Azide treatment enhances cell-to-cell diffusion in staminal hairs ofSetcreasea purpurea

Summary The effect of azide on the diffusion of fluorescent molecular probes was examined in staminal hairs ofSetcreasea purpurea. Staminal hairs were treated with azide before being microinjected with fluorescent molecular probes of different size, charge, and structure. The cell-to-cell movement of these fluorescent molecules was videotaped, analyzed, and coefficients of diffusion through plasmodesmata (D) and coefficients of diffusion across the tonoplast (k₁) were calculated and compared to those of untreated cells. The D was larger and the k₁ was smaller for many fluorescent probes in azide treated cells compared to normal, untreated cells. In addition, the cell-to-cell diffusion selectivity based on molecule structure, size and charge no longer existed in azide treated cells. An average D of 3.3×10^–8cm²/s and an average k₁ of 2.9×10^–7/m²/s was calculated for the molecular probes tested. New size limits for permeation were observed indicating that the plasmodesmata had become enlarged.Abbreviations CF carboxyfluorescein - D diffusion coefficient for molecular probes in intercellular pores - FITC-Ang fluorescein isothiocyanate-angiotensin II - k₁ coefficient of diffusive loss across the tonoplast     

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)d-myo-inositol (I); (2)d-myo-inositol 2-monophosphate (IP₁); (3)d-myo-inositol 1,4-bisphosphate (IP₂); (4)d-myo-inositol 1,4,5-trisphosphate (IP₃); (5)d-fructose 2,6-diphosphate (F-2,6P₂) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP₁ or F-2,6P₂ was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP₂ or IP₃ was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.Abbreviations CF 5(6)Carboxyfluorescein - DG diacylglycerol - F2, 6P₂ d-fructose 2,6-diphosphate - I d-myo-inositol - IP₁ d-myo-inositol 2-monophosphate - IP₂ d-myo-inositol 1,4-bisphosphate - IP₃ d-myo-inositol 1,4,5-trisphosphate     

Fas-associated factor (Faf1) is a novel CD40 interactor that regulates CD40-induced NF-κB activation via a negative feedback loop

CD40-induced signalling through ligation with its natural ligand (CD40L/CD154) is dependent on recruitment of TRAF molecules to the cytoplasmic domain of the receptor. Here, we applied the yeast two-hybrid system to examine whether other proteins can interact with CD40. Fas-Associated Factor 1(FAF1) was isolated from a HeLa cDNA library using the CD40 cytoplasmic tail (216–278 aa) as a bait construct. FAF1 was able to interact with CD40 both in vitro and in vivo. The FAF1 N-terminal domain was sufficient to bind CD40 and required the TRAF6-binding domain within the cytoplasmic tail of CD40 for binding. CD40 ligation induced FAF1 expression in an NFκB-dependent manner. Knockdown of FAF1 prolonged CD40-induced NFκB, whereas overexpression of FAF1 suppressed CD40-induced NFκB activity and this required interaction of FAF1 with the CD40 receptor via its FID domain. Thus, we report a novel role for FAF1in regulating CD40-induced NFκB activation via a negative feedback loop. Loss of FAF1 function in certain human malignancies may contribute to oncogenesis through unchecked NFκB activation, and further understanding of this process may provide a biomarker of NFκB-targeted therapies for such malignancies.     

Analysis of allozyme variability in three Plantago species and a comparison to morphological variability

Summary The level of electrophoretic variability in three Plantago species, P. major, P. coronopus, and P. lanceolata, was analyzed in relation to their breeding systems and compared with their morphological variability. From each species several populations were analyzed. The outcrossing P. lanceolata had the highest level of electrophoretic variability and the lowest population differentiation. The inbreeding P. major showed the opposite: a low level of electrophoretic variability and a high population differentiation. P. coronopus, with an intermediate breeding system, had an intermediate level of variability and differentiation. In comparing the species, it appeared that P. coronopus and P. major showed good concordance in the distribution of both kinds of variability, each having only a slightly higher morphological than electrophoretic differentiation between populations. P. lanceolata showed a higher morphological than electrophoretic differentiation between populations. A comparison of populations, within species, revealed good concordance of electrophoretic and morphological variability only within P. coronopus, while some populations of the other two species had relatively lower morphological variability compared with electrophoretic variability.Grassland Species Research Group Publication No. 182     

ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors

With the existing knowledge of ATM''s role in therapeutic resistance, the present study aimed at identifying the molecular mechanisms that influence ATM to oscillate between chemoresistance and chemosensitivity. We observed that the redox status of tumors functions as a major determinant of ATM-dependent ‘resistance-to-apoptosis'' molecular switch. At a low reactive oxygen species (ROS) condition during genotoxic insult, the ATM/sumoylated-IKKγ interaction induced NFκB activation that resisted JNK-mediated apoptosis, whereas increasing cellular ROS restored ATM/JNK apoptotic signaling. A search for the upstream missing link revealed that high ROS induces oxidation and ubiquitin-mediated degradation of PIASγ, thereby disrupting PIASγ-IKKγ cross talk, a pre-requisite for IKKγ sumoylation and subsequent NFκB activation. Interruption in the PIASγ-mediated resistance pathway channels ATM signaling toward ATM/JNK pro-death circuitry. These in vitro results also translated to sensitive and resistant tumor allograft mouse models in which low ROS-induced resistance was over-ruled in PIASγ knockout tumors, while its overexpression inhibited high ROS-dependent apoptotic cues. Cumulatively, our findings identified an unappreciated yet critical combinatorial function of cellular ROS and PIASγ in regulating ATM-mediated chemosensitization of resistant tumors. Thus, therapeutic strategies employing ROS upregulation to inhibit PIASγ during genotoxic therapy may, in future, help to eliminate the problems of NFκB-mediated tumor drug resistance.     

The absorption, fluorescence and phosphorescence of phalloidin and α-amanitin

The peptides, phalloidin and α-amanitin, contain two unusual indole-derived chromophores, whose absorption, fluorescence and phosphorescence in aqueous solutions are described. Fluorescence is very weak, but phosphorescence at 77 K is intense. Phalloidin seems to undergo adiabatic photodissociation in alkaline solutions and to fluorescence from the indole anion form. In contrast, neither the phenolic nor the indolic protons of α-amanitin undergo photodissociation. The pK_a value of the phenolic hydroxy group of α-amanitin is 9.71 at 23°C.     

Occurrence and genotypes of Giardia isolated from lambs in Spain

Three hundred and eighty six faecal specimens were randomly collected from 1- to 3-month-old lambs from 16 farms in Spain to investigate the presence of different genotypes of Giardia duodenalis. Individual specimens were examined by IFA (Immunofluorescence assay) and β-giardin PCR polymerase chain reaction. Cysts of G. duodenalis were shed by lambs in every flock analyzed, showing a prevalence by farms of 100%. The average prevalence of G. duodenalis for the 386 specimens was 42%, ranging from 8.3 to 80% depending on the farm. β-giardin PCR positive samples were sequenced to determine the genotypes present at each farm and seven new subtypes of β-giardin Assemblage E are reported in this study. In each farm, one to six different β-giardin subtypes were found, showing the high variability of the target. Also, one flock had the zoonotic Assemblage A. This is the first report of Giardia subgenotype A-1 in sheep in Spain.     

Occurrence of 9′Z-β-Echinenone in the Sea Urchin Pseudocentrotus depressus

β-Echinenone is a major carotenoid in the gonad of sea urchins and may play an important role in reproduction and embryonic development. We reinvestigated β-echinenone occurrence in the gonad, viscera, test, and spine of the sea urchin Pseudocentrotus depressus. It was found that β-echinenone fraction consisted of all-E- and 9′Z-β-echinenone. The highest abundance of 9′Z-β-echinenone (76.0–78.2% of the total β-echinenone fraction) was observed in the ovary and testis of the sea urchin. In both females and males, all-E-β-echinenone predominated in the viscera (63.6–75.9%), unlike the 9′Z-β-echinenone, and it was also present in the test and spine (41.3–64.9%). It should be made clear that the work suggests that the Z-carotenoid may have a specific function in the sea urchin, possibly related to reproduction.     

ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα

Gliomas are resistant to radiation therapy, as well as to TNFα induced killing. Radiation-induced TNFα triggers Nuclear factor κB (NFκB)-mediated radioresistance. As inhibition of NFκB activation sensitizes glioma cells to TNFα-induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NFκB, Akt and Erk activation. Abrogation of Akt and NFκB activation further potentiated the death inducing ability of NCS in TNFα cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and ATM phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-IκB abrogated NCS-induced TIGAR both in the presence and absence of TNFα, TIGAR had no effect on NFκB activation. Transfection with TIGAR mutant (i) decreased apoptosis and γH2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNFα. Heightened TIGAR expression was observed in GBM tumors. While NCS induced ATM phosphorylation in a NFκB independent manner, ATM inhibition abrogated TIGAR and NFκB activation. Metabolic gene profiling indicated that TNFα affects NCS-mediated regulation of several genes associated with glycolysis. The existence of ATM-NFκB axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNFα cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death.     

Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Identification, mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis

Ankyrins are a family of adapter molecules mediating linkages between integral membrane and cytoskeletal proteins. Ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. A novel ANK-like gene of Chinese mitten crab Eriocheir japonica sinensis (denoted as EjsANK) was identified and cloned by expressed sequence tag and rapid amplification of cDNA end approaches. The full-length cDNA of EjsANK is 4375 bp and contains an open reading frame of 1095 bp which encodes a 364 amino acids polypeptide (40.23 kD) bearing seven ankyrin repeats. EjsANK cDNA has a 3073 bp uniquely long 3′ untranslated region with three K-box elements, one GY-like box domain and one Brd-like box domain. Sequence alignment and three-dimensional structural analyses revealed that EjsANK should be a novel cytosolic member of the ankyrin family. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsANK mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that the relative level of EjsANK mRNA expression was significantly higher in the abdomen at the first crab stage. Functional bioinformatics prediction analyses indicated that EjsANK has an analogical effect like IκB which is a key component of IκB/NF-κB complex in mammalian cells playing very important roles in the development process. Results suggest that EjsANK gene is involved in the early developmental regulation of Chinese mitten crab, especially brachyurization regulation.     

Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.