Extracellular

The extracellular and intracellular metabolites formed upon exposure of activated sludge microorganisms to a sublethal concentration of N-ethylmaleimide were monitored by liquid chromatography with ion trap mass spectrometry. The metabolite N-ethylsuccinimido-S-glutathione (m/z 433) was converted rapidly to N-(2-oxoethyl)-2,2-(propionylamino)propanamide (m/z 187) and N-ethylmaleamic acid (m/z 144).

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular R-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the R-galactosidases were identical. Intracellular R-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular R-galactosidase presented higher k cat than the intracellular enzyme (7.16 vs 3.29 s-1 , respectively) for the p-nitrophenyl-R-D-galactopyranoside substrate. The K m for hydrolysis of pNPRGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was acompetitively inhibited by galactose (K i) 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55°C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Exposure to hypobaric hypoxia, a condition involving decreased availability of oxygen is known to be associated with oxidative stress, neurodegeneration and memory impairment. The multifactorial response of the brain and the complex signaling pathways involved therewith limits the therapeutic efficacy of several antioxidants in ameliorating hypobaric hypoxia-induced memory impairment. The present study was therefore aimed at investigating the potential of acetyl-L-carnitine (ALCAR), a known antioxidant that has been reported to augment neurotrophin-mediated survival mechanisms, in ameliorating hypoxia-induced neurodegeneration and memory impairment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor involved in the cellular defense mechanism against oxidative stress related to brain injury and neurological disorders. The study was designed to understand the mechanisms involving Nrf2 stabilization following exposure to hypobaric hypoxia. The results displayed reference memory impairment in Sprague-Dawley rats exposed to hypobaric hypoxia (7620 m) for 14 consecutive days which however improved on administration of ALCAR during hypoxic exposure. The study also revealed Nrf2 regulated augmented antioxidant response on administration of ALCAR which was through a novel tyrosine kinase A (TrkA) receptor-mediated mechanism. A decrease in free radical generation, lipid peroxidation and protein oxidation was also observed along with a concomitant increase in thioredoxin and reduced glutathione levels on administration of ALCAR during exposure to hypobaric hypoxia. The present study therefore reveals the therapeutic potential of ALCAR under conditions of hypobaric hypoxia and elucidates a novel mechanism of action of the drug.

Microelectrode array (MEA) technology holds tremendous potential in the fields of bio-detection, lab-on-a-chip applications, and tissue engineering by facilitating non-invasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.

Lactic acid bacteria (LAB) play a key role in the development of the organoleptic and textural qualities of fermented dairy products. The exopolysaccharides (EPS) produced by LAB are of great technological interest since they improve the rheological characteristics of these products and help reduce syneresis. In the present work, an EPS-producing Streptococcus thermophilus strain was isolated from Algerian raw cow milk. Its identity was confirmed by biochemical and molecular methods. The presence in its genome of a priming glycosyltransferase gene was confirmed by PCR. The strain's growth and EPS production in milk was analysed and the molar mass of the EPS produced determined by HPLC.

Blue light and red light can be used to improve the efficiency of photosynthesis and increase the production of extracellular polysaccharide. Growth and extracellular polysaccharide production by Porphyridium cruentum were measured as a function of light wavelength and intensity. Light quality was a key factor for controlling the growth and polysaccharide production. The growth rate of P. cruentum increased with enhanced of light intensity, however a light level beyond the saturation point inhibited the growth of microalgae. A chromatic spectrum of blue and red light was used to enhance the growth and extracellular polysaccharide production due to the characteristics of the photosynthetic process. Efficient light transfer was the most important parameters in optimizing the mass culture of the photosynthetic organism.

The implementation of element-to-element propagation into the Miller-Geselowitz heart model, so as to automatically generate activation isochrones, is described. This implementation was achieved from initiation sites on the endocardial surface of the model via a Huygens' construction, utilizing ellipsoidal propagation wavelets to reflect anisotropic propagation in the myocardium. Isochrones similar to those specified for normal activation of the original Miller-Geselowitz model were obtained, using propagation velocities derived from published propagation velocities measured in isolated tissue. Futther validation of the new model was sought by simulating the Wolff-Parkinson-White syndrome, in which preexcitation of the ventricles of the heart occurs due to an accessory pathway connecting atria and ventricles, resulting in an initial delta wave in the QRS complex of the electrocardiogram. The approximate site of the accessory pathway may be deduced from the subject's body surface potential map pattern during the delta wave, or from the polarities of the delta wave in the 12-lead electrocardiogram, or again from the orientation of the spatial vectorcardiogram during the delta wave. By specifying eight separate accessory pathway initiation sites, followed 40 ms later by normal activation, the isochrones corresponding to preexcitation were simulated. The body surface potential maps, electrocardiograms, and vectorcardiograms were calculated using an inhomogeneous torso model. While agreement between simulated and experimentally reported map patterns was very good for all eight sites, that between delta wave polarities in simulated and clinically expected electrocardiograms was not as good, owing to the sensitivity of these polarities to variations in heart orientation and position within the torso. This was also the case for delta vector orientations in simulated and clinically recorded vectorcardiograms.

Extracellular secretion of over 4 g‚L -1 of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut + and Mut S phenotypes were compared in a bioreactor under closedloop PID control of the methanol level. As expected, the Mut S phenotype has a growth rate lower than that of the Mut + (0.37 vs 1.05 d -1 ) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut + (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g‚L -1 for the Mut S also has a positive effect on the final product concentration. Both Mut + and Mut S phenotypes reach a maximum biomass density around 450 g‚L -1 wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.

Sourdough has been used since ancient times and its ability to improve the quality and increase the shelf-life of bread has been widely described. During sourdough fermentation, lactic acid bacteria (LAB) produce a number of metabolites which have been shown to have a positive effect on the texture and staling of bread, e.g. organic acids, exopolysaccharides (EPS) and/or enzymes. EPS produced by LAB have the potential to replace more expensive hydrocolloids used as bread improvers. Organic acids affect the protein and starch fractions of flour. Additionally, the drop in pH associated with acid production causes an increase in the proteases and amylases activity of the flour, thus leading to a reduction in staling. While improving the textural qualities of bread, sourdough fermentation also results in increased mineral bioavailability and reduced phytate content. In this review we will be discussing the effect of sourdough on wheat and rye bread as well as the potential of sourdough to improve the quality of gluten-free bread.

Aerobic granulation is drawing increasing global interest in a quest for an efficient and innovative technology in wastewater treatment. Developed less than two decades ago, extensive research work on aerobic granulation has been reported. The instability of the granule, which is one of the main problems that hinder practical application of aerobic granulation technology, is still to be resolved. This paper presents a review of the literature in aerobic granulation focusing on factors that influence granule formation, granule development and their stability in the context of sludge granulation. The review attempts to shed light on the potential of developing granules with adequate structural stability for practical applications. The possibilities and perspective of using stored granule as inoculums for rapid startup, and as microbial supplement to enhance treatment of bioreactor systems are also discussed.

As a result of the wide application and utilization of the waste activated sludge process, excess sludge presents a serious disposal problem. Many efforts have been devoted to reduce the excess sludge by treatments such as digestion and dewatering. It has been known for many years that a thermal pre-treatment gives an improvement in the dewaterability of sludges. This paper provides a literature review concerning the optimum treatment conditions to obtain enhanced dewaterability and digestibility of sludge. The main commercial hydrolysis processes (Cambi, Porteous and Zimpro) are discussed. The literature findings concerning the optimum treatment conditions of thermal or thermochemical pre-treatments are reviewed.

Lactic acid bacteria (LAB) that produce exopolysaccharides (EPSs) play an important role in the dairy industry because of their contribution to the consistency and rheology of fermented milk products. The EPS polymers can be considered as natural biothickeners because they are produced in situ by the LAB-starters that have General Recognised As Safe status (GRAS). The physico-chemical properties of EPSs determine their viscosifying efficiency. Hence, the knowledge of the structure-function relationship of these biopolymers is crucial in order to choose or design polymers for a specific technological application. In addition, health benefits have been attributed to some of these EPSs, particularly antitumor and immunomodulating activities. Also a prebiotic role has been suggested. However, almost all studies were performed in vitro and scarce information is available concerning in vivo experiments with oral administration. This overview focuses on the recent information about the functional properties of lactic acid bacterial EPSs, including both technological and health-promoting aspects. r

The observation of biofilm formation is not a new phenomenon. The prevalence and significance of biofilm and aggregate formation in various processes have encouraged extensive research in this field for more than 40 years. In this review, we highlight techniques from different disciplines that have been used to successfully describe the extracellular, surface and intracellular elements that are predominant in understanding biofilm formation. To reduce the complexities involved in studying biofilms, researchers in the past have generally taken a parts-based, disciplinary specific approach to understand the different components of biofilms in isolation from one another. Recently, a few studies have looked into combining the different techniques to achieve a more holistic understanding of biofilms, yet this approach is still in its infancy. In order to attain a global understanding of the processes involved in the formation of biofilms and to formulate effective biofilm control strategies, researchers in the next decade should recognise that the study of biofilms, i.e. biofilmology, has evolved into a discipline in its own right and that mutual cooperation between the various disciplines towards a multidisciplinary research vision is vital in this field.

Fouling characterisation Extracellular polymeric substances Cleaning procedures NOM complexation Metal cations a b s t r a c t In this study, for the first time a full-scale membrane bioreactor (MBR) was investigated with focus on organic compounds in activated sludge over a period of approximately 2 years. Soluble extracellular polymeric substances (EPS) in the sludge supernatant and permeate as well as bound EPS extracted from fouled membranes were determined photospectrometrically and revealed a typical composition of three main components in the order metals4humic acids4carbohydrates4proteins. Results showed an important influence on membrane fouling by soluble humic substances and carbohydrates in complexes with metal cations. It was found that Fe 2+ and Fe 3+ play a decisive role in natural organic matter (NOM) complexation and subsequent membrane blockage. The determination of molar mass distribution in supernatant and permeate by size exclusion chromatography (SEC) revealed a significant retention of macromolecular compounds by the porous membranes in the range of 10-50%.

An exopolysaccharide (EPS) was isolated from Bacillus coagulans RK-02. Time course accumulation of EPS was studied with respect to biomass growth. The probiotic bacterium produces an EPS during exponential and stationary growth phases. Growth kinetics of the producer organism was characterized. The probiotic bacterium exhibited high affinity for the growth limiting substrate and hence grows at a higher specific growth rate. Based on HPLC and FTIR analyses, the EPS was found to be heteropolymer composed of galactose, mannose, fucose, glucose and glucosamine. The EPS showed significant emulsifying activities in different vegetable oils/hydrocarbon substrates. To the best of our knowledge, this is the first report on a bacterial EPS with fucose as a constituent sugar and also the first report on an EPS from B. coagulans.

The optimal temperature, pH and incubation time for production of exopolysaccharide (EPS) by Lactobacillus delbruckii subsp. bulgaricus and Streptococcus thermophilus strains in MRS and M17 media, respectively, were determined. In all strains, the temperature and incubation time for EPS production were 45 °C and 18 h, respectively. At 45 °C, L. delbruckiisubsp. bulgaricus B3 and G12 and S. thermophilus W22 strains produced 263, 238 and 127 mg/l, respectively. At 18 h, B3, G12 and W22 strains produced 220, 152 and 120 mg/l, respectively. While the pH for highest EPS production by L. delbruckii subsp. bulgaricus strains was 6.2 (in B3 strain: 211 mg/l, in G12 strain: 175 mg/l), for highest EPS production byS. thermophilus strain it was 6.8 (114 mg/l).

Microbial polysaccharides have multiple functions and can be divided into intracellular polysaccharides, structural polysaccharides and extracellular polysaccharides or exopolysaccharides (EPS). EPS from both prokaryotes and eukaryotes has a great deal of research interest. Recent approaches are carried out to replace the traditionally used plant gums by their bacterial counterparts. The bacterial EPS represent a huge variety of chemical structures, but have not yet get appreciable significance. Chemically, EPS contains high molecular weight polysaccharides (10-30 kDa) and have homopolymeric as well as heteropolymeric composition. They have new-fangled applications due to their unique properties they possess. Therefore EPS have found multifarious applications in the food, pharmaceutical and other industries. It also possesses potential application on waste water treatment.

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPSproducing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria. There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and highquality yogurt production.

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.

The subunit structures from a number of different species of lactic acid bacteria are now known, with many of the structures being published only in the last five years. The nuclear magnetic resonance spectra to date show around twenty different subunits and molecular mass determinations suggest that these exopolysaccharides occur as very large molecules, frequently greater than 1×106 Da. The physical properties of these molecules will be influenced by their mass and also by the subunit structure. Milk however is a complex medium and studies concerned with effects on rheology of milk as a result of in situ production of exopolysaccharides from these bacteria have to consider many factors. Studies have found that it is not only the nature of the exopolysaccharide, but that the amount produced, the acidity of the milk, the composition of the milk and the length of fermentation, also influence final texture.

The field of nanotechnology has generated great enthusiasm in recent years. The development of reliable, eco-friendly process for the synthesis of nano scale materials is an important aspect of nanotechnology. In this context a wide range of biological organisms such as bacteria, actinomycetes, algae, fungus etc. were listed for the development of clean, nontoxic and environmentally acceptable "green chemistry" procedure. This review covers a brief overview of the worldwide research efforts on the use of fungus in the biosynthesis of inorganic nanoparticles.

The transduction pathway of ampullary electroreceptor organs involves ionic currents. It has been shown that calcium, as well as sodium and potassium play important parts in this process. In this study we examine the stimulus-evoked changes in the Fura-2 ratio in electroreceptor cells. Furthermore, we recorded stimulus-evoked Fura-2 ratio changes while Na q and K q channels were blocked by amiloride and TEA. Simultaneously, extracellular recordings of the afferent spike activity were made. The results show the presence of stimulus evoked fluctuations in the Fura-2 ratio. These fluctuations can be abolished by the application of Cd 2q , TEA, and amiloride. The stimulus-evoked activity of the afferent nerve was decreased due to application of these drugs. We conclude that the transduction current is carried by Na q , K q , and probably Ca 2q. This fits the existing model on transduction in electroreceptors.

The general secretory pathway is routinely concerned with a multitude of extracellular enzymes. By eliminating obstructive competitors the export machinery may transport larger quantities of remaining proteins under circumstances in which the secretion machinery is fully loaded. Hence, in this study, genes encoding efficiently expressed but dispensable exoenzymes were knocked out in Bacillus licheniformis MD1. Single, double, and triple mutants with deletions of celA, chiA, and amyB, respectively, were generated via in vivo recombination by making use of a vector with a temperature sensitive origin of replication. Overexpression of a heterologous amylase gene on a multi-copy plasmid, a common scenario in biotechnological processes, resulted in an articulate reduction of chromosomally encoded extracellular enzyme activities indicating that the secretion machinery works to capacity in such transformants. Deletion mutants with the expression plasmid displayed enhanced amylase activities compared to the strain with the wild type genetic background. In addition, the chromosomally encoded protease activity was clearly higher in transformants with deletions.

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g−1 of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.

We examined the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The EPS obtained from a mid-developmental-stage biofilm (DS1) and a mature biofilm (DS2) were qualitatively and quantitatively compared. More than twice as much EPS was derived from DS2 as from DS1 (approximately 340 and 150 mg of EPS per g [dry weight] for DS2 and DS1, respectively). Composition analyses indicated the presence of carbohydrates, metals, proteins, and minor quantities of DNA and lipids, although the relative concentrations of these components were different for the two EPS samples. EPS from DS2 contained higher concentrations of metals and carbohydrates than EPS from DS1. Fe was the most abundant metal in both samples, accounting for about 73% of the total metal content, followed by Al, Mg, and Zn. The relative concentration profile for these metals resembled that for the AMD solution in which the biofilms grew, except for Si, Mn, and Co. Glycosyl composition analysis indicated that both EPS samples were composed primarily of galactose, glucose, heptose, rhamnose, and mannose, while the relative amounts of individual sugars were substantially different in DS1 and DS2. Additionally, carbohydrate linkage analysis revealed multiply linked heptose, galactose, glucose, mannose, and rhamnose, with some of the glucose in a 4-linked form. These results indicate that the biochemical composition of the EPS from these acidic biofilms is dependent on maturity and is controlled by the microbial communities, as well as the local geochemical environment.

Aims: The selection of exopolysaccharide (EPS)-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. Methods and Results: Improved EPS-overproducing strains of L. delbrueckii subsp. bulgaricus were derived by chemical mutagenesis and selection. Initial screening of the chemically induced mutant pool relied primarily on the selection of strains with raised levels of lactic acid and reduced biomass formation. Supporting selection criteria used were ropiness and colonial mucoidy. Final screening of candidate strains undertaken in a semi-defined medium in batch culture, resulted in the selection of a mutant with a 35% improvement in specific EPS yield relative to the parent strain. Conclusions: Initial selection of mutants of L. delbrueckii subsp. bulgaricus on the basis of enhanced formation of lactate and reduced biomass formation, coupled with a ropy or mucoid phenotype, proved to be a satisfactory means of isolating strains with the potential for a higher level of specific EPS production than the parent strain. Significance and Impact of the Study: The assay protocol allowed for the selection of an EPS-overproducing strain of L. delbrueckii subsp. bulgaricus. Such strains are useful for the purposes of metabolic studies related to EPS-production.

A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.

The steroidal glycoalkaloid α-tomatine of tomato plants has been reported to protect Lycopersicon species against fungal attack. Two isolates from Fusarium solani were found to produce an extracellular enzyme inducible by α-tomatine. TLC showed that the enzyme catalyzes the hydrolysis of the glycoalkaloid into β-lycotetraose and tomatidine. The enzymatic activity was concentrated against polyethylene glycol 35 000, and the enzyme was partially purified by preparative isoelectric focusing, preparative gel electrophoresis and ion-exchange chromatography. The enzyme was found to be a monomer of about 32 kDa by both SDS-PAGE and gel filtration. This molecular mass differs from that of the tomatinase of Fusarium oxysporum (50 kDa). Moreover, polyclonal antibody antitomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani, suggesting that this tomatinase may be a novel enzyme.

A study was made of the partition and purification of α-amylase from a culture supernatant of Bacillus subtilis C10 in a polyethylene glycol/potassium phosphate aqueous two-phase system. Factors that influenced the partition of the proteins in this system, including polyethylene glycol molecular weight and concentration, and potassium phosphate concentration, were investigated. The optimum conditions for primary aqueous two-phase extraction were 40% polyethylene glycol 6000/18% potassium phosphate (pH 7.0) at room temperature. The partition coefficient for α-amylase (K α-amylase ) was 10.51 with a partitioning yield of 93.45% and specific activity of 364.36 unit/mg protein. The α-amylase, which was concentrated in the top phase, was further mixed with 30% potassium phosphate (pH 7.0) at a ratio of 7:3 at room temperature to elute the bottom phase (salt-rich phase). Using these steps, fold-purification of enzyme of 3.56 was achieved, with a recovery yield of 59.37%.

Purpose Nucleoside and base modulation of the cytotoxicity of nucleic acid and folate antimetabolite drugs has been widely discussed. Many investigators have observed reduced toxicity due to circumvention of drug-induced inhibition of de novo purine and pyrimidine synthesis. However, exogenous purine nucleosides and bases may also enhance the cytotoxicity of even moderate concentrations of antifolate drugs (MTX and PTX) which inhibit dihydrofolate reductase. In this study, the effects of nucleosides in the medium on the cytotoxicity and deoxyribonucleoside triphosphate pools after brief exposure of cultured cells to methotrexate have been studied in cultured L1210 murine leukaemia cells. Methods Cell viability was determined by trypan blue exclusion assay. Colony formation was assessed by microtitration cloning assay. The deoxyribonucleotides were measured by a modification of the DNA polymerase assay. Purines were extracted with trioctylamine and 1,1,2trichlorotrifluoroethane buffer and concentrations of purine bases were determined by HPLC.

We examined the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The EPS obtained from a mid-developmental-stage biofilm (DS1) and a mature biofilm (DS2) were qualitatively and quantitatively compared. More than twice as much EPS was derived from DS2 as from DS1 (approximately 340 and 150 mg of EPS per g [dry weight] for DS2 and DS1, respectively). Composition analyses indicated the presence of carbohydrates, metals, proteins, and minor quantities of DNA and lipids, although the relative concentrations of these components were different for the two EPS samples. EPS from DS2 contained higher concentrations of metals and carbohydrates than EPS from DS1. Fe was the most abundant metal in both samples, accounting for about 73% of the total metal content, followed by Al, Mg, and Zn. The relative concentration profile for these metals resembled that for the AMD solution in which the biofilms grew, except for Si, Mn, and Co. Glycosyl composition analysis indicated that both EPS samples were composed primarily of galactose, glucose, heptose, rhamnose, and mannose, while the relative amounts of individual sugars were substantially different in DS1 and DS2. Additionally, carbohydrate linkage analysis revealed multiply linked heptose, galactose, glucose, mannose, and rhamnose, with some of the glucose in a 4-linked form. These results indicate that the biochemical composition of the EPS from these acidic biofilms is dependent on maturity and is controlled by the microbial communities, as well as the local geochemical environment.

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world's most debilitating, costly, and intractable diseases.

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h 21 to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.

A novel biological method for the synthesis of silver nanoparticles using the fungus Verticillium is reported. Exposure of the fungal biomass to aqueous Ag + ions resulted in the intracellular reduction of the metal ions and formation of silver nanoparticles of dimensions 25 ± 12 nm. Electron microscopy analysis of thin sections of the fungal cells indicated that the silver particles were formed below the cell wall surface, possibly due to reduction of the metal ions by enzymes present in the cell wall membrane. The metal ions were not toxic to the fungal cells and the cells continued to multiply after biosynthesis of the silver nanoparticles.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP C ) into an altered disease-associated conformation generally designated PrP Sc , abnormal deposition of PrP Sc aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP C aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP C molecules may trigger its aggregation in neuronal cells. A chimeric PrP C composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP C were created, and transfected in N2a cells. Similar to PrP C , Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP C , both dimerization and oligomerization of PrP C resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP C molecules may constitute a minimal and sufficient molecular event leading to PrP C aggregation and extracellular deposition.

Transfer of CD45RB hi CD4 + naïve T cells into severe combined immunodeficient (SCID) mice induces colitis and skin lesions. Recipients treated with cyclosporin A (CsA), CTLA4-Ig, or vehicle were evaluated for weight loss, skin lesions, and cutaneous blood flow. Necropsy, histological, hematological and cytokine analyses were performed at the conclusion of the experiment to confirm the clinical findings. Vehicle-treated mice lost weight and had 100% incidence of skin lesions by 46days. CsA-treated mice also lost weight, but only 3/8 mice developed mild, clinically evident skin lesions. In contrast, all CTLA4-Ig-treated mice gained weight and did not develop skin lesions. Increase in cutaneous blood flow correlated with the development of skin lesions. Granulocyte numbers, which were high or moderately high in the vehicle-or CsA-treated mice, respectively, remained as low in the CTLA4-Ig-treated group as in untreated mice. IFN-g, IL-1h, and TNF-a levels in the gut and skin correlated with the extent of inflammation in both organs. Histology revealed that CTLA4-Ig but not CsA effectively prevented both autoimmune disorders. The ability of CTLA4-Ig to prevent both colitis and skin lesions suggests that CD28-1567-5769/02/$ -see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 -5 7 6 9 ( 0 1 ) 0 0 2 0 1 -6