Java editor for biological pathways

SUMMARY: A visual Java-based tool for drawing and annotating biological pathways was developed. This tool integrates the possibilities of charting elements with different attributes (size, color, labels), drawing connections between elements in distinct characteristics (color, structure, width, arrows), as well as adding links to molecular biology databases, promoter sequences, information on the function of the genes or gene products, and references. It is easy to use and system independent. The result of the editing process is a PNG (portable network graphics) file for the images and XML (extended markup language) file for the appropriate links.     

Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions

The budding yeast spindle aligns along the mother- bud axis through interactions between cytoplasmic microtubules (CMs) and the cell cortex. Kar9, in complex with the EB1-related protein Bim1, mediates contacts of CMs with the cortex of the daughter cell, the bud. Here we established a novel series of events that target Kar9 to the bud cortex. First, Kar9 binds to spindle pole bodies (SPBs) in G(1) of the cell cycle. Secondly, in G(1)/S the yeast Cdk1, Cdc28, associates with SPBs and phosphorylates Kar9. Thirdly, Kar9 and Cdc28 then move from the SPB to the plus end of CMs directed towards the bud. This movement is dependent upon the microtubule motor protein Kip2. Cdc28 activity is required to concentrate Kar9 at the plus end of CMs and hence to establish contacts with the bud cortex. The Cdc28-regulated localization of Kar9 is therefore an integral part of the program that aligns spindles.     

Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling

The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis-12-oxophytodienoic acid (cis-OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi-phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA-levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA-levels kinetically coinciding with the second, more pronounced peak in wound-induced JA. In SA-treated leaves, the level of cis-OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.     

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.     

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     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Annulus cells release ATP in response to vibratory loading in vitro

Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca(2+)](ic)) and releasing adenosine 5'-triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin-luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP](ec)) in the range of 1-1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP](ec). Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations A_o binding site concentration - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-nor-8-hydroxyfusicoccin - K_D dissociation constant of the FCBP-radioligand complex We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision).     

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.