Über den Nachweis eines Neurohormones beim Laubmooscallus und seine Beeinflussung durch das Phytochrom

Summary The presence of a neurohormone in moss callus could be demonstrated by means of pharmacological experiments on the heart of the frog (Rana temporaria L.) and by chromatography.The hearts react in the same manner as they do to application of acetylcholine and the substance resembles acetylcholine in its R_f-value. Therefore it is suggested that this hormone is identical with acetylcholine. The concentration of the hormone in the callus cells is mediated by the phytochrome. Moss callus cultivated under red and far-red illumination contain less substance than moss callus grown in red light.

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Herrn Dauscher, Institut für Physiologische Zoologie, danke ich für die Durchführung der pharmakologischen Tests am Froschherzen.     

A heme-nonapeptide tracer for electron microscopy

Summary A heme-nonapeptide (H-9-P)¹, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of 50% and purity of >99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, _E(max) ^{pH 7} = 397.5 nm, _{22 °C, pH 7} ^{397.5 nm} = 1.11 × 10⁵ [Liter/Mole x cm]. With the use of a diaminobenzidine-H₂O₂-medium — as applied for cytochemistry — we determined spectrophotometrically a pH_opt=12.5 and an apparent K₅ = 3.14 × 10^{– 3} [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.Abbreviations AAA amino acid analysis - AHC ammoniumhydrogencarbonate - BSA bovine serum albumin - Cyt c cytochrome c - DAB 5,3-diaminobenzidine - GA glutardialdehyde - H-8-P heme-octapeptide - H-9-P heme-nonapeptide - H-11-P heme-undecapeptide - HR-POX horseradish peroxidase - MW molecular weight - PAGE polyacrylamid-gel-electrophoresis - pI isoelectric point - SDS sodiumdodecylsulphate - SG-TLC silicagel-thin-layer-chromatography This work was supported by the Österreichische Forschungsfonds     

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.     

Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

RESTRICTION FRAGMENT ANALYSIS OF CHLOROPLAST DNA AND THE SYSTEMATICS OF VIGUIERA AND RELATED GENERA (ASTERACEAE: HELIANTHEAE)

Phylogenetic relationships among 31 species representing variously Helianthus, Helianthopsis, Heliomeris, Simsia, Viguiera, and Tithonia were assessed by chloroplast DNA restriction site mapping. A total of 147 mutations including 92 informative ones was detected using 16 restriction enzymes, with an estimated sequence divergence within the group of 1.4%. Parsimony analysis produced a single most parsimonious tree with a length of 161 steps and a consistency index of 0.91. Statistically significant clades, as assessed by the bootstrap method, correspond to a number of taxa recognized previously, including Helianthus (3 species), Helianthopsis (5 species), and several groups within Viguiera, including sect. Maculatae (4 species), the Baja California group (6 species), sect. Paradosa (2 species) and V. dentata (3 samples). However, species of Viguiera collectively form a highly paraphyletic group relative to species of other genera. Helianthus and Helianthopsis were separated into different clades, supporting their recent segregation. Placement of H. porteri in Helianthus rather than Heliomeris was confirmed; the single sample of the latter genus was most similar to the Baja California group of Viguiera. An expected relationship between Simsia (2 species) and one member of Viguiera ser. Grammatoglossae was confirmed (although not with two other putatively related members of Viguiera) and an unexpected relationship between Simsia and Tithonia was suggested. The presence of Mexican taxa as the more basal groups in the tree points toward a possible Mexican origin for Viguiera and related genera. A molecular clock hypothesis is rejected in many pairwise comparisons involving woody taxa with herbaceous ones, although it could not be rejected in most pairwise comparisons involving taxa of similar habit.     

External flavonoid aglycones of Viguiera series Viguiera (asteraceae: heliantheae)

Distribution of external flavonoid aglycones supports other data to suggest that Viguiera series Viguiera (series Dentatae Blake) is a mixed taxonomic grouping. Two species, V. dentata and V. potosina, lack external flavonoid aglycones, whereas the remaining species of the series all produce external flavonoid aglycones and appear to form a natural taxonomic group. The latter group, which consists of species from Baja California and nearby xeric areas, exhibits a diversity of flavonoid types, with different diploid species variously producing flavonols, simple flavones, 6-methoxyflavones, 6-hydroxy,7-methoxyflavones and a chalkone as major compounds. Polyploid species exhibit combinations of flavonoid types from possible diploid ancestors. Compounds of the 6-hydroxy,7-methoxyflavone type appear to characterize the group of species from Baja California and nearby areas within Viguiera.     

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.     

Structural Basis for Eliciting a Cytotoxic Effect in HER2-Overexpressing Cancer Cells via Binding to the Extracellular Domain of HER2

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Chemicals from alternative feedstocks in the United States

Abstract: Growing industrial interest in products from renewable alternative feedstocks has resulted in the creation of more industry-led federal research and development programs. The basis of this interest is introduced, followed by an overview of the various federal programs that support basic through applied research relevant to chemical products from renewable resources, and evidence of the increased coordination efforts between programs. The majority of the paper outlines a new program within the U.S. Department of Energy, the Alternative Feedstocks Program, which specifically targets chemicals from renewables which have the potential to become part of the next generation of high-volume chemical feedstocks for the manufacturing industries. The first product of the program, an iterative process technology decision analysis tool, is broadly presented using the first process development project: succinic acid from fermentable sugars.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.