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Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.

Crosses, Genetic↗

Transient osmotic stress facilitates mutant huntingtin aggregation.

Human neuroblastoma SH-SY5Y cell lines stably expressing mutant truncated huntingtin with 82 (mutant) glutamine repeats (N63-82Q) were briefly exposed to hyperosmotic conditions which decrease cell volume and therefore transiently increased the concentration of N63-82Q, as well as activating specific stress-induced pathways. Transient hyperosmotic treatment significantly increased the number of cells with aggregates. When the N63-82Q cells were subsequently returned to iso-osmotic medium after the treatment, the number of cells with aggregates remained constant up to 12 h. However, between 12 and 24 h another significant increase in aggregate frequency was observed, with approximately 55% of the cells containing aggregates after 24 h. This may be due in part to the formation of microaggregates during hyperosmotic conditions that act as seeds for the aggregate formation. Further, treatment of cells with geldanamycin, which activates a heat shock response, significantly attenuated the hyperosmotic-induced increase in aggregate formation.

Benzoquinones↗

Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans.

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.

Agriculture↗

Acid- and pressure-induced (un)folding of yeast glutathione reductase: competition between protein oligomerization and aggregation.

Glutathione reductase (GR) is a homodimeric flavoenzyme involved in cellular defense against oxidative stress. In the present study, we have used a combination of acidic pH and hydrostatic pressure to investigate the (un)folding transition of yeast GR. Our results indicate that at pH 2 a distinct partially folded state is stabilized, as judged by intrinsic fluorescence, bis ANS binding and circular dichroism (CD) analysis. Further characterization of this partially folded state by size exclusion chromatography revealed that it corresponds to expanded GR monomers. CD analysis at pH 2 showed a significant loss of secondary structure. The partially folded GR monomers stabilized at pH 2 were fully and reversibly unfolded using hydrostatic pressure (up to 3.5kbar) as a thermodynamic perturbant. By contrast, return to physiological pH after exposure to acidic pH led to a competing reaction between refolding dimerization and aggregation of GR. These results support the notion that a partially folded intermediate state is not only critical for folding of GR but also appears to be a seed for protein aggregation.

Acids↗

Soft agarose culture human tumour colony forming assay for drug sensitivity testing: [3H]-thymidine incorporation vs colony counting.

In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.

Antineoplastic Agents↗

Computer simulation of electrical conductivity of colloidal dispersions during aggregation.

The computation approach to the simulation of electrical conductivity of colloidal dispersions during aggregation is considered. We use the two-dimensional diffusion-limited aggregation model with multiple-seed growth. The particles execute a random walk, but lose their mobility after contact with the growing clusters or seeds. The two parameters that control the aggregation are the initial concentration of free particles in the system p and the concentration of seeds psi. The case of psi=1, when all the particles are the immobile seeds, corresponds with the usual random percolation problem. The other limiting case of psi=0, when all the particles walk randomly, corresponds to the dynamical percolation problem. The calculation of electrical conductivity and cluster analysis were done with the help of the algorithms of Frank-Lobb and Hoshen-Kopelman. It is shown that the percolation concentration phi c decreases from 0.5927 at psi=1 to 0 at psi --> 0. Scaling analysis was applied to study exponents of correlation length v and of conductivity t. For all psi>0 this model shows universal behavior of classical 2d random percolation with v approximately t approximately 4/3. The electrical conductivity sigma of the system increases during aggregation reaching up to a maximum at the final stage. The concentration dependence of conductivity sigma(phi) obeys the general effective medium equation with apparent exponent ta(psi) that exceeds t. The kinetics of electrical conductivity changes during the aggregation is discussed. In the range of concentration Pc(phi)<p<0.9527 the time of percolation cluster formation pi c decreases with increasing phi.

Journal Article↗

Cerebrovascular P-glycoprotein expression is decreased in Creutzfeldt-Jakob disease.

The abnormal conformation and assembly of proteins in the central nervous system is increasingly thought to be a critical pathogenic mechanism in neurodegenerative disorders such as Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease (AD). CJD is marked primarily by the buildup of misfolded prion protein (PrP(Sc)) in brain, whereas the accrual of beta-amyloid protein (Abeta) and tau protein are characteristic for AD. Prior studies have shown that the ATP-binding cassette transporter P-glycoprotein (P-gp) is a cellular efflux pump for Abeta, and that age-associated deficits in P-gp may be involved in the pathogenesis of Alzheimer's disease. In the present study, we investigated the relationship between P-gp and idiopathic CJD, and found that CJD, like AD, is associated with a decrease in the expression of cerebrovascular P-gp. In some instances, Abeta and PrP deposits coexist in cases of CJD, suggesting the possibility of pathogenic interactions. Since there is, to date, no evidence that PrP itself is a substrate for P-gp, we hypothesize that the age-related deficits in P-gp could promote the accumulation of PrP(Sc) either by promoting the buildup of Abeta (which could act as a seed for the aggregation of PrP(Sc)), or by overloading the ubiquitin-proteasomal catabolic system, and thereby facilitating the accumulation of PrP. Alternatively, the loss of P-gp could be a non-specific response to neurodegenerative changes in the central nervous system. In either case, dysfunction of this critical toxin-elimination pathway in CJD and AD suggests that selectively increasing cerebrovascular P-gp function could open new therapeutic pathways for the prevention and/or treatment of a number of proteopathic disorders of the central nervous system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins.

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.

Amino Acid Sequence↗

Different associational and conformational behaviors between the second and third repeat fragments in the tau microtubule-binding domain.

The third repeat fragment (R3) in the four-repeat microtubule-binding domain of the water-soluble tau protein has been considered to play an essential role in the protein's filamentous assembly. To clarify the associational and conformational features that differentiate R3 from the second repeat, R2, the heparin-induced assembly profiles of these peptide fragments were monitored by the thioflavin fluorescence method and electron microscopy. The trifluoroethanol-induced reversible conformational change from a random structure to an alpha-helical structure, in an aqueous solution, was monitored by CD measurement, and the structure of R2 in trifluoroethanol solution was analyzed by a combination of two-dimensional 1H-NMR measurements and molecular modeling calculations to facilitate comparison with the structure of R3. The speed of R3 assembly was remarkably faster than that of R2, in spite of their similar amino acid sequences. The averaged NMR conformers of R2 exhibited the whole-spanning alpha-helical structure. Similar features observed in R2 and R3 conformers in trifluoroethanol were that the Leu10-Leu20/Lys20 sequence takes a helical structure with the amphipathic-like distribution of the respective side-chains, whereas the C-terminal moieties are both flexible. In contrast, a notable difference was observed at the N-terminal Val1-Lys6 sequence, namely, a helical conformation for R2 and an extended conformation for R3. These conformational behaviors would be associated with the different self-aggregation speeds and seeding reactions between R2 and R3.

Amino Acid Sequence↗

Biological activity and colonization pattern of the bioluminescence-labeled plant growth-promoting bacterium Kluyvera ascorbata SUD165/26.

Kluyvera ascorbata SUD165/26 is a spontaneous siderophore-overproducing mutant of K. ascorbata SUD165, which was previously isolated from nickel-contaminated soil and shown to significantly enhance plant growth in soil contaminated with high levels of heavy metals. To develop a better understanding of the functioning of K. ascorbata SUD165/26 in the environment, and to trace its distribution in the rhizosphere, isolates of this bacterium were labeled with either green fluorescent protein or luciferase. When the plant growth-promoting activities of the labeled strains were assayed and compared with the activities of the unlabeled strain, none of the monitored parameters had changed to any significant extent. When the spatial colonization patterns of the labeled bacteria on canola roots were determined after seed application, it was observed that the bacterium was tightly attached to the surface of both roots and seeds, and formed aggregates. The majority of the bacterial population inhabited the upper two thirds of the roots, with no bacteria detected around the root tips.

Journal Article↗

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering.

BACKGROUND: In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering. METHODS: DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope (SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated. RESULTS: DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs. CONCLUSION: dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.

Bone Marrow Cells↗

[A study of crystallization on urolithiasis in vitro].

The formation, growth and aggregation of calcium oxalate crystals were evaluated with a Coulter counter. CG-120 and sodium pentosan polysulfate (SPP) has inhibitory activity on calcium oxalate crystal formation, growth and aggregation in the seeded crystal system and whole urine system. In continuous crystallizer system, they inhibit the nucleation rate. A new system of observing the crystal formation and growth of fragmented stones after extracorporeal shock wave lithotripsy has been developed. CG-120 has an inhibitory effect on their growth. The deposition of calcium oxalate crystals in the rat kidney was induced, and the, the volume and number of crystals were estimated using a Coulter counter. SPP has an inhibitory effect on calcium oxalate crystal growth in vivo.

Animals↗

Chondrocyte aggregation on micrometric surface topography: a time-lapse study.

Cellular aggregation or mesenchymal "condensation" is a prerequisite in the process of chondrogenesis. It has been observed that during in vitro engineering of cartilage, chondrocytes form aggregates in the initial cell seeding process of polymer scaffolds. However, the exact mechanism behind this aggregation has yet to be elucidated, although cell collision has been implicated. As all polymers have a surface topography, we hypothesized that topography may play a role in chondrocyte aggregation. 1(st) and 2(nd) passage chondrocytes were seeded on micrometric topography ranging from 0.75 to 8 microm in depth and 5 to 12.5 microm in width. Both 1(st) and 2(nd) passage cells formed aggregates as cells collided, and larger aggregates formed as aggregates collided with each other on the grooves. Furthermore, aggregates migrated parallel to the direction of the groove long axis. F-actin organization was altered only in the cellular layer in direct contact with the surface; stress fibers oriented in the direction of the groove long axis. Chondrocytes maintained type II collagen expression on all groove depths. This study shows that micrometric grooves could be an effective means for studying chondrocyte aggregation, and could additionally be utilized in the regeneration of cartilage.

Animals↗

Marker seed migration in prostate localization.

PURPOSE: Marker seed location was analyzed to test the hypothesis that there is no intraseed migration within the prostate, a premise fundamental to the technique of marker seed localization of this organ. Despite increasing interest in the use of implanted seeds as fiducial markers for gland location, there are few data available with which to evaluate the validity of this technique, particularly over the entire course of external beam radiation therapy. METHODS AND MATERIALS: Between May 2001 and December 2001, after obtaining fully informed written consent, 9 patients with early stage prostate cancer were enrolled on an institutionally reviewed protocol. Patients had four to five marker seeds implanted into the prostate under transrectal ultrasound guidance before definitive radiotherapy. The porous gold seeds were each 1.2 x 2.0 mm in dimension. Seed locations from orthogonal radiographs based on the initial simulation and weekly orthogonal films were digitized using a CMS Focus planning system, thereby facilitating the determination of intraseed spacing. The digitization of the isocenter from each orthogonal pair of radiographs was used to determine digitizing error for seed localization. Pubic symphysis, bilateral femoral heads, and isocenter were also digitized and will be analyzed at a later date. RESULTS: Overall, the average migration of all the seeds in the patients was 1.2 +/- 0.2 (SD) mm. The greatest average movement of any seed in any patient was 1.9 mm over the entire 7-week course of radiotherapy. The smallest average movement was 0.6 mm. The greatest change in intraseed spacing in any of the patients during the full course of therapy was 6.6 mm. One seed in 1 patient was lost at the start of the third week of therapy and censored from analysis. Digitizing error in seed localization was calculated to be 0.20 +/- 0.03 (SD) mm. CONCLUSIONS: As an aggregate, there is negligible seed migration within the prostate over the entire course of definitive radiotherapy. However, there are small, detectable movements in individual seed locations, perhaps resulting from topographic changes in the gland secondary to seed placement, anatomic changes in bladder and rectum, or treatment itself. With respect to seed migration, prostate marker seeds represent an accurate and reliable surrogate of gland location during a full course of radiotherapy.

Brachytherapy↗

An ApoE-Abeta inhibition complex in Abeta fibril extension.

BACKGROUND: Literature reports differ dramatically in showing that apolipoprotein E either facilitates or inhibits Abeta aggregate formation in vitro. Resolution of the nature of the ApoE-Abeta interaction is critical for progress towards understanding its possible role in the modulation of Alzheimer's disease. RESULTS: Here, we show that purified ApoE-Abeta co-aggregate is a poor seed of fibril formation. We also demonstrate ApoE inhibition of Abeta fibril growth in four independent aggregation assays, arguing that the poor fibril formation observed under these conditions is real and not an analytical artifact. We also directly show ApoE binding to immobilized Abeta fibrils by surface plasmon resonance. CONCLUSIONS: The results suggest a unifying model in which ApoE binds to Abeta fibril seeds and nascent nuclei to generate stable complexes that inhibit the rapid extension of mono-component Abeta fibrils but at the same time can foster continued slow growth of mixed ApoE-Abeta aggregates. In vivo co-aggregate formation may be important in many examples of pathological protein misassembly.

Amyloid beta-Peptides↗

Surface-induced aggregation of beta amyloid peptide by co-substituted alkanethiol monolayers supported on gold.

The primary pathological characteristic of Alzheimer's disease is the presence in the brain of self-assembled beta amyloid (Abeta) protein fibrils, consisting of 35-43 amino acid residues. The toxicity of the aggregated protein structures has previously been proposed to be related to the interaction of Abeta fibrils with neuronal membranes (phospholipid bilayers). Here, surfaces consisting of self-assembled alkanethiol monolayers with different end groups--supported on Au--are used to test the effect of surface chemistry on the structure and morphology of aggregates formed from an active fragment (Abeta10-35) of the Abeta peptide. The influence of monolayer nature (end group) on the aggregation of Abeta10-35 was examined using reflection-absorption infrared spectroscopy (RAIRS) and scanning force microscopy (SFM). Evaluation of the SFM and RAIRS data reveals the presence of Abeta10-35 protein on the various monolayer surfaces, with the surface protein possessing predominantly beta-sheet and random-coil conformations. Time-dependent studies of the extent of Abeta10-35 aggregation and deposition on the various surfaces and the effect of the monolayers on seeding of Abeta10-35 aggregates in solution are also discussed.

Alkanes↗

Hematological studies on black cumin oil from the seeds of Nigella sativa L.

The methanol soluble portion of black cumin oil, which is prepared by compression of seeds of Nigella sativa L., showed inhibitory effects on arachidonic acid (AA)-induced platelet aggregation and blood coagulation. By bioactive assay of AA-induced platelet aggregation, the methanol soluble part was purified to isolate a new compound 2-(2-methoxypropyl)-5-methyl-1,4-benzenediol (1) and two known compounds, thymol (2), carvacrol (3), having very strong inhibitory activity. Further, we then examined the isolated compounds (1-3) and eight related compounds by the screening test for AA-induced platelet aggregation. Compounds possessing aromatic hydroxyl and acetoxyl group had more potent activity than aspirin, which is well known as a remedy for thrombosis.

Animals↗

Effects of initial cell seeding density for the tissue engineering of the temporomandibular joint disc.

Tissue engineering may provide a better treatment modality for postoperative discectomy patients. The TMJ disc is an ideal candidate for tissue engineering approaches because of its lack of an intrinsic regenerative ability. Unfortunately, basic knowledge related to TMJ disc tissue engineering is still at an infancy level and not on par to that related to articular cartilage tissue engineering. The objective of this study was to examine the effects of initial cell density of TMJ disc cells seeded in nonwoven poly-glycolic acid (PGA) scaffolds on the biochemical and biomechanical properties of constructs examined at 0, 3, and 6 weeks after seeding. Low, medium, and high seeding densities were chosen to be 15, 30, and 120 million cells per ml of scaffold, which were seeded using a spinner flask. Significant differences were found temporally and as a function of seeding density in morphology, total collagen, GAG content, and permeability of the constructs, but not in aggregate modulus. The high seeding density group outperformed the low and medium groups in collagen and GAG content at all time points measured. The high-density group produced a total of 55.37 +/- 3.56 microg of collagen per construct, maintained 15.77 +/- 1.86 microg of GAG per construct, and only shrunk to 50% of the original scaffold size. Permeability of the constructs at 6 weeks was decreased by 70% compared to 0 weeks.

Animals↗