Membrane Filter Assay Research Articles

Agglutination of Human Polyomaviruses by Using a Tetravalent Glycocluster as a Cross-Linker.

Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand–virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, γ-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

An approach using a novel phage mix for detecting Pseudomonas aeruginosa in water

AbstractThe present study aims to integrate the benefits of plaque assay using a novel phage mix with phylogenetic and molecular analysis for detecting Pseudomonas aeruginosa in water. Three phages were isolated and the transmission electron microscope related their morphological resemblance to those of Siphoviridae and Podoviridae families, while molecular analysis showed different cp‐gene sizes. The Phage mix was highly specific (86.0%), and data misleading didn't exceed 14.0% compared to membrane filter assay (39.2%). Time elapsed for test completion required 24 h. Identified P. aeruginosa were verified using 16S‐rDNA. Nucleotide sequence data for both phages and bacteria were submitted to the NCBI GenBank database, USA and gained their accession numbers. Concluding remarks highlight the potential of plaque assay as specific, simple and rapid method. The study recommended future efforts to isolate and characterize new phages for detecting other bacterial pathogens of public health concern to control water pollution and maintain adequate hygiene.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.

Atividade antimicrobiana in vitro de produtos antissépticos por meio de técnica time kill

Many chemicals (alcohol, iodophor, chlorhexidine, etc) are used in laboratories and industries. Unlike disinfectants, no specific norms and criteria have been standardized for evaluating the antiseptics activity. This study analyzed the antimicrobial activity of antiseptics using Time Kill Test (Hobson & Bolsen). This assay assessed the evolution of a population of aerobic microorganisms in a specific period of time when tested against antimicrobial agents. Two different recovery methodologies were evaluated: membrane filtration and pour plate technique The membrane filtration assay was less sensitive. Pour plate technique showed high sensitivity with high colonies counts. Of 25 samples of products analyzed, only the chlorhexidine digluconate-based antiseptics were unsatisfactory, showing no efficacy on all reference micro-organisms strains, and corresponded to 20 % of analyzed samples. However, they were efficacious against clinical strains. Therefore, these products should be used with caution and further studies are needed, as data on its efficacy have still been scarce. These findings might give support to the health surveillance and public health in establishing the future legislation, as these products have been available on the market, but without following any specific legislation.

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Probing the Catalytic Sites and Activation Mechanism of Photoreceptor Phosphodiesterase Using Radiolabeled Phosphodiesterase Inhibitors

Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory gamma-subunits (Pgamma) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pgamma inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [(3)H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [(3)H]vardenafil binds stoichiometrically to both the alpha- and beta-subunits of the activated PDE6 heterodimer. [(3)H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pgamma, consistent with Pgamma blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [(3)H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [(3)H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pgamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.

Detection and quantification of tau aggregation using a membrane filter assay

Aggregation of the microtubule-associated protein tau contributes to the formation of neurofibrillary lesions in Alzheimer’s disease and is a useful marker of disease progression. Although filter trap assays have been employed to assess the extent of tau aggregation in cells and tissues as well as in vitro, their performance relative to other assay modalities has not been reported. To clarify this issue, the ability of the filter trap approach to quantify aggregation of purified recombinant full-length tau protein in vitro was examined as a function of membrane chemistry in a 96-well format. Results showed that nitrocellulose yielded the greatest assay sensitivity relative to polyvinylidene fluoride or cellulose acetate at equal membrane porosity. However, all combinations of filter chemistries, porosities, and monoclonal detection antibodies yielded nonlinear correlations between signal intensity and analyte concentration. When corrected for nonlinearity, the filter trap assay determined a value for the critical monomer concentration for tau aggregation that was statistically identical to determinations made by electron microscopy assay. The data suggest conditions under which filter trap assays can be used to estimate tau aggregation kinetics.

Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

The utility of the AC/TC ratio for watershed management: a case study

A human-impacted watershed was monitored during the dry summer seasons in 2002 and 2003 to investigate the impact of providing access to sewer mains to local village residences. Faecal coliform concentrations were monitored at select sites along the 30-mile stretch of creek, together with faecal streptococci, enterococci and total coliforms. Analysis of the results found that levels of faecal coliforms were inadequate at identifying significant known influxes of human and animal sewage established by sanitary survey. However, the bacterial ratio of atypical colonies to total coliform colonies (AC/TC), obtained from the total coliform membrane filter assay on m-Endo media, correctly indexed human faecal impact of inadequately sewered villages located along the creek. In addition, the AC/TC ratio correctly classified the predominant source of faecal runoff in the creek headwaters as agricultural, and indicated when aged agricultural faecal material was introduced by tributaries. An approach for watershed management that uses the AC/TC ratio in addition to levels of bacteria is proposed.

Blood polymorphonuclear leukocyte migration as a predictive marker for infections in severe trauma: comparison with various inflammation parameters.

To assess in patients with multiple trauma the relevance of the following as predictive markers for infections: the inflammation parameters white blood count, body temperature, blood polymorphonuclear leukocyte (PMN) migration; blood levels of C-reactive protein, PMN elastase, procalcitonin, neopterin, interleukin 6, interleukin 8, malondialdehyde, total antioxidative status; the stress parameters cortisol and lactate. Prospective observational cohort study. Intensive Care Unit of a university surgical department. Twenty-six patients with multiple trauma of differing severity. Trauma severity was estimated by the ISS. PMN migration upon F-Met-Leu-Phe stimulation was determined in fresh whole blood in a ready-for-use, one-way membrane filter assay and evaluated by automated image analysis. The other parameters were measured with commercially available tests. During hospitalization, nine patients developed infections, and 17 patients were free of infection. PMN migration below a critical minimum preceded infections in eight of the infected, but occurred in only three of the non-infected patients (positive/negative predictive values 0.72/0.93; sensitivity/specificity 0.88/0.82; likelihood ratio 5.0). Fever (> or =38.0 degrees C) had predictive values of 0.83/0.80 and a high likelihood ratio of 9.4, but a low sensitivity/specificity of 0.55/0.94. The other parameters were without significance. Procalcitonin, elastase, C-reactive protein, neopterin and lactate correlated positively with the injury severity score. PMN migration proved to be a highly sensitive predictive marker for infections. The whole-blood PMN migration test may facilitate early aggressive antimicrobial therapy.

Impaired blood polymorphonuclear leukocyte migration and infection risk in severe trauma.

We investigated the association of impaired blood polymorphonuclear leukocyte (PMN) migration with the incidence of bacterial infections in patients with severe trauma. Twenty-six intensive-care patients with different injury severity scores were enrolled in a prospective study. PMN migration was measured daily using 300 microl fresh whole blood in a membrane filter assay. Migration was evaluated in an automated image analyzer that recorded numbers and distribution of the immigrant PMNs within a filter. The relevant parameter was the percentage of PMNs that migrated from the blood samples into the filters upon f-Met-Leu-Phe stimulation. Nine patients developed posttraumatic infections verified microbiologically. These patients showed a reduced PMN migratory capacity in comparison with the 17 patients without infections. A migrating portion of six per cent or less at least three days in succession preceded infections by one to 19 days and indicated infection in eight true positive versus three false positive cases, and 14 true negative versus one false negative case, i.e. specificity was 82.3% and sensitivity 88.8%, p=0.0008. Trauma severity had no influence on PMN migration. Trauma patients with impaired PMN migration are at risk for bacterial infections. Whole-blood migration tests can define the infection risk and thus may be useful predictive markers for infections.

Bacterial responses to ultraviolet irradiation

The UV dose-response behavior of laboratory cultures of waterborne bacteria were examined for UV doses ranging from ca. 0-100 mW.s/cm2 using a collimated-beam reactor. Specific physiological responses measured in these tests included viability (ability to reproduce) and respiration (oxygen uptake rate). The results of these exposures indicated that resistance to UV-imposed loss of viability in E. coli cultures can be partially attributed to agglomeration during the irradiation process. From these results, it is conjectured that a bacterial population may be comprised of two sub-populations: one with low resistance (discrete or paired cells) and a second with high resistance (bacterial aggregates). A small fraction of the high-resistance portion of the population appears to be essentially unaffected by UV irradiation, thereby causing a discontinuity in the measured dose-response behavior. Moreover, the dose-response behavior of the highly resistant fraction is variable and difficult to describe quantitatively. The basis of these statements and most information in the literature is microbial viability as quantified by the membrane filtration assay. In contrast to these findings, the results of analyses for bacterial activity (respiration) suggest that comparatively little change in the population can be found to result from UV irradiation. This suggests that UV radiation accomplishes inactivation of the bacteria, but does not "kill" the bacterial cells per se, thereby highlighting the importance of considering bacterial repair processes in the design of UV disinfection systems.

Altered migratory capacity of polymorphonuclear leucocytes as an effect of TNF-alpha blockade in patients with rheumatoid arthritis

Rheumatoid Arthritis (RA) is associated with progressive joint destruction, functional disability and decreased life expectancy. Althought the underlying cause of RA is unknown, TNF-alpha, a proinflammatory cytokine, contributes to the pathogenesis of synovitis and joint destruction. Anti-TNF-biological response modifiers, such as Etanercept a recombinant human TNF receptor fusion protein, suggest that TNF-alpha-inhibition is a viable approach to control disease activity in RA. Considering the pivotal role TNF alpha plays in the first line defense against bacterial and fungal infections by polymorphonuclear leucocytes (PMN) it seems important to focus on the PMN function in RA patients, treated with Etanercept. Since a 29% increase in infections of the upper respiratory tract under TNF-alpha blockade has been reported, we investigated inflammatory parameters related to PMN-activity in 6 RA-patients, before and after 3-months of Etanercept treatment without altering their methotrexate and/or glucocorti-coid-medication. The migration of the neutrophils was measured with a standardized whole blood membrane filter assay with and without stimulation by the chemoattractant fMLP. The percentage of migrating PMN (total migration index,TMI) and the relative penetration depth into the filters (distribution characteristics, DC) served to characterize the migratory behaviour of neutrophils. To estimate in vivo granulocyte activation, neutrophil elastase was measured in plasma from RA patients. In addition, PMN-blood count and C-reactive protein as a marker of disease activity, were analysed. TNF-alpha blockade significantly (p < 0,03) reduced the spontaneous and fMLP stimulated median TMI (15.2 vs. 10.9 and 16.5 vs. 11,4, respectively). Moreover the spontaneous and the fMLP-stimulated median DC was reduced under Etanercept-treatment (21.1 vs. 5.9, P < 0.03 and 20.5 vs. 6.6, P < 0.06 respectively). Interestingly, these values were still within the normal range of migratory activity. Furthermore, TNF blockade significantly reduced the median plasma elastase levels (252 vs. 108, p <0,04), as well as the median C reactive protein levels (21 vs. 7 P < 0,035) and the median number of polymorphonuclear leucocytes (7.73 vs. 4.58, P < 0.0001). Of note, plama elastase levels as a sign of systemic PMN-activation were still above the normal range. During the observed treatment period all patients showed an improvement in the inflammatory symptoms of RA (2 had a 70% ACR-response, 3 a 50% and 1 a 20% ACR response). No patients had signs of bacterial or fungal infections. In conclusions, TNF alpha blockade did not suppress PMN migratory activities to levels that are assosciated with higher incidences of infections.

Impaired whole-blood polymorphonuclear leukocyte migration as a possible predictive marker for infections in preterm premature rupture of membranes.

Objectives: Steroids, used in pretermpremature rupture of membranes (pPROM), to reduce the risk of morbidity and mortality of the preterm neonate, impair the maternal polymorphonuclear leukocyte (PMN)-based immune system. In spite of combination with antibiotics, prenatal and postnatal bacterial infections of mother and child are frequent. This pilot study focuses on the influence of steroids in pPROM on maternal PMN functional capacity and subsequent infections. Methods: After opting for expectant management, eight women with pPROM and no signs of infection were treated by steroids (betamethasone 5.7 mg, i.m. every 24 hours, for three days) and antibiotic therapy with either amoxicillin and clavulanic acid, piperacillin or ampicillin i.v. up to delivery. The conventional inflammation parameters of PMN blood count and C-reactive protein (CRP) were measured daily in parallel with PMN migratory capacity towards N-formyl-methionyl-leucyI-phenylalanine stimulation and under blank conditions, estimated by a whole blood membrane filter assay. Results: In all patients PMN migration decreased during the application of steroids. Three patients showed a decrease in PMN migration below critical values and in spite of antibiotic prophylaxis acute pyelonephritis developed 2–6 days later. PMN count and CRP were not predictive of maternal infection. Conclusion: Reduced PMN function, caused by steroid treatment in pPROM, is suggested to be a reason for serious bacterial infections in spite of antibiotic prophylaxis. PMN migration reflects individual PMN defensive capacity.

Evaluation of the bactericidal efficiency of a 2% alkaline glutaraldehyde solution on Mycobacterium xenopi

Mycobacterium xenopi (M. xenopi) has been implicated in hospital-acquired infections associated with colonization of hospital water systems. M. xenopi is considered to be as resistant as other atypical mycobacteria, which are known to be resistant to many disinfecting treatments. However, the efficacy of disinfectants on this organism has not yet been studied. Therefore we decided to evaluate its susceptibility to 2% alkaline glutaraldehyde solution, which is commonly used in hospitals. Tests were conducted using five strains of M. xenopi: three isolated from human samples, an environmental strain and a collection strain. We used a membrane filtration assay and counted surviving bacteria before and after several exposure times (5, 15, 30 and 60 min) with the disinfecting solution. The log<->10 reduction factor of organisms achieved within 60 min contact ranged from 2.5 to 7.5. This showed M. xenopi to be more resistant to disinfectants thanM. tuberculosis or M. smegmatis and suggested that environmental strains may be more resistant to alkaline glutaraldehyde than those isolated from human samples.

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.

24] Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates

Publisher Summary The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1, 3, and 7. Furthermore, it has been shown in vitro that the proteolytic cleavage of fusion proteins of glutathione S-transferase (GST) and the polyglutamine-containing huntingtin peptide coded for by the first exon of the HD gene s leads to the formation of insoluble high molecular weight protein aggregates with a fibrillar or ribbonlike morphology reminiscent of β -amyloid fibrils in Alzheimer's disease and scrapie prion rods. This chapter demonstrates that the cellulose acetate filter retardation assay can be a useful tool for the identification, structural characterization, and quantification of SDS-insoluble polyglutamine-containing protein aggregates formed in vitro and in vivo. In addition to the histochemical identification of amyloids, it useful in detecting insoluble protein aggregates in all types of human and animal amyloidoses, including the polyglutamine diseases, and also in screening compound libraries for potential aggregation inhibitors. Currently, attempts to develop a microtiter plate-based high-throughput filter retardation assay to identify chemical compounds that slow down the rate of formation of polyglutamine-containing fibrils in vitro are in progress. The amyloid-binding agents arising from this screen then will be tested in a HD cell culture model system and in the HD animal model for their therapeutic potential.

Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA

In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit. L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay. In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture. Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one. Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner. Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA. However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule. L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4×10 6/M. L17 bound the pre-rRNA molecule preferentially ( K a=17×10 6/M) but also bound the mature 25S rRNA species ( K a=10×10 6/M). L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well ( K a=11–12×10 6/M). L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with K a=3.1 vs. 2.5×10 6/M, respectively. We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.

Effect of steroid treatment on the migration behaviour of neutrophils in patients with early rheumatoid arthritis.

The influence of methylprednisolone on the migratory characteristics of neutrophil granulocytes was investigated in 10 patients with early rheumatoid arthritis (RA) and compared to 12 controls. The migration of neutrophils was measured with a whole-blood membrane filter assay with and without stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Total migration index (TMI), distribution characteristics (DC) and the product of TMI and DC (neutrophil migratory activity; NMA) served to characterize the migratory behaviour of neutrophils. The data demonstrated an increased polymorphonuclear leucocyte (PMN) migration in patients with early RA, indicating a bystander role of PMNs in inflammatory joint injury. Treatment with methylprednisolone reduced significantly the penetration depth (DC) of neutrophils, but did not influence the number of migrating cells (TMI). The unstimulated NMA was significantly reduced due to the marked DC reduction, whereas steroids did not influence the stimulated NMA of neutrophils. A significant reduction in PMN penetration depth was demonstrated only after a steroid therapy of at least 10 days, suggesting that a longer period of steroid therapy is necessary to provide effective inflammatory control.