Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25

The 3-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).HGM symbol: RAP1GDS1     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Selected contribution: Effects of aging on cerebrovascular tone and [Ca2+]i.

The lower limits of cerebral blood flow autoregulation shift toward high pressures in aged compared with young rats. Intraluminal pressure stimulates contractile mechanisms in cerebral arteries that might, in part, cause an age-dependent shift in autoregulation. The present project tested two hypotheses. First, cerebral artery tone is greater in isolated arteries from aged compared with mature adult rats. Second, aging decreases the modulatory effect of endothelium-derived nitric oxide (NO) and increases vascular smooth muscle Ca2+ sensitivity. Isolated segments of middle cerebral arteries from male 6-, 12-, 20-, and 24-mo-old Fischer 344 rats were cannulated and loaded with fura-2. Diameter and Ca2+ responses to increasing pressure were measured in HEPES, during NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)], and after removal of the endothelium. Cerebral artery tone (with endothelium) increased with age. Only at the lowest pressure (20 and 40 mmHg) was intracellular Ca2+ concentration ([Ca2+]i) greater in arteries from 24-mo-old rats compared with the other age groups. l-NAME-sensitive constriction increased significantly in arteries from 6- to 20-mo-old rats but declined significantly thereafter in arteries from 24-mo-old rats. [Ca2+]i was less in arteries from 24-mo-old rats compared with the other groups after treatment with l-NAME. Another endothelial-derived factor, insensitive to l-NAME, also decreased significantly with age. For example, at 60 mmHg, the l-NAME-insensitive constriction decreased from 47 +/- 10, 42 +/- 5, 21 +/- 2, and 3 +/- 1 microm in 6-, 12-, 20-, and 24-mo-old rats, respectively. Our data suggest that aging alters cerebral artery tone and [Ca2+]i responses through endothelial-derived NO synthase-sensitive and -insensitive mechanisms. The combined effect of greater cerebral artery tone with less endothelium-dependent modulation may in part contribute to the age-dependent shift in cerebral blood flow autoregulation.     

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the gall constriction hypothesis is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.We thank Dr. Zs. Koncz (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) for Agrobacterium strains and the Deutsche Forschungsgemeinschaft (SFB 199) for financial support to C.I.U.     

Characterization of a rice gene family encoding root-specific proteins

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (<70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5-upstream sequence of RCg2 was translationally fused to a -glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis

Fifteen Frankia strains from five different plant species were analyzed by two-dimensional polyacryl-amide gel electrophoresis to determine their relatedness by comparing the polypeptide patterns obtained. Three major subgroups (A, C, and D) were found in the Alnus-Comptonia-Myrica cross-inoculation group. An isolate from Purshia tridentata had a unique protein pattern and represents a distinct group of frankiae. Members of group A were isolated from root nodules of Alnus incana subsp. rugosa and Alnus viridis subsp. crispa. Group C organisms were from A. incana subsp. rugosa and Comptonia peregrina nodules, and group D organisms were from A. incana subsp. rugosa, A. viridis subsp. cripsa, and Myrica pensylvanica root nodules. Isolates from each gel group were obtained at several widely separated geographical locations. The results indicate that two-dimensional polyacrylamide gel electrophoresis is useful for identifying Frankia isolates.