The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans-membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelength is obtained with valinomycin-induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.     

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well. An improved method of lipid extraction from chlorophyll-containing membranes is presented. This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction. The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography. The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography. The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis. None of the separated lipids alone was able to produce a well-coupled system. The best results were obtained with the native lipid mixture. The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol. Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included. Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C. The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.     

Prejudice in the former Soviet Union

Research in the United States and Europe has focused on the prejudice of majority groups towards minority groups, the implication somehow being that majority groups were more prejudiced than minority groups. In the former Soviet Union, ethnic environments were more complex; the same ethnic group could be a majority in one region but a minority in others. Using a sample of 1,459 first‐ and fourth‐year university students from eight regions of the former USSR, this study focuses on Russian, Tatar and Ukrainian respondents (n = 821) to test the hypothesis that the status of an ethnic group (majority/minority) or in‐group bias explains members’ prejudice. According to in‐group bias, all ethnic groups are equally prejudiced, minority and majority alike, whereas group status posits that groups in a majority position are more prejudiced. Findings show that group status has greater impact on prejudice than does in‐group bias. This applies, however, only to Russians. Interpretations of the findings rest on Soviet history and the rise of nationalism during the breakup of the Soviet Union.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

Occurrence of polygodial in plant organs and tissue culture of Polygonum hydropiper

Shoots of Polygonum hydropiper L. (waterpepper), especially in the leaves and flower-heads, contain significant amounts of the sesquiterpenoid polygodial, a compound with a potential use as a natural pesticide. The polygodial content of the tepals is particularly high: up to 8.5% of the dry weight. Roots and seeds do not contain detectable amounts of polygodial. Polygodial containing organs e.g. leaves and tepals, were found to contain cavities. Fourier transform infra-red (FTIR) microspectroscopy demonstrated that polygodial or its congeners are found in these cavities but not in other tissues or cells. Comparable cavities containing polygodial-like compounds were absent in the closely related species Polygonum persicaria L.
Callus cultures and cell suspensions as well as root- and shoot cultures were initiated from mature P. hydropiper plants. Polygodial could be detected only in shoot cultures. Our results indicate that functioning plastids may be essential for polygodial production and cavities for its accumulation.     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

Mesenterical lymphangiomatosis causing volvulus and intestinal obstruction

Lymphangiomas are benign tumors consisting of lymphatic vasculature that generally occur in the skin and soft tissues. Rarely, lymphangiomas occur in the gastrointestinal tract. Here, we report a case of a 13-year-old girl presenting with an intestinal obstruction. Upon laparotomy, multiple cystic masses in the mesentery causing a volvulus were resected and histologically identified as multiple lymphangiomas, or lymphangiomatosis. Mesenteric lymphangioma is a rare entity, but should be considered as cause of bowel obstruction without other known abdominal disease. As the etiology of lymphangiomas remains elusive, further research is directed at unravelling the mechanistic and molecular factors contributing to this disease.     

Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction

Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N′-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with N,N′-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N′-dicyclohexylcarbodiimide, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis[3-propyl-5-oxoisoxazol-4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H⁺-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.