Formation Of Cell Aggregates Research Articles

RNAi Screening in Drosophila Cells Identifies New Modifiers of Mutant Huntingtin Aggregation

The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.

6507 Catumaxomab treatment in gastric-cancer patients with malignant ascites – subgroup-analysis of a pivotal trial

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10 mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells.

WITHDRAWN: Rheological and morphological characterization of the culture broth during exopolysaccharide production by Enterobacter sp.

Enterobacter sp. was grown on glycerol byproduct from the biodiesel industry for the production of a value-added exopolysaccharide (EPS). The culture broth was characterized in terms of its morphological and rheological properties throughout the cultivation run. Microscopic observations revealed the formation of cell aggregates surrounded by the EPS at the beginning of the cultivation run, while, at the end, aggregates were reduced and an EPS matrix with the cells embedded in it was observed. The apparent viscosity of the culture broth increased over time, which was attributed to the increase of the EPS concentration in the first period of the cultivation run. However, in the final stage, the creation of new polymer interactions within the complex culture broth was likely the reason for the viscosity increase observed, since there was not a significant variation of the EPS concentration, average molecular weight or chemical composition. The broth presented a Newtonian behavior at the beginning of the run, changing to pseudoplastic as the EPS concentration increased, and revealed to follow the Cox–Merz rule.

The in vitro biological activity of the HLA-DR-binding clinical IgG4 antibody 1D09C3 is a consequence of the disruption of cell aggregates and can be abrogated by Fab arm exchange

Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is still controversial. Apoptosis induction following HLA-DR engagement has been proposed. However, the validity of these results has been questioned by the observation that antibodies may induce formation of cell aggregates and cell death is induced upon dispersion of these aggregates prior to the quantification of cell death by flow cytometry. Here we address the capacity of 1D09C3 to induce apoptosis in vitro, also taking account of the recently reported Fab arm exchange of IgG4 antibodies. 1D09C3 induces formation of tight cellular aggregates that can only be dispersed at the expense of massive cell damage and death. Using dual color fluorescence cross-correlation spectroscopy (FCCS) we demonstrate that also this antibody undergoes Fab arm exchange in the presence of IgG4. FCCS is a powerful technique to investigate the molecular mechanism of Fab arm exchange using minute amounts of reagents. Following exchange, the functionally monovalent 1D09C3 chimeras loose their ability to induce aggregate formation of HLA-DR-positive cells. Neither functionally monovalent nor bivalent 1D09C3 antibodies induce cell death or apoptosis in myeloma target cells, when microscopy instead of flow cytometry is employed as the analytical technique. Our results indicate that the activity of 1D09C3 in vitro may have been a consequence of assay design rather than an ability to induce HLA-DR-dependent cell death.

Structure of Loricae and Stalks of Several Bacterivorous Chrysomonads (Chrysophyceae): Taxonomical Importance and Possible Ecological Significance

Stalks and loricae of chrysomonads were studied by fluorescence microscopy employing Calcoflor White-Evans blue (CW-Eb) staining, and by uranyl acetate staining of dried, whole mount preparations for electron microscopy. These structures were composed of microfibrils approximately 4 nm in diameter embedded in a matrix. The organization of the loricae of Poterioochromonas malhamenesis, "Amimonas minuta", Poterioochromonas stipitata and Ochromonas gloeopara showed a similar structural plan, consisting of a foot, stalk and cup region that together resemble a wine glass. CW-Eb-stained, microfibrillar stalks were identified also in Paraphysomonas vestita, Anthophysa vegetans and "Felimonas flocculans". These results suggest that CW-Eb-stained structures composed of microfibrils approximately 4 nm in diameter may be more common in chrysomonads than previously recognized. In cultures, these structures participate in the formation of cell aggregates and attachment of cells to substrates, and thus may be of ecological importance. Additionally, non-siliceous, CW-Eb-stained cysts were identified for the first time in P. malhamensis.

Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation

Superoxide dismutase-1 (SOD1) and ataxin-3 are two neurodegenerative disease proteins in association with familial amyotrophic lateral sclerosis and Machado-Joseph disease/spinocerebellar ataxia type 3. Both normal and mutant types of SOD1 and ataxin-3 are degraded by the proteasome. It was recently reported that these two proteins are associated with the endoplasmic reticulum (ER). Mammalian gp78 is an E3 ubiquitin ligase involved in ER-associated degradation (ERAD). Here, we show that gp78 interacts with both SOD1 and ataxin-3. Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins, whereas knockdown of gp78 stabilizes them. Moreover, gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death. Furthermore, gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice. Thus, our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.

Computational fluid dynamics of aggregating red blood cells in postcapillary venules

Aggregate formation of red blood cells (RBCs) in a postcapillary venular bifurcation is investigated with three-dimensional computer simulations using the Chimera grid method. Interaction energy between the RBCs is modelled by a depletion interaction theory; RBCs are modelled as rigid oblate ellipsoids. The cell–cell interactions of RBCs are strongly dependent on vessel geometry and shear rates. The experimental data on vessel geometry, pseudoshear rates, and Dextran concentration obtained in our previous in vivo RBC aggregation study in postcapillary venules of the rat spinotrapezius muscle were used to simulate RBC aggregation. The computational results were compared to the experimental results from the in vivo study. The results show that cells have a larger tendency to form an aggregate under reduced flows. Aggregate formation also depends on the angle and location of the cells before they enter the bifurcation region. Comparisons with experimental data are discussed.

Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease

Recent evidence suggests that red blood cell aggregation and the ratio of hematocrit to blood viscosity (HVR), an index of the oxygen transport potential of blood, might considerably modulate blood flow dynamics in the microcirculation. It thus seems likely that these factors could play a role in sickle cell disease. We compared red blood cell aggregation characteristics, blood viscosity and HVR at different shear rates between sickle cell anemia and sickle cell hemoglobin C disease (SCC) patients, sickle cell trait carriers (AS) and control individuals (AA). Blood viscosity determined at high shear rate was lower in sickle cell anemia (n=21) than in AA (n=52), AS (n=33) or SCC (n=21), and was markedly increased in both SCC and AS. Despite differences in blood viscosity, both sickle cell anemia and SCC had similar low HVR values compared to both AA and AS. Sickle cell anemia (n=21) and SCC (n=19) subjects had a lower red blood cell aggregation index and longer time for red blood cell aggregates formation than AA (n=16) and AS (n=15), and a 2 to 3 fold greater shear rate required to disperse red blood cell aggregates. The low HVR levels found in sickle cell anemia and SCC indicates a comparable low oxygen transport potential of blood in both genotypes. Red blood cell aggregation properties are likely to be involved in the pathophysiology of sickle cell disease: the increased shear forces needed to disperse red blood cell aggregates may disturb blood flow, especially at the microcirculatory level, since red blood cell are only able to pass through narrow capillaries as single cells rather than as aggregates.

Methylene blue and dimebon inhibit aggregation of TDP-43 in cellular models

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are major neurodegenerative diseases with TDP-43 pathology. Here we investigated the effects of methylene blue (MB) and dimebon, two compounds that have been reported to be beneficial in phase II clinical trials of Alzheimer’s disease (AD), on the formation of TDP-43 aggregates in SH-SY5Y cells. Following treatment with 0.05 μM MB or 5 μM dimebon, the number of TDP-43 aggregates was reduced by 50% and 45%, respectively. The combined use of MB and dimebon resulted in a 80% reduction in the number. These findings were confirmed by immunoblot analysis. The results indicate that MB and dimebon may be useful for the treatment of ALS, FTLD-U and other TDP-43 proteinopathies.

Spatial patterning of cell proliferation and differentiation depends on mechanical stress magnitude

Mechanical stress has been proposed as a major regulator of tissue morphogenesis; however, it remains unclear what is the exact mechanical signal that leads to local tissue pattern formation. We explored this question by using a micropatterned cell aggregate model in which NIH 3T3 fibroblasts were cultured on micropatterned adhesive islands and formed cell aggregates (or “cell islands”) of triangular, square, and circular shapes. We found that the cell islands generated high levels of mechanical stresses at their perimeters compared to their inner regions. Regardless of the shape of cell islands, the mechanical stress patterns corresponded to both cell proliferation and differentiation patterns, meaning that high level of cell proliferation and differentiation occurred at the locations where mechanical stresses were also high. When mechanical stretching was applied to cell islands to elevate overall mechanical stress magnitudes, cell proliferation and differentiation generally increased with the relatively higher mechanical stresses, but neither cell proliferation nor differentiation patterns followed the new mechanical stress pattern. Thus, our findings indicate that a certain range of mechanical stress magnitudes, termed window stress threshold, drives formation of cell proliferation and differentiation patterns and hence possibly functions as a morphogenetic cue for local tissue pattern formation in vivo.

Somatic embryogenesis and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.) Marechal]: a recalcitrant grain legume

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with 0.75 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 6-benzylaminopurine (BA) and with various additives (50 mg l−1 ascorbic acid and 25 mg l−1 each of adenine sulphate, citric acid and l-arginine). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with 0.25 mg l−1 2,4-D and 0.5 mg l−1 of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing 0.2 mg l−1 BA and 2.0 mg l−1 gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.

A Human Folliculoid Microsphere Assay for Exploring Epithelial– Mesenchymal Interactions in the Human Hair Follicle

The search for more effective drugs for the management of common hair growth disorders remains a top priority, both for clinical dermatology and industry. In this pilot study, we report a pragmatic organotypic assay for basic and applied hair research. The patented technique produces microdroplets, which generate human folliculoid microspheres (HFMs), consisting of human dermal papilla fibroblasts and outer root sheath keratinocytes within an extracellular matrix that simulates elements of the hair follicle mesenchyme. Studying a number of different markers (for example, proliferation, apoptosis, cytokeratin-6, versican), we show that these HFMs, cultured under well-defined conditions, retain several essential epithelial-mesenchymal interactions characteristic for human scalp hair follicle. Selected, recognized hair growth-modulatory agents modulate these parameters in a manner that suggests that HFMs allow the standardized preclinical assessment of test agents on relevant human hair growth markers under substantially simplified in vitro conditions that approximate the in vivo situation. Furthermore, we show by immunohistochemistry, reverse transcriptase-PCR, and DNA microarray techniques that HFMs also offer a useful discovery tool for the identification of target genes and their products for candidate hair drugs. HFM thus represent an instructive modern experimental and screening tool for basic and applied hair research in the human system.

Investigating the Role of Proteases in Aggressiveness of Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer with a three‐year survival rate of 42% versus 85% for other breast cancers. IBC is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer that invades into lymphatics where it forms large cellular aggregates. Here we performed live‐cell imaging using a SUM149 model of human inflammatory breast cancer. We hypothesize that the invasion of IBC is dependent on proteolytic activity at the tumor cell surface and that proteases contribute to the formation of tumor cell aggregates within the lymphatics. We assessed proteolytic activity using our functional assay for proteolysis. Quantification on a per cell basis throughout the volume of IBC structures was performed. Briefly, cells were grown in 3D reconstituted basement membrane (rBM) overlay cultures in which the lower layer of rBM is mixed with DQ‐collagen IV. Fluorescent cleavage products arising from the degradation of the collagen IV substrate were imaged on a Leica TCS SP5 laser scanning confocal microscope. Fluorescence intensities from 3D reconstructions of optical sections were quantified using Metamorph and Volocity software. Incubating the 3D rBM overlay cultures of SUM149 with inhibitors that target different classes of protease indicated that matrix metalloproteases and perhaps the cysteine protease cathepsin B play roles in the invasiveness of IBC.

Bioremediation of n-alkanes and the formation of biofloccules by Rhodococcus erythropolis NTU-1 under various saline conditions and sea water

From our previous report, the speedy removal of some n-alkanes or branched-alkanes by Rhodococcus erythropolis NTU-1 was already demonstrated in aqueous MS medium. Therefore, this strain was further tested under saline conditions, as well as in pure sea water. Hexadecane of 2000 ppmv was used as a model carbon source in this report. Under saline conditions, the biodegradation was again confirmed with the cell aggregates formation during the cultivation. The formation of biofloccules, trapping significant amount of alkane, provides an easy physical removal of alkane, in addition to biodegradation. At an initial NaCl concentration 1.2% and 2.4%, 80–90% removal of alkane was achieved within 56 h and 65% removal at 3.6% NaCl concentration within 68 h. Addition of 0.24 g/L nutrient broth (NB) in the medium would further promote the biodegradation rate and cell aggregation phenomenon during the process and more than 80–95% removal of alkane was achieved within 56–68 h. In the case of sea water, alkane removal was about 50% after 140 h and 65% with NB addition. The results suggest that strain NTU-1 has considerable ability for the bioremediation processes of n-hexadecane in a marine environment.

Enhanced insulin production from murine islet beta cells incubated on poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate)

Islet transplantation represents an important alternative for the treatment of diabetes. However, the selection of suitable materials is critical for the success of such an implantation application. In this study, cellular migration, aggregation, and insulin production of a murine islet beta-cell line, NIT-1 cells on microbially produced polyesters poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) or polylactic acid (PLA) films were investigated. Spherical islet-like structures were only detected on PHBHHx films after 48 h cultivation. To understand the mechanism underlying the formation of cell aggregates, NIT-1-GFP, a stable transfectant of the green fluorescent protein was used in a time-lapse imaging study. Cell aggregation began on PHBHHx at 2 h, and became obvious at 4 h. Furthermore, cells on PHBHHx displayed higher metabolic activities measured by MTT assay than that on tissue culture plate. More importantly, insulin gene expression as well as extracellular secretion was upregulated after growth on PHBHHx for 72 h. Thus, PHBHHx can be a strong candidate for islet transplantation.

Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease

Event Abstract Back to Event Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease Rita Perfeito1*, Catarina R. Oliveira1, 2 and Cristina A. Rego1, 2 1 University of Coimbra, Center for Neuroscience and Cell Biology, Portugal 2 University of Coimbra, Institute of Biochemistry, Faculty of Medicine, Portugal Accumulation of alpha-synuclein (alpha-syn) as intracellular filamentous aggregates is a pathological feature of Parkinson´s disease (PD). These aggregates localize in neuronal cytoplasm as Lewy bodies (LBs), mainly composed of alpha-syn, ubiquitin and synphilin-1, and are associated with degeneration of dopaminergic neurons in both sporadic and familial forms of PD. Furthermore, changes in alpha-syn post-translational modifications, as well as mitochondrial dysfunction (through inhibition of complex I) and oxidative stress constitute key pathogenic events of this disorder. Indeed, brains of PD patients contain more iron in the substantia nigra than normal controls, and LBs from surviving neurons display oxidative damage. Since mitochondrial dysfunction and oxidative stress have been linked to PD, and alpha-syn includes several sites susceptible to undergo post-translational modifications, the aim of this work was to analyse the role of FeSO4 and rotenone (complex I inhibitor) on alpha-syn phosphorylation and aggregation, and the formation of reactive oxygen species. We used a human neuroblastoma cell line (SH-SY5Y) transiently transfected with wild type (WT) and mutant A53T alpha-syn, linked to eGFP, as a PD model. Results showed that prolonged exposure (4 days) to FeSO4 increased phosphorylation at Ser129, particularly of mutant A53T alpha-syn. Similar results were observed in the presence of rotenone, whereas phosphorylation at Tyr125 was not affected. Analysis of cell death with propidium iodide indicated that the chosen concentrations of iron caused approximately 1% necrotic cell death, whereas rotenone caused very little cell death. Aggregate formation was also examined by confocal microscopy. Prolonged iron and rotenone exposure did not cause the formation of Ub-labelled alpha-syn aggregates in SH-SY5Y cells, suggesting that alpha-syn phosphorylation does not correlate with protein aggregation under these experimental conditions. However, in cells expressing WT or A53T alpha-synGFP, and further exposed to iron or rotenone, we observed a decrease in MitoTracker Red fluorescence, suggesting mitochondrial depolarization. Preliminary data using the fluorescent probe dichlorodihydrofluorescein-diacetate (DCFH2-DA) showed that short incubations (up to 2h) with FeSO4 increased the formation of endogenous hydroperoxides in SH-SY5Y cells expressing WT alpha-synGFP and this effect was more pronounced in cells expressing A53T alpha-synGFP. These data suggest that early ROS formation may be important for defining subsequently alpha-syn phosphorylation, which precede alphasyn aggregation and prominent cell death in PD.Supported by: SFRH/BD/25515/2005 FCT Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neurodegenerative Disorders Citation: Perfeito R, Oliveira CR and Rego CA (2009). Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.061 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 07 Aug 2009; Published Online: 07 Aug 2009. * Correspondence: Rita Perfeito, University of Coimbra, Center for Neuroscience and Cell Biology, Alicante, Portugal, rita_perfeito@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rita Perfeito Catarina R Oliveira Cristina A Rego Google Rita Perfeito Catarina R Oliveira Cristina A Rego Google Scholar Rita Perfeito Catarina R Oliveira Cristina A Rego PubMed Rita Perfeito Catarina R Oliveira Cristina A Rego Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effects of Poly(L-lysine), Poly(acrylic acid) and Poly(ethylene glycol) on the Adhesion, Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

Microenvironments, composed of many kinds of cytokines and growth factors plus extracellular matrices with diverse electrostatic properties, play key roles in controlling cell functions in vivo. In this study, three kinds of water-soluble polymers, positively charged poly(L-lysine) (PLL), negatively charged poly(acrylic acid) (PAAc) and neutral poly(ethylene glycol) (PEG), were compared based on their effects on the adhesion, spread, proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). The MSCs were seeded and cultured in the presence of polymers of different concentrations applied by methods using coating, mixing or covering. The effects of the water-soluble polymers depended on their electrostatic properties and method of application. The methods were in the order of coating, mixing and covering in terms of high to low influence. A low concentration of PLL promoted MSC adhesion, spread, proliferation and chondrogenic differentiation, while a high concentration of PLL was toxic. The PEG-coated surface facilitated cell aggregation and spheroid formation by inhibiting cell adhesion. A high concentration of mixed PEG (10 μg/ml) promoted cell proliferation in serum-free medium. PAAc showed no obvious effects on MSC adhesion, spread, proliferation, or chondrogenic differentiation.

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.

A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice

Vimentin is the main intermediate filament (IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin-/- mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a (R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans.

Isolation and characterization of biosurfactant-producing Alcanivorax strains: hydrocarbon accession strategies and alkane hydroxylase gene analysis

Biosurfactant-producing bacteria belonging to the genera Alcanivorax, Cobetia and Halomonas were isolated from marine sediments with a history of hydrocarbon exposure (Aristizábal and Gravina Peninsulas, Argentina). Two Alcanivorax isolates were found to form naturally occurring consortia with strains closely related to Pseudomonas putida and Microbacterium esteraromaticum. Alkane hydroxylase gene analysis in these two Alcanivorax strains resulted in the identification of two novel alkB genes, showing 86% and 60% deduced amino acid sequence identity with those of Alcanivorax sp. A-11-3 and Alcanivorax dieselolei P40, respectively. In addition, a gene homologous to alkB2 from Alcanivorax borkumensis was present in one of the strains. The consortium formed by this strain, Alcanivorax sp. PA2 (98.9% 16S rRNA gene sequence identity with A. borkumensis SK2 T) and P. putida PA1 was characterized in detail. These strains form cell aggregates when growing as mixed culture, though only PA2 was responsible for biosurfactant activity. During exponential growth phase of PA2, cells showed high hydrophobicity and adherence to hydrocarbon droplets. Biosurfactant production was only detectable at late growth and stationary phases, suggesting that it is not involved in initiating oil degradation and that direct interfacial adhesion is the main hydrocarbon accession mode of PA2. This strain could be useful for biotechnological applications due to its biosurfactant production, catabolic and aggregation properties.