N-acetyl-β-D-glucosaminidaasi-entsyymiaktiivisuus normaalimaidossa sekä utaretulehdusta sairastavien lypsylehmien maidossa.

N-acetyl-β-D-glucosaminidaasi (NAGaasi) on glykosidaaseihin kuuluva, solujen lysosomeissa esiintyvä entsyymi, jota vapautuu maitoon utaretulehduksen aikana vaurioituneista utareen epiteelisoluista, neutrofiileistä ja makrofageista. NAGaasientsyymiaktiivisuuden on useissa tutkimuksissa havaittu korreloivan utareen tulehdustilan ja maidon soluluvun (SCC) kanssa ja sitä on ehdotettu käytettäväksi utareen epiteelisolutuhon mittaamiseen yksinään tai yhdistettynä SCC:n määritykseen. Koska saostuminen ei häiritse NAGaasi-entsyymiaktiivisuuden mittausta maidosta, entsyymiaktiivisuus ei muutu maitoa säilytettäessä ja entsyymin mittaaminen on melko yksinkertaista ja nopeaa, menetelmä vaikuttaisi sopivan hyvin seulontatestiksi piileville utaretulehduksille. NAGaasin käyttö on toistaiseksi rajoittunut tutkimuskäyttöön. Sen hyödyntämistä vaikeuttaa se, että terveille lehmille eri tutkimuksissa määritetyissä NAGaasi-entsyymiaktiivisuuden viitearvoissa on suurta vaihtelua. NAGaasi-entsyymiaktiivisuus maidossa on useiden tutkimusten mukaan korkeampi silloin, kun tulehduksen on aiheuttanut jokin merkittävä patogeeni kuin silloin, kun tulehduksen taustalla on vähäpätöinen patogeeni. Lypsykauden vaiheen on havaittu vaikuttavan maidon NAGaasi-entsyymiaktiivisuuteen siten, että aktiivisuudet ovat korkeampia heti poikimisen jälkeen ja lypsykauden lopulla. On myös havaittu, että normaalimaidossa NAGaasi-entsyymiaktiivisuus on hieman korkeampi loppumaidossa kuin alkumaidossa. Poikimakerran vaikutuksista NAGaasi-entsyymiaktiivisuuteen on ristiriitaisia tutkimustuloksia. Tämän tutkimuksen tavoitteena oli määrittää NAGaasi-entsyymiaktiivisuuden viitearvot terveen sekä utaretulehdusta sairastavan lypsylehmän maidossa, sekä selvittää tulehduksen voimakkuuden, aiheuttajapatogeenin, poikimakerran ja lypsykauden vaiheen vaikutusta kyseisen entsyymin aktiivisuuteen maidossa. Tutkimusaineistossa oli mukana kaikkiaan 838 vuosina 2000–2010 otettua maitonäytettä 62 eri lypsykarjatilalta Suomesta ja Virosta. Normaalimaidon NAGaasi-entsyymiaktiivisuuden viitearvot määritettiin yhdeksältä suomalaiselta lypsykarjatilalta kerätyistä 196 maitonäytteestä, jotka täyttivät asettamamme normaalimaidon kriteerit. Normaalimaidon kriteerit olivat seuraavat: SCC < 100 000, lehmällä ei ole utaretulehduksen oireita, poikimisesta on kulunut aikaa yli 30 vuorokautta ja edellisestä lypsystä yli 6 tuntia. NAGaasi-entsyymiaktiivisuus mitattiin modifioidulla Mattilan menetelmällä (Mattila 1985) vakioiduissa olosuhteissa. Aineisto analysoitiin käyttäen Stata Intercooler tilasto-ohjelman versiota 11.0 (Stata Corporation, Texas, USA). Maidon NAGaasientsyymiaktiivisuuteen terveessä neljänneksessä vaikuttavia tekijöitä tutkittiin lineaarisella sekamallilla, jossa sekoittavana tekijänä oli tila. SCC:n ja NAGaasi-entsyymiaktiivisuuden korrelaatiota arvioitiin terveillä lehmillä, piilevää utaretulehdusta sairastaneilla lehmillä ja koko aineistossa. Korrelaatiot laskettiin Pearsonin korrelaatiokertoimella. Tilastollisesti merkitsevänä raja-arvona kaikissa analyyseissä pidettiin p < 0.05. Normaalimaidon NAGaasi-entsyymiaktiivisuuden viitearvoiksi lehmillä, joilla poikimisesta oli kulunut yli 30 vrk, saatiin 0,09–1,04 pmol/min/μl maitoa. Verrattuna normaalimaidon NAGaasi-entsyymiaktiivisuuksien keskiarvoon (0,56) ja piilevää utaretulehdusta sairastaneiden lehmien NAGaasi-entsyymiaktiivisuuksien keskiarvoon (2,49), kliinistä utaretulehdusta sairastavien lehmien maidon NAGaasi-entsyymiaktiivisuus oli keskimäärin selvästi korkeampi (16,65). Keskiarvoissa oli selvä ero paikallisoireisten (12,24) ja yleisoireisten (17,74) lehmien välillä. Terveiden neljännesten maitonäytteistä määritetyn NAGaasi-entsyymiaktiivisuuden ja SCC:n välillä ei havaittu korrelaatiota. Piilevässä utaretulehduksessa havaittiin positiivinen korrelaatio (0,74) maidon NAGaasientsyymiaktiivisuuden ja SCC:n välillä. NAGaasi-entsyymiaktiivisuuteen vaikuttivat tilastollisesti merkitsevästi SCC, poikimisesta kulunut aika ja poikimakerta. Eri patogeeniryhmien osalta havaitsimme, että neljänneksissä, joista eristettiin vähäpätöinen patogeeni, NAGaasi-entsyymiaktiivisuus oli selvästi matalampi kuin neljänneksissä, joista eristettiin merkittävä patogeeni. NAGaasi-entsyymiaktiivisuuden keskiarvoksi vähäpätöisille patogeeneille (KNS, koryneformi) saatiin 2,82 ja merkittäville patogeeneille (S. aureus, Str. uberis, Str, agalactiae, Str. dysgalactiae, E.coli) 16,87.

Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine

Radioactivity from S-adenosyl-L-[methyl-H-3] methionine ([methyl-H-3]AdoMet) was bound to the EcoP15 DNA methyltransferase (M.EcoP15) following short-wave ultraviolet (UV) irradiation. The labeled protein was subjected to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and detected by fluorography and autoradiography. Labeling was found to be dependent on the concentration of AdoMet and time of UV irradiation. The photolabeling by [methyl-H-3]AdoMet was specific and blocked by S-adenosyl-L-homocysteine (AdoHcy) and sinefungin which are known to function as competitive inhibitors. Limited digestion of the M EcoP15-AdoMet adduct by Staphylococcus aureus protease V8 generated three peptides of approx. 50, 32 and 30 kDa; Interestingly, only the 30-kDa peptide fragment contained radioactivity, as detected by SDS-PAGE, followed by fluorography and autoradiography. Further, sequencing of a few amino acids at the N-terminus of these peptides showed that the 30-kDa fragment was the N-terminal portion of M.EcoP15, These results suggest that photolabeling is at the AdoMet-binding site and that the N-terminal half of M.EcoP15 may be involved in substrate binding.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Role of Spectral Studies in Detection of Antibacterial Phytoelements and Phytochemicals of Moringa oleifera

This paper reports, the Laser Induced Breakdown Spectroscopy (LIBS) studies and structure elucidation of compounds isolated from the fruit extract of Moringa oleifera and also deals with their possible effects on some bacterial strains viz. Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa. The extract was found to be active against all four microorganisms used. Extent of inhibitory effect of extract was assessed at different concentrations of 25, 50, 75 mg/ml by measuring diameter of inhibition zone (DIZ). Our results clearly showed that the 75 mg/ml concentration of the extract had 14, 12 and 18 mm of the DIZ against Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa and 14 mm with 50 mg/ml concentration against Escherichia coli. The results were compared with the standard antibiotic `ampicillin' of 1 mg/ml concentration. LIBS was recorded with high power pulsed laser beam from Nd: YAG Laser (Continuum Surelite III-10), focused on the surface of the material, which was in liquid form, to generate plasma on the surface of the sample. LIBS data clearly demonstrate the presence of trace elements, magnesium and iron, in high concentration in the extract. Whereas, from the phytochemical profile reveals the presence of two new compounds, S-ethyl-N-{4-[(alpha-L-rhamnosyloxy) benzyl]} thiocarbamate and 2-acetoxy {4-[(2',3',4'-tri-O-acetyl-alpha-L-rhamnosyloxy) benzyl]} acetonitrile as the major constituents. This study is the first report on synergetic effect of the phytoconstituents and certain set of elements present in their defined role in bacterial management against different bacterial strains.

Antimicrobial Properties of Nanostructured TiO2 Plus Fe Additive Thin Films Synthesized by a Cost-Effective Sol-Gel Process

We prepared thin films composed of pure TiO2 or TiO2 with an Fe additive (at concentrations of 0.2-0.8 wt%) via a simple and cost effective sol gel process, and tested their antifungal properties (against Candida albicans (MTCC-1637), Candida tropicalis (MTCC-184), Candida parapsilosis (MTCC-2509), and Candida glabrata (MTCC-3019) and antibacterial properties (against Staphylococcus faecalis (NCIM-2604) Staphylococcus epidermidis (NCIM-2493), Staphylococcus aureus (NCIL-2122), and Bacillus subtilis (NCIM-2549)). The films were deposited on glass and Si substrates and subjected to annealing at 400 degrees C for 3 h in ambient air. The film structural and morphological properties were investigated by X-ray photoelectron spectroscopy profilometry and scanning electron microscopy, respectively. Antifungal and antibacterial tests were conducted using the drop test method. Among the species examined, Candida albicans (MTCC-1637), and Staphylococcus aureus (NCIL-2122) showed complete colony formation inhibition after exposure for 4 h for the TiO2 loaded with 0.8 wt% Fe thin films. These results indicate that increasing the Fe concentration increased the antimicrobial activity, with complete inhibition of colony formation after 4 h exposure.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid: Synthesis, Characterization, and Antimicrobial Activity

A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@ AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV-visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria.

Differential modulation of intracellular survival of cytosolic and vacuolar pathogens by curcumin

Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.

Process Development for Functionalization of Cotton with Silver Nanoparticles Synthesized by Bio-based Approaches

Cotton is a widely used raw material for textiles but drawbacks regarding their poor mechanical properties often limit their applications as functional materials. The present investigation involved process development for one step coating of cotton with silver nanoparticles (SNP) synthesized using Azadirachta indica and Citrus limon extract to develop functional textiles. Addition of starch to functional textiles led to efficient binding of nanoparticles to fabric and led to drastic decrease in release of silver from fabricated textiles after ten washing cycles enhancing their environment friendliness. Differential scanning calorimetry, scanning electron microscopy, FT-IR analysis and mechanical studies demonstrated efficient binding of nanoparticles to fabric through bio-based processes. The functionalized textiles developed by the bio-based methods showed significant antibacterial activity against E. coli and S. aureus (with 99% microbial reduction). Present work offers a simple procedure for coating SNP using bio-based approaches with promising applications in specialized functions.

Design and synthesis of positional isomers of 5 and 6-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzimida zoles as possible antimicrobial and antitubercular agents

In this Letter, we report the structure activity relationship (SAR) studies on series of positional isomers of 5(6)-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzim idazoles derivatives 7(a-j) and 8(a j) synthesized in good yields and characterized by H-1 NMR, C-13 NMR and mass spectral analyses. The crystal structure of 7a was evidenced by X-ray diffraction study. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, (Gram-positive), Escherichia coil and Klebsiella pneumoniae (Gram-negative), antifungal activity against Candida albicans, Aspergillus flavus and Rhizopus sp. and antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis, Mycobacterium fortuitum and MDR-TB strains. The synthesized compounds displayed interesting antimicrobial activity. The compounds 7b, 7e and 7h displayed significant activity against Mycobacterium tuberculosis H37Rv strain.

Polyelectrolyte/silver nanocomposite multilayer films as multifunctional thin film platforms for remote activated protein and drug delivery

We demonstrate a nanoparticle loading protocol to develop a transparent, multifunctional polyelectrolyte multilayer film for externally activated drug and protein delivery. The composite film was designed by alternate adsorption of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on a glass substrate followed by nanoparticle synthesis through a polyol reduction method. The films showed a uniform distribution of spherical silver nanoparticles with an average diameter of 50 +/- 20 nm, which increased to 80 +/- 20 nm when the AgNO3 concentration was increased from 25 to 50 mM. The porous and supramolecular structure of the polyelectrolyte multilayer film was used to immobilize ciprofloxacin hydrochloride (CH) and bovine serum albumin (BSA) within the polymeric network of the film. When exposed to external triggers such as ultrasonication and laser light the loaded films were ruptured and released the loaded BSA and CH. The release of CH is faster than that of BSA due to a higher diffusion rate. Circular dichroism measurements confirmed that there was no significant change in the conformation of released BSA in comparison with native BSA. The fabricated films showed significant antibacterial activity against the bacterial pathogen Staphylococcus aureus. Applications envisioned for such drug-loaded films include drug and vaccine delivery through the transdermal route, antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Synthesis, antimicrobial and cytotoxic activities of pyrimidinyl benzoxazole, benzothiazole and benzimidazole

A variety of pyrimidinyl benzoxazoles, benzothiazoles and benzimidazoles linked by thio, methylthio and amino moieties were prepared and studied their antimicrobial and cytotoxic activities. The compound pyrimidinyl bis methylthio benzimidazole 22 was a potent antimicrobial agent particularly against Staphylococcus aureus (29 mm, MIC 12.5 mu g/mL) and Penicillium chrysogenum (38 mm, MIC 12.5 mu g/mL). The amino linked pyrimidinyl bis benzothiazole 24 exhibited cytotoxic activity on A549 cells with IC50 value of 10.5 mu M. (C) 2014 Elsevier Masson SAS. All rights reserved.

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (C-protein ca 1 ng/mu l). Reproducible measurements on protein samples at this concentration (I-p,I-a of 80 +/- 1.2 mu A) could be achieved using a sample volume of ca 30 mu l. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments. (C) 2014 Elsevier B.V. All rights reserved.

Biogenic nanosilver incorporated reverse osmosis membrane for antibacterial and antifungal activities against selected pathogenic strains: An enhanced eco-friendly water disinfection approach

Reverse osmosis (RO) membranes have been used extensively in water desalination plants, waste water treatment in industries, agricultural farms and drinking water production applications. The objective of this work is to impart antibacterial and antifungal activities to commercially available RO membrane used in water purification systems by incorporating biogenic silver nanoparticles (AgNPs) synthesized using Rosa indica wichuriana hybrid leaf extract. The morphology and surface topography of uncoated and AgNPs-coated RO membrane were studied using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Elemental composition of the AgNPs-coated RO membrane was analyzed by energy-dispersive X-ray spectroscopy (EDAX). The functional groups were identified by Fourier Transform Infrared spectroscopy (FT-IR). Hydrophilicity of the uncoated and AgNPs-coated RO membrane was analyzed using water contact angle measurements. The thermal properties were studied by thermogravimetric analysis (TGA). The AgNPs incorporated RO membrane exhibited good antibacterial and antifungal activities against pathogenic bacterial strains such as E. coli, S. aureus, M. luteus, K. pneumoniae, and P. aeruginosa and fungal strains such as Candida tropicalis, C. krusei, C. glabrata, and C. albicans.