Single-molecule anisotropy imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.     

Butyrate-synchronized cloned T cells retain their dependence on interleukin-2 for growth induction : A model system for growth regulation

Treatment of ST2/K9 cells, a cloned mouse T-cell line, with 1 mM sodium butyrate for 24 h leads to complete growth arrest in G1. This block is completely reversible and restimulation of cellular growth is entirely dependent on the presence of interleukin-2 (Il-2) in the culture medium. Additional as yet undefined serum factors are necessary for maintenance of further proliferation. After release from butyrate-induced growth arrest, Il-2 is required only during the induction phase of DNA replication. At the onset of thymidine incorporation, the growth factor can be removed, after which DNA replication occurs and the cells are able to complete only one cycle of duplication. The data presented here show that synchronization with sodium butyrate promotes cellular accumulation in the lymphokine-sensitive phase of the cell cycle. On the basis of the parameters established for restimulation of these cells, the detailed characterization of the molecular events involved in Il-2-mediated growth is possible.     

Using an evolutionary algorithm and parallel computing for haplotyping in a general complex pedigree with multiple marker loci

Background  

Haplotype reconstruction is important in linkage mapping and association mapping of quantitative trait loci (QTL). One widely used statistical approach for haplotype reconstruction is simulated annealing (SA), implemented in SimWalk2. However, the algorithm needs a very large number of sequential iterations, and it does not clearly show if convergence of the likelihood is obtained.     

Failure to Use Cubicles and Concentrate Dispenser by Heifers after Transfer from Rearing Accommodation to Milking Herd

Thirty-three dairy farms in the Norwegian counties of ?stfold and Akershus in which cubicle sheds had been in use for at least one year and with a herd size of less than 60 cows, were contacted and asked to participate in a study. The study focused on heifers' use of cubicles and concentrate dispenser just after being transferred from rearing accommodation to the milking herd. For each heifer, the farmer recorded cubicle use once nightly between 9 and 11 pm. The daily amount of concentrate released in the dispenser and the allotted daily ration were also recorded. The recording period was 15 consecutive days for cubicle use and 7 days for concentrate dispenser use. Cubicle refusal behaviour, i.e. lying outside the cubicles, was analysed by logistic regression using rearing accommodation of heifers, herd size, heifer age, and housing layout as independent variables, and herd as a clustering variable. On Day 2 after transfer, 34% of the heifers were showing cubicle refusal behaviour (N = 340). By Day 15 this percentage had dropped to 23. Cubicle refusal was lower throughout the whole period among heifers which used the cubicles on the 3 first days after transfer compared to those which did not. This tendency could also be detected several months later. The analysis showed cubicle refusal to be significantly associated with rearing accommodation (OR = 6.1, c.i._95%OR = 1.5–24.3, P = 0.01) and cubicle layout in the shed (OR = 0.2, c.i._95%OR = 0.0–0.7, P = 0.01). None of the tested variables were found to be significant for failure to use the concentrate dispenser, a behaviour which was less frequent than cubicle refusal. However, 8 percent of the heifers did not visit the dispenser at all throughout the 7 days of observation.     

Changes in Triphasic Mechanical Properties of Proteoglycan-Depleted Articular Cartilage Extracted from Osmotic Swelling Behavior Monitored Using High-Frequency Ultrasound

This study aims to obtain osmosis-induced swelling strains of normal and proteoglycan (PG) depleted articular cartilage using an ultrasound system and to investigate the changes in its mechanical properties due to the PG depletion using a layered triphasic model. The swelling strains of 20 cylindrical cartilage-bone samples collected from different bovine patellae were induced by decreasing the concentration of bath saline and monitored by the ultrasound system. The samples were subsequently digested by a trypsin solution for approximately 20 min to deplete proteoglycans, and the swelling behaviors of the digested samples were measured again. The bi-layered triphasic model proposed in our previous study (Wang et al., J Biomech Eng-Trans ASME 2007; 129: 413-422) was used to predict the layered aggregate modulus Hafrom the data of depth-dependent swelling strain, fixed charge density and water content. It was found that the region near the bone, for the normal specimens, had a significantly higher aggregate modulus (Ha₁= 20.6±18.2 MPa) in comparison with the middle zone and the surface layer (Ha₂= 7.8±14.5 MPa and Ha₃= 3.6±3.2 MPa, respectively) (p < 0.001). The normalized thickness of the deep layer h₁ was 0.68±0.20. After the trypsin digestion, the parametric values decreased to Ha₁ = 13.6±9.6 MPa, Ha₂ = 6.7±11.5 MPa, Ha₃ = 2.7±3.2 MPa, and h₁ = 0.57±0.28. Other models were also used to analyze data and the results were compared. This study showed that high-frequency ultrasound measurement combined with the triphasic modeling was capable of nondestructively quantifying the alterations in the layered mechanical properties of the proteoglycan-depleted articular cartilage.     

Mathematical design of prokaryotic clone-based microarrays

Background  

Clone-based microarrays, on which each spot represents a random genomic fragment, are a good alternative to open reading frame-based microarrays, especially for microorganisms for which the complete genome sequence is not available. Since the generation of a genomic DNA library is a random process, it is beforehand uncertain which genes are represented. Nevertheless, the genome coverage of such an array, which depends on different variables like the insert size and the number of clones in the library, can be predicted by mathematical approaches. When applying the classical formulas that determine the probability that a certain sequence is represented in a DNA library at the nucleotide level, massive amounts of clones would be necessary to obtain a proper coverage of the genome.     

Anti-α-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-α-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sjögren's syndrome (SjS). We looked (in Nijmegen) for anti-α-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS [6], rheumatoid arthritis [RA] [12], systemic lupus erythematosus [SLE] [6], and scleroderma [6]) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-α-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The ELISAs for α-fodrin showed six (Nijmegen) and four (Hanover) anti-α-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-α-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-α-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-α-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-α-fodrin autoantibodies does not add much to the diagnosis of Sjögren's syndrome.     

Synthesis of amphiphilic photochromic benzo-15(18)-crown-5(6)-ethers and their properties in monolayers

New amphiphilic photochromic benzo-15(18)-crown-5(6) ethers (APC) differing in the position of the octadecyl substituent and the size of the crown cavity were synthesized. The compounds form stable monolayers in the air/water and air/alkaline metal salt solution interfaces. The results of the pressure isotherm measurements, atomic force microscopy (AFM), and electronic spectroscopy show that the structure of the monolayers formed depends on the structure of the parent APC and the nature of the cation in salt solutions. The area per molecule of APC in the monolayer (specific area) is the smallest on the water surface and increases by 20-40% on the aqueous subphase surface with an increasing concentration of salts therein to indicate the formation of APC complexes with the metal cations. When the hydrophobic aliphatic substituent is displaced from position 3 to position 5 of the benzothiazole ring, the specific area on the surface of water and subphases decreases twofold, which indicates the compactization of the monolayer on this modification. A reversible E-Z-photoisomerization of APC was found in the monolayers formed in the salt solution/air interface. The features of the reaction are defined by the specific organization of the amphiphilic molecules in the monolayer and by the nature of the cation.     

No interaction of memantine with acetylcholinesterase inhibitors approved for clinical use

The loss of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may underlie aspects of the dementia. Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie the degeneration of cholinergic cells. New drug therapies have been designed to either enhance cholinergic function by inhibition acetylcholinesterase (AChE), e.g. galanthamine, tetrahydroaminoacridine or donepezil, or by attenuation of NMDA receptor function, e.g. memantine. A combination of these two therapeutic approaches may be more beneficial at slowing the progression of the AD. The current study investigated whether memantine would attenuate the inhibition of AChE produced by these three drugs. The results indicate that these AChE inhibitors do not lose their therapeutic efficacy in combination with memantine. Our in vitro data suggest that the clinical combination of memantine with a reversible AChE inhibitor should be a valuable pharmacotherapeutic approach to dementia.     

Bestimmung von Fumonisinen in komplexen Lebensmittelmatrices mittels Accelerated Solvent Extraktion (ASE)

Accelerated solvent extraction (ASE) is an alternative sample extraction procedure for fumonisins in corn and corn products. ASE gave results comparable to that of a draft CEN method, but required less extraction time. Furthermore, ASE gave significantly higher quantitative values than another method reported for extraction of fumonisins (Trucksess et al., 1995).