The nature of stability of yeast cells to drying]

The residual water and dry matter condition in the lyophilized biomass of the yeast Saccharomyces cerevisiae was studied by NMR-relaxation technique. It was shown that the slow component of the transverse magnetization NMR signal spectrum corresponding to the so-called "isolated mobile water" was caused in fact by the interaction of the disaccharide trehalose with the cell biopolymers. The big amount of hydrogen bonds formed by trehalose and their three-dimensional orientation closed to the orientation in water clusters assure the valuable functioning of this disaccharide during the process of removing water out of cells. When stationary phase yeast biomass containing a lot of trehalose was dried the cell organelles condition remained practically unchanged what led to the high resistance of such cells to dehydration.     

Immobilization of yeast cells on various supports for ethanol production

An immobilization technique has been developed for a packed bed fermenter which is being considered as one stage of a process for the production of fuel-grade ethanol from sugar solutions. Relatively inexpensive beech wood chips have been successfully used as the support material and relatively high cell loadings of 188 mg DW cells/g DW support have been achieved for a test system of Saccharomyces cerevisiae cultures.No washout of adsorbed cells occurs below a superficial liquid velocity of 8.9 × 10^-2 cm/s which can be increased to 9.7 × 10^-2 cm/s by including up to 1% Hercofloc solution in the reactor medium during the immobilization procedure. The immobilization procedure is practically unaffected by pH and temperature in the range 3.5 to 5.0 and 22 °C to 37 °C respectively.Typical ethanol productivity of 21.8g/l·hr has been obtained with wood-chip-adsorbed cells, which compares well with optimal values of 18 to 32g/l·hr obtained using free-suspension cultures in stirred-tank fermenters with cell recycle.     

Physical and chemical immobilization of choline oxidase onto different porous solid supports: Adsorption studies

This work carries out for the first time the comparison between the physical and chemical immobilization of choline oxidase onto aminated silica-based porous supports. The influence on the immobilization efficiency of concentration, pH, temperature and contact time between the support and choline oxidase, was evaluated. The immobilization efficiency was estimated taking into consideration the choline oxidase activity, which was assessed by using cadmium telluride (CdTe) quantum dots (QDs), obtained by hydrothermal synthesis, as photoluminescent probes. Hydrogen peroxide produced by enzyme activity was capable of quenching CdTe QDs photoluminescence. The magnitude of the PL quenching process was directly related with the enzyme activity.By comparing the chemical process with the physical adsorption, it was observed that the latter provided the highest choline oxidase immobilization. The equilibrium data were analyzed using Langmuir and Freundlich isotherms and kinetic data were fitted to the pseudo-first-order and pseudo-second-order models. Thermodynamic parameters, such as Gibbs free energy and entropy were also calculated. These results will certainly contribute to the development of new sensing schemes for choline, taking into account the growing demand for its quantification in biological samples.     

Adhesion,spreading, and proliferation of cells on protein carpets: Effects of stability of a carpet

Summary In the present report we have investigated the role that the physical properties of substrata play in modulating the effects which components of extracellular matrix (ECM) exert on adhesion, spreading, and growth of retinal pigmented epithelial cells. By simple modifications of conditions for protein adsorption on glass we obtained a set of substrata all coated with proteins of ECM (protein carpets) but with different physical properties. Using these protein carpets we have shown that their stability (desorption rate) in tissue culture conditions varies according to the technique with which they were prepared. Both semiremovable and immobilized carpets are stable, whereas removable protein carpets desorb readily. Therefore, the protein concentration or composition or both may change with time in tissue culture depending on the technique used to prepare the carpet. In addition, efficacy of cell attachment to given protein may vary depending on whether a technique used to prepare the protein carpet involves denaturation of the protein. Adherent cells quickly remove (clear) weakly adsorbed protein carpets and it seems that the carpet removal is a mechanical process. During the carpet removal cells are rounded, which indicates that a spread cell phenotype normally associated with stress fibers and focal contacts occurs when the substratum is rigid enough to sustain cell traction. In addition, substrata lacking the rigidity to support the spread phenotype do not support cell proliferation either.     

PTPD1 supports receptor stability and mitogenic signaling in bladder cancer cells

PTPD1, a cytosolic non-receptor protein-tyrosine phosphatase, stimulates the Src-EGF transduction pathway. Localization of PTPD1 at actin cytoskeleton and adhesion sites is required for cell scattering and migration. Here, we show that during EGF stimulation, PTPD1 is rapidly recruited to endocytic vesicles containing the EGF receptor. Endosomal localization of PTPD1 is mediated by interaction with KIF16B, an endosomal kinesin that modulates receptor recycling at the plasma membrane. Silencing of PTPD1 promotes degradation of EGF receptor and inhibits downstream ERK signaling. We also found that PTPD1 is markedly increased in bladder cancer tissue samples. PTPD1 levels positively correlated with the grading and invasiveness potential of these tumors. Transgenic expression of an inactive PTPD1 mutant or genetic knockdown of the endogenous PTPD1 severely inhibited both growth and motility of human bladder cancer cells. These findings identify PTPD1 as a novel component of the endocytic machinery that impacts on EGF receptor stability and on growth and motility of bladder cancer cells.     

Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems

Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies.     

Preparation of high-capacity supports containing protein G immobilized to porous silica

This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 Å pore size silica, followed by 33 mg/g for 50 Å silica and 9.3-24 mg/g for 300-4000 Å silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 μmol/m² being obtained for 4000 Å silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 Å silica.     

Adhesion of algal cells to surfaces

This paper reports the cell–substratum interactions of planktonic (Chlorella vulgaris) and benthic (Botryococcus sudeticus) freshwater green algae with hydrophilic (glass) and hydrophobic (indium tin oxide) substrata to determine the critical parameters controlling the adhesion of algal cells to surfaces. The surface properties of the algae and substrata were quantified by measuring contact angle, electrophoretic mobility, and streaming potential. Using these data, the cell–substratum interactions were modeled using thermodynamic, DLVO, and XDLVO approaches. Finally, the rate of attachment and the strength of adhesion of the algal cells were quantified using a parallel-plate flow chamber. The results indicated that (1) acid–base interactions played a critical role in the adhesion of algae, (2) the hydrophobic alga attached at a higher density and with a higher strength of adhesion on both substrata, and (3) the XDLVO model was the most accurate in predicting the density of cells and their strength of adhesion. These results can be used to select substrata to promote/inhibit the adhesion of algal cells to surfaces.     

Apical to basolateral surface area ratio and polarity of MDCK cells grown on different supports.

We have established conditions under which Madin-Darby canine kidney cells develop a well-polarized monolayer on polycarbonate filters and on transparent filters. These filters have biochemical and mechanical advantages over the nitrocellulose filters which have been widely used. Transepithelial resistance was established 10 h after plating and stabilized after 24 h. The distribution of protein antigens was followed by surface immunofluorescence and quantitated by a surface immunoassay that we developed. Uvomorulin was localized to the lateral membrane, with low amounts detectable on the basal membrane. The 58-kDa antigen was distributed over the entire basolateral domain, including cell processes extending into the filter pores. This distribution was confirmed by immunogold labeling of frozen sections. The 114-kDa antigen was found to be present at similar surface densities on both the apical and the basolateral domain. The support used for growth had profound effects on the cell morphology. A morphometric analysis of the plasma membrane of both strains of the cell line showed an increase in the number and size of the microvilli, and a smoother basal membrane as compared to published data on nitrocellulose filters. The apical to basolateral surface area ratio was therefore modified.     

Adhesion of cells to polystyrene surfaces

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.     

Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states

Enterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health.     

Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.     

Influence of physicochemical bacterial surface properties on adsorption to inorganic porous supports

Summary The effect of the hydrophobicity and the electrostatic charge of bacterial cell surfaces on the initial phase of adsorption to inorganic porous supports with SiO₂ or Al₂O₃ as the main components was investigated. The physicochemical surface properties of various Gram-positive and Gram-negative bacteria were characterized by water contact angle and zeta-potential measurements. The influence of microbial charge on adsorption was investigated by varying the ionic strength of the suspending liquid. The amount of Escherichia coli cells adsorbed to Siran and B supports increased with increasing electrolyte concentration. The effect of cell surface hydrophobicity on the extent of adsorption was demonstrated at high ionic strength (0.15 m NaCl) where charge effects were reduced. The supports applied in this study promoted the adsorption of hydrophilic bacteria. Offprint requests to: H. Ziehr     

By-product formation by a novel glycerol-producing yeast,Candida glycerinogenes,with different O2 supplies

Candida glycerinogenes is an aerobe which does not depend on sulphite for production of glycerol. With a sufficient O₂ supply, up to 130 g glycerol l^–1 was produced with 2.6 g acetic acid l^–1 as by-product. However, with an insufficient O₂ supply – with higher volumes of medium or at higher corn steep liquid concentrations – the glycerol concentration was lower because the by-products, ethanol, pyruvate and lactic acid, were produced in greater amounts, up to 45 g l^–1, 4.3 g l^–1, 1.6 g l^–1, respectively, whereas, less acetic acid (0.6 g l^–1) was produced. In addition, ethanol decreased to 0.4 g l^–1 and the glycerol yield improved from 34 to 50% (w/w) by adding 50 g sulphite l^–1, nevertheless, acetic acid increased to 7.8 g l^–1.     

Organic transformations catalyzed by engineered yeast cells and related systems

Asymmetric ketone reductions remain the most popular application of baker's yeast (Saccharomyces cerevisiae) in organic synthesis and data from the genome sequencing project is beginning to have an impact on improving the stereoselectivities of these reactions, augmenting traditional approaches based on selective inhibition. In addition, the catalytic repertoire of yeast has been expanded to include chiral ketone oxidations by overexpression of a bacterial Baeyer-Villiger monooxygenase.     

Adhesion of culture cells to their substratum

The attachment of cells to culture dishes has been investigated by replica techniques and scanning electron microscopy. Cells were removed from their substratum either in a stream of medium or by micromanipulation. In the most usual case of interphase cells one finds many small points of attachment uniformly distributed over the whole cell underside. Attachment sites are also found under the lamellipodia and at the trailing edge of the cells. A large portion of the cell underside is sometimes left behind. This is probably because the sole plate is reinforced by cytoskeletal elements, and does not necessarily indicate the presence of large adhesion points. The distal ends of the retraction fibers formed as the cells round-up in trypsin, or in the cold, represent the attachment points of the cell to the substratum. Agents which tend to stabilize microtubules greatly slow cell detachment by proteolytic agents. The primary effect of trypsin is not on the glue which holds the cells to the substratum but rather on the cell shape itself, affecting the rigidity of cytoskeletal elements.     

不同酿酒酵母(Schromycese cerovisea)对黄酒挥发性香气物质的影响

研究不同酵母菌株对黄酒挥发性香气物质形成的影响,采用4种不同酿酒酵母(黄酒活性干酵母、工业生产用黄酒酵母HJ-1,HJ-2、耐高温活性干酵母(NGW))菌株进行实验室黄酒酿造试验,采用固相微萃取-气质联用技术测定了发酵后四种黄酒中挥发性香气物质含量,检测出与酵母相关的27种挥发性成分.研究结果表明不同酵母菌株酿制黄酒样品挥发性香气物质存在明显的差别.其中,HJ-2菌株β-苯乙醇,总酯含量较高,香气较为纯正;NGW活性干酵母挥发性酸含量较高;黄酒活性干酵母挥发性酯含量较低.本研究发现通过选用不同酵母菌株能够有效的调节黄酒生产风味物质的形成.     

Adhesion of different bifidobacteria strains to human enterocyte-like Caco-2 cells and comparison with in vivo study

The validity of the in vitro adhesion tests performed with cultured cell lines, was determined in this study by comparison with results obtained in vivo , in a previous study. To make this experiment the in vitro adhesion tests were performed during a long period by utilization of an appropriate medium, to determine the capacity of the adhered strain to colonize the intestinal tract. It was demonstrated that the ability of the strain to adhere and colonize the intestinal cell in vivo or the cultured intestinal cells in intro was similar.