In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

α-Isopropylmalate synthase as a marker for the leucine biosynthetic pathway in several clostridia and in Bacteroides fragilis

-Isopropylmalate synthase (EC 4.1.3.12) is present in extracts of Bacteroides fragilis, Clostridium thermoaceticum, Clostridium formicoacetium, Clostridium pasteurianum, and Clostridium kluyveri with specific activities (mol -isopropylmalate formed per min and g protein) of 8.6, 8.9, 2.4, 1.9, and 0.3, respectively. The product -isopropylmalate was identified by gas chromatography combined with mass spectroscopy. The presence of 5 mM leucine in the growth medium represses the synthesis of -isopropylmalate synthase in C. thermoaceticum by 40 and 70 %. The enzyme from C. pasteurianum was partially purified to a specific activity of 1413. All studied enzyme properties are similar to those of the enzymes from aerobic bacteria. It is suggested that in these anaerobic bacteria the -isopropylmalate pathway is present in addition to the pathway via the ferrodoxin-dependent, reductive carboxylation of branched chain fatty acids.Abbreviations used -KIV -Ketoisovalerate - -IPM -Isopropylmalate - CoA Coenzyme A     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

Einfluß von Phospholipiden auf den Stärkemetabolismus

Summary The presence of phospholipids reduces the breakdown of amylose catalyzed by -amylase, phosphorylase and -amylase. The activities of the -amylases of sweet potato (Ipomoea batatas) and barley (Hordeum vulgare L.) disminish to less than 10% of the activity in the control without the phospholipids. When the amylose was complexed with phospholipids the activity of the -amylase of Bacillus subtilis was reduced to about 25% of the control value. A similar effect was observed for the amylases of Zea mays leaves. The phosphorylase effected almost no phosphorolysis of the complexed amylose, but starch synthesis from glucose-1-phosphate proceeded at a rate that was about 60% of that with pure amylose. The activity of the synthetase from bundle sheath cells of maize leaves was not influenced much by the presence of phospholipids, whereas the branching enzyme of maize endosperm did not produce any amylopectin from the complexed amylose. —These facts could explain the simultaneous deposition of amylose and amylopectin in the starch granules. Some of the newly formed glucan chains may be protected by formation of a complex with the phospholipids. This protected amylose can not undergo branching or breakdown, but it can be elongated owing to the activity of synthetase or phosphorylase. Amylopectin is formed from the chains that are not complexed.     

Glycogen phosphorylase ‘b’ in Dictyostelium: stability and endogenous phosphorylation

Summary The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase b form requires 5 AMP for activity and is present in early development. The active phosphorylase a form is 5 AMP independent and occurs during later development. We here show that the 92 kd b enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn²⁺ and ATP, the phosphorylase b shows apparent conversion into a 5 AMP independent form as measured by enzyme activity. In addition, Mn²⁺ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase b subunit. We also demonstrate phosphorylation of the b enzyme subunit in vivo by ³²-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase b subunit is characterized.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Development of a new method for testing strength of cells against fluid shear stress

A new method for testing the strength of cells against fluid shear stress by using a long capillary column was proposed. The trajectories of cells in the column were simulated by introducing the Brownian motion model. The Brownian motion was performed by the generation of random numbers. The mean exposure time to shear stress and the mean shear stress acting on the surface of cells were discussed by the result of computer simulation. The mean shear stress acting on the surface of cells flowing in the capillary column was estimated as 4/3-fold of the shear stress at the column wall provided that the ratio of the cell radius to the column radius does not exceed 0.08. The effectiveness of this new method for testing the strength of cells against fluid shear stress was shown.List of Symbols a m radius of cell - c constant - E distribution function - L m length of capillary column - M number of division - N number of division - p probability - Q m³/s flow rate - R m radius of capillary column - r m radial position - t s time - T s exposure time - T _m s mean exposure time - T ₀ s mean residence time - m/s axial velocity - u _m m/s cross-sectional flow velocity - z m axial position - s^–1 shear rate - _w s^–1 shear rate at wall - Pa s viscosity - spherical coordinate - spherical coordinate - Pa shear stress - _m Pa mean shear stress - _w Pa shear stress at wall     

A cytokinin-sensitive mutant of the moss,Physcomitrella patens,defective in chloroplast division

Summary An X-ray induced mutant (PC22) of the moss,Physcomitrella patens was analysed with respect to its morphology, physiology and suitability for microculture techniques. The mutant protonemata are defective in bud formation and in chloroplast division. As a consequence of the latter, giant chloroplasts are formed which disturb the development of the phragmoplast, the formation of regular cross walls, and cell division. Abnormal cross walls are rich in callose. The actin cytoskeleton was found to be less regularly developed in the mutant than in the wild type. Three-dimensional analysis of the microtubular arrangement with confocal laser scan microscopy demonstrates a close association between spindle- or phragmoplast- and interphase-microtubules. The deficiencies in chloroplast division and in bud formation can partly be compensated for by exogeneously applied cytokinin. The suitability of this particular developmental mutant for further studies was shown by regeneration of protoplasts in microculture and microinjection of the fluorochrome Lucifer yellow into the chloroplast.Abbreviations CLSM confocal laser scan microscope - DAPI diamidinophenyl indole - DiOC 3,3-dihexyloxacarbocyanine iodide - EGTA ethylene glycol-bis-(-amino-ethylether-N,N,N,N-tetraacetic acid - i⁶Ade N⁶-(²-isopentenyladenine) - PIPES piperazine-N, N-bis-2-ethanesulfonic acid - ptDNA chloroplast DNA Devoted to the memory of Prof. Dr. O. Kiermayer, our colleague and friend.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Purification and some properties of Α-amylase from an ectomycorrhizal fungus, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

-Amylase from a still culture filtrate of Tricholoma matsutake, an ectomycorrhizal fungus, was isolated and characterized. The enzyme was purified to a homogeneous preparation with Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The -amylase was highly purified (3580 fold) with a recovery of 10.5% and showed a single protein band by SDS-PAGE. The enzyme was most active at pH 5.0–6.0 toward soluble starch and stable within the broad pH range 4.0–10.0. This -amylase was a relatively thermostable enzyme (optimum temperature, 60°C; thermal stability, 50°C). The molecular mass was 34kDa by size-exclusion chromatography and 46kDa by SDS-PAGE. This enzyme was not inhibited by the Hg²⁺ ion. Measurement of viscosity and TLC and HPLC analysis of the hydrolysates obtained from amylose showed that the amylase from T. matsutake is an endo-type (-amylase). Substrate specificity was tested using amylose with different polysaccharides. This -amylase readily hydrolyzed the -1,4 glucoside bond in soluble starch and amylose A (MW, 2900), but did not hydrolyze the -1,6 bond and cyclic polysaccharides such as - and -cyclodextrin.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Involvement of growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in oxLDL-induced apoptosis of human macrophages in vitro and in arteriosclerotic lesions

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-) superfamily, which has most recently been found in activated macrophages (M). We have now investigated GDF-15/MIC-1 in human M after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C₆-ceramide, tumor necrosis factor (TNF) and hydrogen peroxide (H₂O₂) was found in cultured human M. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) M, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human M by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated M.The last two authors are senior authors.