeasure the electrophoretic velocity of erythrocyte for ob-servation onthe biological effect of magnetic field.1Experi mental Material and Method1 .1Experi mental materialUsing permanent magnet was made of alloys fromCe .Co.Cu.Fe .,of which the force of magnetic field is500Gs ,formseems cylinder andthe weight is 0 .5 mg.1 .2Ani mals and groupingThere were eighteen mice that were choosed on ran-dom,theirs weight was 18-22gto divide equallyinthr…     

Characterization of Novel β-Galactosidase Activity That Contributes to Glycoprotein Degradation and Virulence in Streptococcus pneumoniae

The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a β-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished β-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal β-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total β-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but β-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.Streptococcus pneumoniae is the leading cause of bacterial pneumonia, otitis media, bacterial meningitis, and septicemia (21). Furthermore, a high percentage of the population carries the bacterium in the nasopharynx, asymptomatically or as a prelude to disease (53). The upsurge in antibiotic-resistant strains and the search for new vaccines highlight the need to understand more completely the nature of pneumococcal virulence. A survey of pneumococcal virulence studies indicates a clear asymmetry in favor of research focusing on factors involved in attachment and damage to host tissues and in immune evasion (2, 11, 15, 22, 38). Although these studies have revealed useful information about pneumococcal virulence, there is a severe lack of knowledge on the basic physiology of S. pneumoniae, such that little information is available on how the pneumococcus fulfils its nutritional requirements for the generation of energy.Carbohydrates are the principal energy sources for the pneumococcus, and these must be obtained exclusively from its host (16). Although the pneumococcus is known to utilize a variety of free sugars through at least 14 sugar utilization operons (10), in the upper respiratory tract the concentration of free sugars is low (39). However, monosaccharides are copious within the structures of O- and N-linked oligosaccharides of glycoproteins, which are found predominantly in the structures of mucins (Fig. ​(Fig.1)1) and in circulatory glycoproteins, respectively. Previous studies have shown that the sequential deglycosylation of N-linked glycan structures by pneumococcal glycosidases mediates in vitro growth of S. pneumoniae (5).Open in a separate windowFIG. 1.Schematic representation of the oligosaccharide structure of mucins. The carbohydrate chain is O-glycosidically linked to the apomucin through N-acetylgalactosamine (GalNAc). Galactose (Gal), N-acetylglucosamine (GlcNAc), sialic acid (Neu5Ac), sulfate (SO₃²⁻), and fucose (Fuc) residues are indicated. The structure of 9-O-acetylated sialic acid is shown as a structural formula (constructed from data presented in reference 49).The apical surfaces of the respiratory tract epithelium are covered with mucus, which plays a vital role in the removal of pathogens and environmental toxins via the mucocilliary escalator (42). Paradoxically, mucus is a potentially rich source of carbon and nitrogen for bacteria with a sufficient array of glycosidase activities (43). Mucin is the major macromolecular compound of mucus, and the heavily glycosylated side chains of this glycoprotein may be utilized by bacteria as a carbohydrate source. Although the detailed structure of mucins in different organs exhibits variation, the majority of mucin glycans contain N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfated sugars, and sialic acids in various proportions (Fig. ​(Fig.1)1) (41, 43). The acetylation of terminal sialic acids and sulfation of other monosaccharides, to a certain degree, protect carbohydrate side chains against the cleavage by microbial glycosidases (7).Pneumococci degrade mucin for growth, and NanA is essential for the mucin degradation ability of the pathogen (52), presumably for removal of terminal sialic acid residues to expose the carbohydrate core of oligosaccharides to further degradation by other glycosidases. Recently it was also shown that NanB plays a role in the deglycosylation of human glycoconjugates by some pneumococcal strains (5). It has been demonstrated that mucin stimulates expression of the neuraminidase in the pneumococcus (49), thus enhancing its own degradation. Using isogenic nanA and nanB mutant strains, the involvement of neuraminidases in the pathology of pneumococcal otitis media, meningitis, and pneumonia has been demonstrated by us and others (30, 48). In addition to neuraminidases NanA (SPD_1504) and NanB (SPD_1499), BgaA (SPD_0562) and StrH (SPD0063), which catalyze the hydrolysis of terminal nonreducing β-d-galactose and N-acetyl-β-d-glucosamine residues, respectively, have been shown to be involved in the deglycosylation of human glycoconjugates and to contribute to pneumococcal colonization and pathogenesis of disease (24).The roles of other glycosidases remain unknown, yet at least one-third of the pneumococcal genome encodes proteins involved in sugar transport, degradation, and processing, suggesting that carbohydrate metabolism has a central impact on pneumococcal biology (16, 27, 47). In the sequenced pneumococcal genome, several genes are annotated as sugar hydrolases (otherwise known as glycosidases); however, the importance of these in glycoprotein degradation is not known. In this study we explored the role in glycoprotein degradation and virulence of SPD_0065, annotated as β-galactosidase 3 in the D39 genome (27). During preparation of this paper, Jeong et al. (20) reported some properties of SPD_0065. Because pneumococcal β-galactosidase activity previously had been attributed exclusively to SPD_0562 (54), we investigated the contribution of this newly identified galactosidase, SPD_0065, to total β-galactosidase activity by constructing a pneumococcal strain with both genes mutated. Using an isogenic mutant, it was shown that this glycosidase is important in mucin degradation. The involvement of SPD_0065 in pneumococcal virulence was also tested using this mutant strain in a mouse model of pneumonia and bacteremia, where it was found that SPD_0065 is important primarily for survival in the nasopharynx.     

Preparation and Biological Evaluation of Heat-press Glass Ceramics

INTRODUCTION　　The theory and clinical performance of conventional metal ceramic systems tendto be proficiency and familiar to dentist.However,peeling off and crack of ceramicsresulting in failure of restoration,in the meantime,the inside metal bring aboutbadesthetic results.With more and more esthetic demands,the all- ceramics crown sys-tems have become widespread for restoration of teeth〔1〕.Castable glass ceramics de-veloped in 1 980 s was attached extensive importance forits mechani…     

Two Different PDGF β-Receptor Cohorts in Human Pericytes Mediate Distinct Biological Endpoints

How activation of a specific growth factor receptor selectively results in either cell proliferation or cytoskeletal reorganization is of central importance to the field of pathophysiology. In this study, we report on a novel mechanism that explains how this process is accomplished. Our current investigation demonstrates that soluble platelet derived growth factor- (PDGF)-BB activates a cohort of PDGF-β receptors primarily confined to the lipid raft component of the cell membrane, specifically caveolae. In contrast, cell-bound PDGF-BB delivered via cell–cell contact results in activation and the subsequent up-regulation of a cohort of PDGF β-receptors primarily confined to the non-lipid raft component of the cell membrane. Individual activation of these two receptor cohorts results in distinct biological endpoints, cytoskeletal reorganization or cell proliferation. Mechanistically, our evidence suggests that PDGF-BB-bearing cells preferentially stimulate the non-lipid raft receptor cohort through interleukin 1β-mediated inhibition of the lipid raft cohort of receptors, leaving the non-raft receptor cohort operational and preferentially stimulated. In human skin injected with PDGF-BB and in tissue reparative processes PDGF β-receptors colocalize with the caveolae/lipid raft marker caveolin-1. In contrast, in human skin injected with PDGF-BB-bearing tumor cells and in colorectal adenocarcinoma, activated PDGF β-receptors do not colocalize with caveolin-1. Thus, growth factor receptors are segregated into specific cell membrane compartments that are preferentially activated through different mechanisms of ligand delivery, resulting in distinct biological endpoints.Lipid rafts are cellular membrane domains that contain high concentrations of cholesterol and sphingolipids. These domains include the flat and related vesicular structures referred to as caveolae. Caveolae, which are formed by the macromolecular oligomerization of the 22-kDa caveolin protein are enriched in a number of vital signal transduction molecules, and contain smaller cohorts of many others.^1,2,3,4,5,6 Furthermore, caveolin itself directly binds and/or regulates the activities of a number of these signaling molecules.¹ With specific regards to the platelet derived growth factor (PDGF) signaling axis, PDGF-induced signaling occurs in caveolae of many mesenchymal cells,^5,6,7,8 and PDGF-receptors are functional in isolated caveolae.⁸ Based on the apparent signaling events occurring in lipid rafts, and the abundance of molecules involved in multiple signaling pathways, it is inferred that lipid rafts are important loci for signal amplification and cross talk between signaling pathways.^1,2,5,6,7,8 Recently emerging evidence shows that lipid rafts also have important specific roles in regulating the activity of cytoskeleton-regulating GTPases, in cytoskeletal organization, in the formation of cell extensions, and in cell motility.⁹The PDGF-B chain contains a retention motif that mediates binding to heparan sulfate proteoglycans on cell surfaces.¹⁰ This affords PDGF-BB-producing cells alternate modes of ligand delivery to PDGF β-receptor bearing cells, through heterotypic cell-to-cell contacts or as a secreted soluble ligand.^11,12 In mesenchymal cell–tumor cell co-cultures, activation of PDGF β-receptors is a consequence of cell–cell contacts, and is not accomplished via soluble PDGF-BB.¹³ The biological consequences of cell–cell versus secreted ligand remain unknown. Given the central role of PDGF β-receptor activation in pericyte biology during embryogenesis and reactive conditions in the adult organism,^{14,15,16,17,18,19,20} we chose to investigate the role of caveolae in PDGF β-receptor signaling in primary human pericytes. Activation of PDGF β-receptors in mesenchymal cells leads to several biological endpoints, eg, proliferation and reorganization of the actin cytoskeleton.²⁰ How cells are able to orchestrate signal transduction events leading to different biological endpoints, in response to stimulation by a specific ligand, is not known. Here we demonstrate one mechanism by which context-specific ligand stimulation of a growth factor receptor results in distinct biological endpoints.     

Hydrophobic Determinants of α-Defensin Bactericidal Activity

Mammalian α-defensins are approximately 4- to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic α-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of α-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys^III-Cys^V disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse α-defensin 4 (Crp-4) and rhesus myeloid α-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil α-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic α-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.     

IL-1β Biological Treatment of Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is a recessive, autosomal, auto-inflammatory disorder characterised by brief, recurring, self-limited episodes of fever and serositis resulting in abdominal, chest, joint and muscular pain; it is the most common of the periodic hereditary fevers and mostly affects Mediterranean populations. Daily administration of colchicine, a tricyclic alkaloid with anti-microtubule and anti-inflammatory properties, prevents the recurrence of FMF attacks and the development of secondary (AA) amyloidosis, the major long-tem complication of FMF. Colchicine is generally safe and well-tolerated; nevertheless, 5–10 % of FMF patients do not respond to conventional treatment, while another 2–5 % of patients are colchicine-intolerant because of toxicity issues, leading physicians to search for alternative therapeutic strategies. Recent new insights into the mechanisms of auto-inflammation add further proof to the efficacy of IL-1 targeting drugs in colchicine non-responder/intolerant FMF patients. A systematic study of relevant literature through PubMed/Medline was performed in order to identify publications reporting IL-1β biological treatment of FMF. Treatment methods, comorbidities, clinical response and side effects in literature case reports were analysed, as well as recent advances in the pathogenesis of auto-inflammation mechanisms in FMF and the causes of colchicine resistance or toxicity in common clinical practice. The paradigmatic experience of an FMF patient with severe FMF mutations (M694V/M694V) suffering from colchicine toxicity and successfully treated with anakinra is also reported. The present data show that anti-IL-1β biological treatment is actually a therapeutic option for FMF patients unresponsive or intolerant to colchicine or in FMF patients with concomitant vasculitis.     

Construction of Biological Polygonal Surface Models from Volume Data

1.IntroductionPolygonalsurfacemodelsplayanimportantroleinbiologicalvirtualreality.Withinteractivevisualization,manipulation,andmeasurementofmulti-modality3Dmedicalimagesinvirttlalreality,techniquesinminimallyinvasivesurgery,telepresencesurgery,andcomputerassistedsurgicalplanning,simulationandrehearsalarenowbeingintroducedan(Jaredramaticallyaugmentingthemethodofsurgery.Inadditiontoirtlprovingthetherapeuticresult,thesenewtechniquesaretargetedatreducingthf3risksandcostsofsurgicalprocedures.Robbe…     

Utilization of spectrins βI and βIII in diagnosis of hepatocellular carcinoma

Spectrins are a group of cytoskeletal proteins which participate in many important cellular functions. It has been suggested that loss of spectrin isoforms may be associated with tumorigenesis of lymphoma, leukemia, gastric cancer and hepatocellular carcinoma (HCC). We recently reported that βI spectrin expression was present in normal hepatocytes but lost in HCC cells, which suggested that spectrins may be helpful markers in diagnosis of HCC. In this study, using immunohistochemical staining, we further investigated the expression pattern of four spectrin isoforms (αII, βI-III) on different benign and malignant liver tumors including focal nodular hyperplasia (FNH), hepatic adenoma (HA), HCC, and cholangiocarcinoma (CC). The results revealed that βI spectrin was moderately to strongly positive in FNH and HA tissues, but was only weakly positive or lost in HCC cases and was weakly positive in all CC cases. In addition, the βIII spectrin, majority of which was moderately positive in both FNH and HA tissues, was mostly lost in poorly differentiated HCC but remained at least moderately positive in most CC cases. These results suggest that spectrins βI and βIII may be used to differentiate well differentiated HCC from FNH or HA, and poorly differentiated HCC from CC, respectively.