Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10 mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.     

The Ca2+-Activated K+ Channel KCa3.1 as a Potential New Target for the Prevention of Allograft Vasculopathy

Allograft vasculopathy (AV) remains one of the major challenges to the long-term functioning of solid organ transplants. Although its exact pathogenesis remains unclear, AV is characterized by both fibromuscular proliferation and infiltration of CD4⁺ memory T cells. We here tested whether two experimental immunosuppressants targeting K⁺ channels might be useful for preventing AV. PAP-1 inhibits the voltage-gated Kv1.3 channel, which is overexpressed on CCR7⁻ memory T cells and we therefore hypothesize that it should suppress the memory T cell component of AV. Based on its previous efficacy in restenosis and kidney fibrosis we expected that the KCa3.1 blocker TRAM-34 would primarily affect smooth muscle and fibroblast proliferation and thus reduce intimal hyperplasia. Using immunohistochemistry we demonstrated the presence of Kv1.3 on infiltrating T cells and of KCa3.1 on lymphocytes as well as on proliferating neointimal smooth muscle cells in human vasculopathy samples and in a rat aorta transplant model developing chronic AV. Treatment of PVG rats receiving orthotopically transplanted aortas from ACI rats with TRAM-34 dose-dependently reduced aortic luminal occlusion, intimal hyperplasia, mononuclear cell infiltration and collagen deposition 120 days after transplantation. The Kv1.3 blocker PAP-1 in contrast did not reduce intima hyperplasia despite drastically reducing plasma IFN-γ levels and inhibiting lymphocyte infiltration. Our findings suggest that KCa3.1 channels play an important role in the pathogenesis of chronic AV and constitute an attractive target for the prevention of arteriopathy.     

INTRASPECIFIC VARIATION IN GERMINATION REQUIREMENTS AND GROWTH IN AMARANTHUS DUBIUS

Seeds collected from five Amaranthus dubius plants of the same population were weighed and tested in continuous light or darkness at three temperatures in controlled conditions. Different seed families differed in weight, germination percentages and rate, and responded differently to an increase in temperature from 25 to 38 C. Plants derived from these seed families and grown outdoors, either singly or in mixtures, differed in several growth characteristics and in time to flowering. Significant interactions between growth characteristics and growth conditions, as measured with a two-way ANOVA, suggest that plants derived from different individuals differ in their competitive ability. The progeny of different individuals may thus be favored in different environmental conditions.     

Cytologische Untersuchungen an zwergwüchsigen Gartenrosen

Zusammenfassung Die in der Rosenzüchtung auftretenden monströsen Zwergpflanzen haben neben der veränderten Morphologie eine verminderte Vitalität und Blühwilligkeit. Im Extremfall kommt es zu einer Hemmungssterilität. Sie zeigen gegenüber normalwüchsigen Gartenrosen keine Abweichungen in der Chromosomenzahl (2n-28). Auch überschreiten die meiotischen Unregelmäßigkeiten, die im Auftreten von Uni- und Multivalenten bestehen, nicht das von normalwüchsigen Gartenrosen bekannte Ausmaß. Der monströse Zwergwuchs ist somit wahrscheinlich genisch bedingt, beruht jedoch nicht auf der Wirkung dominanter Gene.Die abstammungsmäßig aufR. chinensis var.minima zurückgehenden Zwergbengalrosen sind diploid (2n=14), soweit nicht in neuerer Zeit tetraploide Gartenrosen eingekreutzt worden sind. Der Zwergwuchs dieser Rosen zeigt gegenüber normalem Wuchs Dominanz. Bei einem diploiden Bastard zwischen der Polyantharose Mignonnette und der Zwergbengalrose Peón treten in der Meiosis in geringer Frequenz Uni- und Multivalente auf, bei R. Roulettii wurde Synapsis zu 711 oder 611+21 beobachtet.Sowohl bei den monströsen Zwergpflanzen als auch bei den Zwergbengalrosen beruht die Verkleinerung der Organe nicht auf einer Verringerung der Zellgröße, sondern auf einer Herabsetzung der Zellteilungsrate.Mit 12 Textabbildungen     

Probing the Diameter Limit of Single Walled Carbon Nanotubes in SWCNT: Fullerene Solar Cells

In this work, for the first time, the diameter limit of surfactant wrapped single walled carbon nanotubes (SWCNTs) in SWCNT:C₆₀ solar cells is determined through preparation of monochiral small and large diameter nanotube devices as well as those from polychiral mixtures. Through assignment of the different nanotube chiralities by photoluminescence and optical density measurements a diameter limit yielding 0% internal quantum efficiency (IQE) is determined. This work provides insights into the required net driving energy for SWCNT exciton dissociation onto C₆₀ and establishes a family of (n,m) species which can efficiently be utilized in polymer‐free SWCNT:C₆₀ solar cells. Using this approach the largest diameter nanotube with an IQE > 0% is found to be (8,6) with a diameter of 0.95 nm. Possible strategies to extend this diameter limit are then discussed.     

The cleavage specificity of RNase III.

We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.     

Early Synthesis of Membrane Protein After Bacteriophage T4 Infection

Proteins that associate with cellular membrane during the first 5 min after infection with bacteriophage T4 were examined. Several procedures, including electrophoretic separations in three sodium dodecyl sulfate polyacrylamide gel systems and inhibition of host protein synthesis by UV irradiation, were employed to distinguish host-specified proteins from those induced by T4. Residual host protein synthesis was found to account for much of the new protein in preparations of the total membrane and for almost all of the newly synthesized protein in the outer membrane. Preliminary evidence indicates that the synthesis of some host membrane proteins is shut off less rapidly than is host synthesis of soluble protein. One host-directed polypeptide of the outer membrane was unique in that its synthesis or incorporation into the membrane was preferentially inhibited by infection. Also, it was found that the detergent Sarkosyl solubilizes all early T4 membrane proteins; this observation provides the basis for a simple procedure for distinguishing phage proteins from host outer membrane proteins.     

Mapping of the fabA Locus for Unsaturated Fatty Acid Biosynthesis in Escherichia coli

fabA mutants of Escherichia coli require an appropriate unsaturated fatty acid for growth. The fabA locus has now been mapped at minute 21.5 of the linkage map of E. coli. The locus is cotransduced with pyrD and aroA but not with pyrC, purB, or pdxC. The clockwise order of markers in the region is pdxC, aroA, cmlB, pyrD, fabA, pyrC.