935 resultados para Broad-spectrum


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Bacterial flora associated with tail rot/fin rot of Carassius auratus, Xiphophorus helleri and hemorrhagic ulcers of Clarias spp were studied. Sensitivity pattern of 33 isolates comprising Aeromonas spp, Pseudomonas spp and Gram-positive rods from diseased C. auratus, X. helleri and Clarias spp were screened against six broad-spectrum antibiotics viz. ciprofloxacin, chloramphenicol, co-trimoxazole, gentamycin, nitro-furantoin and oxytetracycline. Ciprofloxacin was the most effective in inhibiting bacteria at 0.05-0.10 µg/ml level. About 44% of Pseudomonas spp. was resistant to nitrofurantoin. Resistance to oxytetracycline was seen in 27% of Aeromonas spp Gram-positive rods were comparatively more resistant to antibiotics. The multiple antibiotic resistances were seen in 21% of the bacterial isolates of diseased fish.

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Two antimicrobial peptides manifested a broad spectrum of anti microbial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary Stru

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Industrial emergence is a broad and complex domain, with relevant perspectives ranging in scale from the individual entrepreneur and firm with the business decisions and actions they make to the policies of nations and global patterns of industrialisation. The research described in this article has adopted a holistic approach, based on structured mapping methods, in an attempt to depict and understand the dynamics and patterns of industrial emergence across a broad spectrum from early scientific discovery to large-scale industrialisation. The breadth of scope and application has enabled a framework and set of four tools to be developed that have wide applicability. The utility of the approaches has been demonstrated through case studies and trials in a diverse range of industrial contexts. The adoption of such a broad scope also presents substantial challenges and limitations, with these providing an opportunity for further research. © IMechE 2013.

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5-氟尿嘧啶(5-Fluorouracil, 5-FU)是一种抗代谢药物,广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症,但其首过代谢显著、亲脂性较低,选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作,包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin,简称CD),可被结肠中的糖苷酶特异性地降解成小分子糖,而胃和小肠中由于缺乏相应的酶而使环糊精不被降解,这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基,易进行化学修饰,将药物与环糊精通过共价键结合制成前药,使其在胃和小肠中不降解,而在盲结肠中被特异性的酶降解释出药物,达到结肠靶向释药的目的。研究表明,环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体,通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联,合成了系列5-FU-β-CD前体药物,并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时,还研究了前体药物的体外释药性质。具体内容包括: 1. 含有羧基的5-FU衍生物中间体的合成:(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联:分别通过以6-OTs-β-CD为中间体的取代法和活化酯法,合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中,通过改变原料的比例,合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究:分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为,并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

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研究背景与目的:近二十年来,抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌,所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙(Odorrana grahami)皮肤抗菌肽具有广谱抗菌活性,但对真核细胞没有毒性,因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C,为大量获取抗菌肽资源提供技术支撑。 方法:依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后,与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接,并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测,鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母(Pichia pastoris)GS115宿主菌,用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测,鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达,SDS - PAGE电泳及反相层析检测表达产物,并做抑菌活性检测。 成果:PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高(>94%)的抑制率。 结论:无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功,并且都在毕赤酵母系统中获得了成功表达。 Background & Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

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Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

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Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

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Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

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Durbin, J. & Urquhart, C. (2003). Qualitative evaluation of KA24 (Knowledge Access 24). Aberystwyth: Department of Information Studies, University of Wales Aberystwyth. Sponsorship: Knowledge Access 24 (NHS)

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

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The development of procedures and media for the micropropagation of B. rex are described. Media for the production of plantlets from a number of other Begonia hybrids are also provided. Growth analysis data is given for plants produced in vivo from leaf cuttings and in vitro from mature leaf petioles and immature leaves derived from singly and multiply recycled axenic plantlets. No significant difference was found in phenotype or quantitative vegetative characters for any of the populations assessed. The results presented from studies on the development of broad spectrum media for the propagation of a number of B. rex cultivars using axenic leaf explants on factorial combinations of hormones illustrate the major influence played by the genotype on explant response in vitro and suggest media on which a range of B. rex cultivars may be propagated. Procedures for in vitro irradiation and colchicine treatments to destabilize the B. rex genome have also been described. Variants produced from these treatments indicate the utility of in vitro procedures for the expression of induced somatic variation. Colour variants produced from irradiation treatment have been cultured and prove stable. Polyploids produced as variants from irradiation treatment have been subcultured but prove unstable. Media for the induction and proliferation of callus are outlined. The influence of callus subculture and aging on the stability of the B. rex genome is assessed by chromosomal analysis of cells, in vitro and in regenerants. The B. rex genome is destabilized in callus culture but attenuation of variation occurs on regeneration. Diploid cell lines are maintained in callus subcultures and supplementation of regenerative media with high cytokinin concentrations, casein hydrolysate or adenine failed to produce variants. Callus aging however resulted in the production of polyploids. The presence and expression of pre-existing somatic variation in B. rex pith and root tissue is assessed and polyploids have been produced from pith tissues cultured in vitro. The stability of the B. rex genome and the application of tissue culture to micropropagation and breeding of B. rex are discussed.

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Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

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Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibioticassociated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. The main etiological agent of C. difficile-associated diarrhea (CDAD) is perturbations to the gut microbiota by broad-spectrum antibiotics. Recently, thuricin CD, a two-peptide narrow spectrum sactibiotic bacteriocin with potent activity against C. difficile has been discovered. It is produced by Bacillus thuringiensis DPC6431. The efficacy of thuricin CD against a range of C. difficile clinical isolates has been determined in the form of minimum inhibitory concentration (MIC) values and compared to metronidazole, vancomycin, ramoplanin and actagardine in this thesis. Furthermore, by assessing paired combinations of the above-mentioned antimicrobials, it was determined that ramoplanin and actagardine function in a synergistic manner against the majority of C. difficile isolates. The functions of the genes in the thuricin CD gene cluster have also been elucidated by cloning the cluster and expressing thuricin CD in a heterologous Bacillus subtilis host and are described herein. In addition, the immunity mechanisms employed by the B. thuringiensis DPC6431 producer to protect itself from the antimicrobial actions of thuricin CD have also been elucidated. It has been shown that a small immunity peptide, TrnI, is involved in thuricin CD immunity, most likely by intercepting the thuricin CD peptides and/or blocking their access to the thuricin CD receptor. This immunity peptide and also the ABC-transporter system TrnFG serve to protect the B. thuringiensis host against thuricin CD.

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BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

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Ag+- and Zn2+-exchanged zeolites zeolites and clays have been used as coatings and in composites to confer broad-spectrum antimicrobial properties on a range of technical and biomedical materials. 11 angstrom tobermorite is a bioactive layer lattice ion exchanger whose potential as a carrier for Ag+ and Zn2+ ions in antimicrobial formulations has not yet been explored. In view of this, batch Ag+- and Zn2+-exchange kinetics of two structurally distinct synthetic 11 angstrom tobermorites and their subsequent bactericidal action against Staphylococcus aureus and Pseudomonas aeruginosa are reported. During the exchange reactions, Ag+ ions were found to replace labile interlayer cations; whereas, Zn2+ ions also displaced structural Ca2+ ions from the tobermorite lattice. In spite of these different mechanisms, a simple pseudo-second-order model provided a suitable description of both exchange processes (R-2 >= 0.996). The Ag+- and Zn2+-exchanged tobermorite phases exhibited marked bacteriostatic effects against both bacteria, and accordingly, their potential for use as antimicrobial materials for in situ bone tissue regeneration is discussed. (C) 2008 Elsevier Ltd. All rights reserved.