Characterization of recombinant human factor VIII

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.     

Variation in the Fine Structure of a Marine Achromobacter and a Marine Pseudomonad Grown Under Selected Nutritional and Temperature Regimes

Certain features of the fine structure of a marine achromobacter and a marine pseudomonad were dependent upon the conditions of growth. Cells of achromobacter grown at 10 C in a low peptone-seawater (SW) medium displayed the characteristic morphology of the achromobacter: a regularly undulant outer element of the cell wall and a planar inner element, tightly packed ribonucleoprotein (RNP) particles in the cytoplasm, deoxyribonucleic acid (DNA) disposed in a lobate manner, and dense inclusion bodies. Few mesosomes, however, were seen. Cells of achromobacter grown at 10 C in a high peptone-SW medium had larger and more highly organized mesosomes. At 22 C, in a low peptone-SW medium, no mesosomes were seen, but the inclusions were more frequently seen and were larger in the achromobacter cells. At 22 C, in a high peptone-SW medium, these cells revealed the greatest variation in cellular morphology. They contained both small and large mesosomes, or no mesosomes, and both small and large inclusions, or no inclusions. Pseudomonad cells at 10 C in a low peptone-SW medium revealed a typical gram-negative morphology: double-layered, irregularly undulant cell wall; more nearly planar cytoplasmic membrane; densely stained, lightly packed RNP particles; finely fibrillar, axially disposed DNA; simple mesosomes. At 10 C, in a high peptone-SW medium, pseudomonad cells revealed associated strands of material and intracytoplasmic ringlike structures. At 22 C, in a low peptone-SW medium, pseudomonad cells had a more undulant cell-wall and a more nearly planar cytoplasmic membrane. At 22 C, in a high peptone-SW medium, these cells revealed prominent blebs of the cell wall.     

Effect of choline on the morphology, growth and phospholipid composition of Fusarium graminearum

Studies were made of the growth kinetics, morphology and phospholipid composition of two strains of Fusarium graminearum, a wild-type strain (A3/5) and a highly branched variant (C106) which arose spontaneously during cultivation of A3/5. No significant difference was observed between the hyphal diameters of the two strains and therefore increased branching of C106 could not be explained in the terms of an increase in hyphal radius in the absence of a change in hyphal growth unit volume. The two strains had the same specific growth rate in batch culture and this was not affected by the addition of up to 1.5 mM-choline to the medium. However, choline increased the mean hyphal extension rate and colony radial growth rate of both strains and this response was correlated with the formation of mycelia which were more sparsely branched than mycelia grown on medium lacking choline. Addition of betaine, choline, ethanolamine, monomethylethanolamine or dimethylethanolamine (but not serine, glycine, dimethylglycine, methylamine, hydroxylamine or beta-hydroxyethylhydrazine) to the medium also resulted in appreciable increases in the colony radial growth rates of A3/5 (increased by about 130% for choline) and C106 (increased by about 25% for choline). No significant difference was observed between the phospholipid compositions of the two strains, and the addition of 100 microM-choline to the medium had no significant effect on the phospholipid composition of either strain.     

Plant-derived compounds and extracts with potential as aphid repellents

We devised a method for screening various substances for possible aphid repellency. Corn leaf aphids ( Rhopalosiphum maidis ) were released in an arena and allowed to select paired green tiles coated with petroleum jelly alone or petroleum jelly containing 1% of the substance being tested. Aphids adhering to tiles were counted 24 h later. If significantly fewer aphids landed on treated tiles, the substance was considered to have potential for repelling aphids. Fifty-five substances were tested. Most showed no activity, but several including β-citronellol, farnesol, geraniol, linalool, oils distilled from several species of Artemisia and Achillea millefolia (yarrow) oil showed some promise.     

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay

Introduction

Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.

Methods

The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.

Results

Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).

Conclusion

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.     

Extensive linkage disequilibrium in small human populations in Eurasia

The extent of linkage disequilibrium (LD) was studied in two small food-gathering populations—Evenki and Saami—and two larger food-producing populations—Finns and Swedes—in northern Eurasia. In total, 50 single-nucleotide polymorphisms (SNPs) from five genes were genotyped using real-time pyrophosphate DNA sequencing, whereas 14 microsatellites were genotyped in two X-chromosomal regions. In addition, hypervariable region I of the mtDNA was sequenced to shed light on the demographic history of the populations. The SNP data, as well as the microsatellite data, reveal extensive levels of LD in Evenki and Saami when compared to Finns and Swedes. mtDNA-sequence variation is compatible with constant population size over time in Evenki and Saami but indicates population expansion in Finns and Swedes. Furthermore, the similarity between Finns and Swedes in SNP allele- and haplotype-frequency distributions indicate that these two populations may share a recent common origin. These findings suggest that populations such as the Evenki and the Saami, rather than the Finns, may be particularly suited for the initial coarse mapping of common complex diseases.     

Out of Africa and into an ice age: on the role of global climate change in the late Pleistocene migration of early modern humans out of Africa

The results from two climate model simulations are used to explore the relationship between North Atlantic sea surface temperatures and the development of African aridity around 100,000 years ago. Through the use of illustrative simulations with an Earth System Climate Model, it is shown that freshwater fluxes associated with ice sheet surges into the North Atlantic, known as Heinrich events, lead to the southward shift of the intertropical convergence zone over Africa. This, combined with the overall increased aridity in the cooler mean climate, leads to substantial changes in simulated African vegetation cover, particularly in the Sahel. We suggest that Heinrich events, which occurred episodically throughout the last glacial cycle, led to abrupt changes in climate that may have rendered large parts of North, East, and West Africa unsuitable for hominin occupation, thus compelling early Homo sapiens to migrate out of Africa.