Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

An improved method to detect β- galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections

The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection     

Modifications of the EEG Activity upon Lateralized Stimulation of the Visual Inputs to the Right and to the Left Brain Hemispheres by Light with Different Wavelengths

In 80 healthy humans, we studied changes in the frequency spectrum and values of the relative spectral coefficients for subsequent 1-Hz-wide frequency bands under conditions of simultaneous lateralized stimulation of the retinal zones, which form receptive fields for the right- and for the left-brain hemispheres; light of different colors was used for stimulation. We found that reactions of the right and left hemispheres to such stimulations demonstrated obvious specificity; spatial characteristics of these reactions were obtained. Changes in the values of most corresponding spectral coefficients in the hemispheres usually had similar directions and demonstrated similarity in their intensities, which is indicative of a complementary pattern of the interhemispheric interaction. Reciprocal changes in the corresponding spectral coefficients in the hemispheres were observed more rarely. Modifications of EEG upon red-green stimulations of different polarity were found to be similar, which can be a manifestation of the moderating influence of the right hemisphere on the left one.     

The dual location of β-oxidation enzymes in germinating pea cotyledons

-Oxidation enzymes were detected both in the mitochondria and microbodies of pea cotyledons. Intact mitochondria did not show -oxidation enzyme activity but in ruptured mitochondria this activity was high. It is apparent that the mitochondrial membrane barrier prevents rapid access of acyl-CoA substrates to matrix -oxidation sites. Removal of the membrane barrier permits rapid access of acyl-CoAs and these enzyme activities may then be measured.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Degradation products of myelin proteins in a light CNS subcellular fraction

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from beavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Snake venom hyaluronidase: a therapeutic target

The diffusion of toxins from the site of a bite into the circulation is essential for successful envenomation. Degradation of hyaluronic acid in the extracellular matrix (ECM) by venom hyaluronidase is a key factor in this diffusion. Hyaluronidase not only increases the potency of other toxins but also damages the local tissue. In spite of its important role, little attention has been paid to this enzyme. Hyaluronidase exists in various isoforms and generates a wide range of hyaluronic acid degradation products. This suggests that beyond its role as a spreading factor venom hyaluronidase deserves to be explored as a possible therapeutic target for inhibiting the systemic distribution of venom and also for minimizing local tissue destruction at the site of the bite.     

Transfection of rabbit articular chondrocytes by the calcium phosphate procedure: Improvement of efficiency and reproducibility

The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding -galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (-galactosidase assay).Results revealed that addition of 4 U ml^–1 of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a giant calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.Abbreviations CPRG Chlorophenol red--D-galactopyranoside - EDTA Ethylenediaminetetraacetic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - HBS Hepes buffered saline - RAC Rabbit articular chondrocytes - X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside     

Isozymes in nutrient medium of suspension-cultured apple cells (Malus sylvestris Mill.)

The time course of the activities of esterase, -galactosidase, and -glucosidase in cell sap and nutrient medium in in vitro cultured apple cells (Malus sylvestris Mill.) was studied. The corresponding isozyme patterns and the intracellular and extracellular isozyme patterns of acid phosphatase and polyphenol oxidase were compared using isozyme visualization methods adapted to ultra-thin-layer isoelectric focusing. Neither quantitative (total activity) nor qualitative (isozyme pattern) data were congruent for cell saps and nutrient media. Malate dehydrogenase, malic enzyme, and glutamate dehydrogenase occurred in cell sap only. The extracellular activities probably originate to a great part from a programmed release by intact cells. Nutrient media of plant cell cultures constitute a rich source of active plant isozymes.     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

Biochemical and Physiological Aspects of Developmental Cycle of Colchicum autumnale L.

This study was conducted to examine the individual developmental stages of Colchicum autumnale. We identified the sclerenchymatic tissue in the middle part of the protuberance. This tissue supports the function of protuberance as a kind of hollow diverticulum. On the boundary of the new corm and the shoot a meristematic layer was recognized. We assume that this abscission zone-like structure can initiate dying back of the above-ground part regularly at the end of annual life-cycle. The major part of starch is reutilized in the mother corm during the autumnal stage, supporting sprouting which takes place in the soil. Decline of starch content is paralleled by increasing of total amylolytic activity. From amylolytic enzymes -amylase, -amylase and -glucosidase have been identified. The presence of pullulanase and starch phosphorylase was not observed. From free sugars glucose, fructose and sucrose were identified in corms. The level of sucrose increased significantly during winter season.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Role of α-galactosidase in cell wall metabolism of date palm (Phoenix dactylifera) endosperm

Summary The endosperm of developing date palm (Phoenix dactylifera) seeds was sampled at regular intervals from pollination to mature fruit. The galactose content of the cell wall mannans was assessed. Accumulation of -galactosidase, a cell wall hydrolase, during endosperm development was analyzed by isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with Western blotting and immunolocalization on tissue sections. N-terminal amino acid sequence of the first 15 amino acids showed homology with amino acids 71 to 85 of the sequence reported for the mature guar protein. Four forms of the enzyme with isoelectric points ranging from 4.4 to 5.2 appeared by 11 weeks after pollination, and all forms remained until maturity. A major band of 41 kDa and several lower M_r, lightly staining bands cross reacted with the anti--galactosidase antiserum. The major band remained until maturity while the lightly staining bands gradually disappeared. In the mobilizing endosperm of germinated seeds, two darkly staining bands were observed at 41 and 40 kDa. At 9 weeks after pollination, the endosperm was cellular and the silver enhanced gold label localizing -galactosidase occurred predominantly in the cell periphery. By 11 weeks, the label was present in the cytoplasm, but lacking on the thickening cell wall. -Galactosidase accumulated in the protein bodies along with the storage protein. At 13 to 17 weeks, the label accumulated and then was lost in a centrifugal pattern (from the middle lamella inward) from the cell walls as they matured and was lost in the cytoplasm. The mature endosperm cells had intense label present only over the protein bodies and over the inner cell wall. These observations suggest that -galactosidase is synthesized during endosperm development and unique forms of the enzyme are associated with cell wall maturation and cell wall mobilization in this species.     

Afferent bursting activity of ruff lateral line induced by background noise stimulation

Summary Single unit resting activities were recorded from fibres innervating a neuromast of the supra-orbital canal of the lateral line system of the ruff (Acerina cernua). The interval histogram of 1 of the 4 types of resting activity had a bimodal distribution (bursting activity). The resting activity of these fibres was compared with the measured vibration of the experimental table. The conclusion that the bursting activity is not spontaneous but is caused by small background vibrations of the table was supported by recording of extracellular hair cell responses.Abbreviation ISI Interspike interval     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.