Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion.

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

Early and stable upregulation of collagen type II,collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond–Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.     

GMFilter and SXTestPlate: software tools for improving the SNPlex? genotyping system

Background  

Genotyping of single-nucleotide polymorphisms (SNPs) is a fundamental technology in modern genetics. The SNPlex™ mid-throughput genotyping system (Applied Biosystems, Foster City, CA, USA) enables the multiplexed genotyping of up to 48 SNPs simultaneously in a single DNA sample. The high level of automation and the large amount of data produced in a high-throughput laboratory require advanced software tools for quality control and workflow management.     

An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L-like cysteine proteases.

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved alpha-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.     

Vorkommen und Nachweis von Fusarinsäure in Weizen

An analytical method for the detection of fusaric acid in wheat and wheat products has been developed as well as employed for the survey of randomly selected samples (n=33). While the application of GC-FID proved to be not sensitive enough for this purpose, the sensitivity and selectivity of GC-MS permitted the quantification of the mycotoxin in 78% of the wheat samples.The measured concentrations ranged from 7 to 570 μg/kg (average value 134 μg/kg). However, clear differences were recognizable regarding the type of sample: While field samples showed 332 μg/kg on an average, cleaned grains designated for human consumption contained averaged 138 μg/kg, and wholemeal products 68 μg/kg.Although the limited number of samples does not permit final conclusions, the toxicological relevance of fusaric acid alone may probably be classified as small. To what extent the well known cumulative effects with deoxynivalenol and fumonisin B1 impair this evaluation has to remain elusive.     

Image Quality of 3rd Generation Spiral Cranial Dual-Source CT in Combination with an Advanced Model Iterative Reconstruction Technique: A Prospective Intra-Individual Comparison Study to Standard Sequential Cranial CT Using Identical Radiation Dose

Objectives

To prospectively intra-individually compare image quality of a 3^rd generation Dual-Source-CT (DSCT) spiral cranial CT (cCT) to a sequential 4-slice Multi-Slice-CT (MSCT) while maintaining identical intra-individual radiation dose levels.

Methods

35 patients, who had a non-contrast enhanced sequential cCT examination on a 4-slice MDCT within the past 12 months, underwent a spiral cCT scan on a 3^rd generation DSCT. CTDI_vol identical to initial 4-slice MDCT was applied. Data was reconstructed using filtered backward projection (FBP) and 3^rd-generation iterative reconstruction (IR) algorithm at 5 different IR strength levels. Two neuroradiologists independently evaluated subjective image quality using a 4-point Likert-scale and objective image quality was assessed in white matter and nucleus caudatus with signal-to-noise ratios (SNR) being subsequently calculated.

Results

Subjective image quality of all spiral cCT datasets was rated significantly higher compared to the 4-slice MDCT sequential acquisitions (p<0.05). Mean SNR was significantly higher in all spiral compared to sequential cCT datasets with mean SNR improvement of 61.65% (p*_{Bonferroni0.05}<0.0024). Subjective image quality improved with increasing IR levels.

Conclusion

Combination of 3^rd-generation DSCT spiral cCT with an advanced model IR technique significantly improves subjective and objective image quality compared to a standard sequential cCT acquisition acquired at identical dose levels.