Extracellular polymeric substances of the marine fouling diatom amphora rostrata Wm.Sm

Amphora rostrata was grown under continuous illumination at 27°C in batch cultures using f/2 medium. Cell biomass (measured as chllorophyll a and cell counts) reached a maximum on day 7. Thereafter, cell biomass as chl a showed a small decrease. Planktonic('free') and biofilm extracellular polymeric substances (EPS) from the adherent cells of A. rostrata were studied. Both types of EPS were produced during the logarithmic phase of growth. However, production was higher during the stationary growth phase. Enhanced EPS production was associated with nutrient deficient conditions. Planktonic and biofilm EPS were purified by gel filtration using Sephadex G‐200 and ion exchange chromatography using DEAE‐cellulose. Both polymers showed the presence of a single peak. Capillary gas Chromatographie analysis of both planktonic and biofilm EPS showed that fucose (36.7%) and galactose (27.6%) were the most abundant monosaccharides, with small quantities of rhamnose, xylose, arabinose, mannose and glucose. Other chemical analysis showed the presence of sulphate, uronic acids, hexoamines, pyruvate and proteins in both the planktonic and bio‐film EPS. Uronic acid, pyruvate and sulphate together were found to contribute ～50 to 60% (W/W) to the EPS of A. rostrata. Such a high content of non‐sugar components indicates their importance to the diatom in metal binding, desiccation prevention and flexibility.     

A comparative study of induction of pneumonia in mice with planktonic and biofilm cells of Klebsiella pneumoniae

In the present study, the course of acute pneumonia in normal BALB/c mice infected by intranasal inoculation of planktonic and preformed biofilm cells (3 days old) of Klebsiella pneumoniae B5055 was studied and compared. With both cell forms the peak of infection was observed on the third post infection day, as assessed on the basis of lung bacterial load and corresponding pathology. There was an intense neutrophil infiltration in bronchoalveolar lavage fluid. Tissue damage was assessed on the basis of increased amounts of nitrite, malondialdehyde and lactate dehydrogenase in lung homogenates. The phagocytic potential of alveolar macrophages was lower in biofilm cell-induced infection than in that induced by planktonic cells. Biofilm cell induced infection generated significantly greater production of tumor necrosis factor-α and interleukin-1β on the third and fifth days of infection, respectively. Production of interleukin-10 was, however, variable. There was no significant difference in the ability of planktonic and biofilm cell forms of K. pneumoniae to induce acute pneumonia in mice in terms of bacterial counts and histopathological changes. However, biofilm cell-induced infection showed delayed clearance as compared to infection induced with the planktonic form.     

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.     

Biological activity of roots and aerial parts of Zinnia peruviana on pathogenic micro-organisms in planktonic state and biofilm forming

Microbial resistance to antibiotics affects the control of clinical infections and is a growing concern in global public health. One important mechanism whereby micro-organisms acquire resistance is biofilm formation. This context has led to the investigation of new antimicrobial substances from plants popularly used in folk medicine. In this work, we studied the antimicrobial and antibiofilm activity of Zinnia peruviana roots, ziniolide (major root metabolite) and aerial parts against Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The minimum inhibitory and minimum microbicidal concentration and inhibition of biofilm production was determined. All Z. peruviana extracts showed antimicrobial activity, but that corresponding to the roots was the most active one. The best inhibitory and microbicidal activity was detected against Gram-positive bacteria (0·039–0·078 mg ml⁻¹). The acetonic extract from Z. peruviana leaves showed moderate activity against Gram-positive bacteria (0·625 mg ml⁻¹). Acetonic extract of Z. peruviana flowers showed weak activity (1·25–5 mg ml⁻¹). All the extracts tested showed inhibition of biofilm formation, as well as the ziniolide, however, roots and flowers extracts showed higher antibiofilm activity particularly against Staphylococcus, Listeria and Candida. The extracts tested may be a promising natural alternative for the control of microbial infections.     

Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures

Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro‐organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug‐resistance of S. aureus both in suspension and in biofilm.     

Destruction of planktonic, adherent and biofilm cells of Staphylococcus epidermidis using a gliding discharge in humid air

AIMS: To determine the efficiency of an electric discharge of the gliding arc type for the destruction of Staphylococcus epidermidis planktonic, adherent and biofilm cells. METHODS AND RESULTS: Bacterial cells were treated in humid air and at atmospheric pressure by a nonthermal quenched plasma of the glidarc type. The kinetics of destruction (followed by plating) were modelled by an Add-inn for Microsoft Excel, GInaFiT. For planktonic cells, log-linear destruction was obtained, whereas biphasic kinetics were observed for sessile cells. An increased resistance of biofilm cells was observed: the reduction of 6 logarithm units of the population was obtained in 15, 30 and 70 min for planktonic, adherent and biofilm cells, respectively. The experiments also show that the cells destruction did not depend on the adhesion surface but was governed by the gap between the target and the plasma source. CONCLUSION: The complete destruction of planktonic, adherent and more resistant biofilm cells of Staph. epidermidis is achieved by a glidarc air plasma at atmospheric pressure. SIGNIFICANCE AND IMPACT OF THE STUDY: The glidarc plasma technology is a promising candidate among the emerging nonthermal techniques for decontamination, as it can destroy even biofilms that are known as particularly resistant to various antimicrobials.     

Elucidation of metabolism in hybridoma cells grown in fed‐batch culture by genome‐scale modeling

Genome‐scale modeling of mouse hybridoma cells producing monoclonal antibodies (mAb) was performed to elucidate their physiological and metabolic states during fed‐batch cell culture. Initially, feed media nutrients were monitored to identify key components among carbon sources and amino acids with significant impact on the desired outcome, for example, cell growth and antibody production. The monitored profiles indicated rapid assimilation of glucose and glutamine during the exponential growth phase. Significant increase in mAb concentration was also observed when glutamine concentration was controlled at 0.5 mM as a feeding strategy. Based on the reconstructed genome‐scale metabolic network of mouse hybridoma cells and fed‐batch profiles, flux analysis was then implemented to investigate the cellular behavior and changes in internal fluxes during the cell culture. The simulated profile of the cell growth was consistent with experimentally measured specific growth rate. The in silico simulation results indicated (i) predominant utilization of glycolytic pathway for ATP production, (ii) importance of pyruvate node in metabolic shifting, and (iii) characteristic pattern in lactate to glucose ratio during the exponential phase. In future, experimental and in silico analyses can serve as a promising approach to identifying optimal feeding strategies and potential cell engineering targets as well as facilitate media optimization for the enhanced production of mAb or recombinant proteins in mammalian cells. Biotechnol. Bioeng. 2009;102: 1494–1504. © 2008 Wiley Periodicals, Inc.     

Investigation of the effectiveness of disinfectants against planktonic and biofilm forms of P. aeruginosa and E. faecalis cells using a compilation of cultivation,microscopic and flow cytometric techniques

This study evaluated the effectiveness of selected disinfectants against bacterial cells within a biofilm using flow cytometry, the conventional total viable count test and scanning electron microscopy (SEM). A flow cytometric procedure based on measurement of the cellular redox potential (CRP) was demonstrated to have potential for the rapid evaluation of activity against biofilm and planktonic forms of microbes. Quaternary ammonium compound-based disinfectant (QACB) demonstrated a higher level of anti-microbial activity than a performic acid preparation (PAP), with mean CRP values against P. aeruginosa cells of 2 and 1.33 relative fluorescence units (RFU) vs 63.33 and 61.33 RFU for 8 and 24 h cultures respectively. Flow cytometric evaluation of the anti-biofilm activity demonstrated a higher efficacy of QACB compared to PAP for P. aeruginosa cells of 1 and 0.66 RFU vs 18.33 and 22.66 RFU for 8 and 24 h cultures respectively. SEM images of treated P. aeruginosa cells demonstrated disinfectant-specific effects on cell morphology.     

Glycoprotein and protein precursors to plasma membranes in vesicular stomatitis virus infected HeLa cells

Vesicular stomatitis virus is known to mature at HeLa cell plasma membranes. To study the process, cells, infected with vesicular stomatitis virus, were fractionated after short term labeling studies (1 min pulse, 1 min chase) to determine the assembly kinetics of G protein and M protein into plasma membranes. Newly synthesized M protein was found released in the supernatant from which free polysomes were sedimented during sucrose gradient analysis of these polysomes. If this M protein is particle bound, it must have a density of less than 1.08 g/ml. About 40% of this M protein so labeled was not sedimentable at 165,000 X g for 16 h. This newly synthesized M protein had not yet assembled into plasma membrane and thus must represent an internal pool. This and previous studies show that it has a subsequent transit time to the plasma membrane of about 2 min. Once associated with plasma membranes, M protein decayed in an approximately logarithmic fashion indicating that newly synthesized M randomly mixes (and turns over) with preexisting M protein. G protein was particle bound in a 1 min pulse, 1 min chase, and was never found released in a soluble form. At the later time when fucose is added to G protein, the oligosaccharide moiety is near to complete, and on completion is about 2,000 in molecular weight. Evidence is presented showing that fucose is probably attached to the N-acetylglucosamine of the protein carbohydrate linkage. G protein to which fucose had just been added was located internally on a membranous fraction of density 1.14 g/ml in sucrose; its subsequent transit time from this pool (which in uninfected cells is between 1–2% of the total cell fucosyl glycoprotein) was about 15 min. Because their densities were different and their transit times were different, internal newly synthesized M and fucosyl G protein which assemble into plasma membranes were not on the same internal membranous component. Association of M protein with the plasma membranes may thus occur from a nonsedimentable soluble cytoplasmic pool by a process of direct adsorption.     

CHO工程细胞无血清悬浮分批培养的生长代谢特征及动力学模型

以悬浮适应的表达尿激酶原CHO工程细胞为研究对象,在100mL的摇瓶中进行无血清悬浮培养,以细胞密度、细胞活力、Pro-UK活性、葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、乳酸对葡萄糖的得率系数(Ylac/glc)为观察指标,同时以细胞有血清悬浮培养作为参照,考察CHO工程细胞无血清悬浮培养生长和代谢特征。观察结果表明,CHO工程细胞在无血清及有血清悬浮培养条件下表现为大致相似的细胞生长和代谢特征。在此基础上,依据实际检测的数据,应用MATLAB软件对细胞对数生长期的细胞生长、乳酸生成及葡萄糖消耗的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了细胞在无血清培养条件下的生长及代谢动力学模型。     

Secretion of ribonucleases by normal and immortalized cells grown in serum-free culture conditions

Summary The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts, bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin. Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase family secreted by normal versus tumor cells in culture.     

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.     

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.     

Development and characterization of cell lines of normal mouse bladder epithelial cells and 2-Acetylaminoflourene-induced urothelial carcinoma cells grown in monolayer tissue culture

Summary Normal urinary bladder epithelial cells and cells derived from 2-acetylaminofluorene-induced urothelial carcinomas from male Balb/c mice were grown in monolayer culture and were characterized. Cell lines of normal bladder epithelium were mononucleated, sheet-forming cells, with a modal chromosome number of 40. Bladder epithelial carcinoma cells induced by 2-acetylaminofluorene were polynucleate, relatively fast growing, grew in soft agar, demonstrated a higher cloning efficiency than normal cells and formed tumors when inoculated into syngeneic hosts. Differences in morphology were recorded by photomicrography using phase optics and scanning electron microscopy.