ζ-Carotene<Emphasis Type="Italic"> cis</Emphasis> isomers as products and substrates in the plant poly-<Emphasis Type="Italic">cis</Emphasis> carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and -carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9-tricis--carotene with 15,9-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by -carotene desaturase, the 15-cis double bond of 9,15,9-tricis--carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9-dicis--carotene is utilized by -carotene desaturase via 7,9,9-tricis-neurosporene to 7,9,7,9-tetracis-lycopene. Other -carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis--carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to -carotene. The 5-cis-lycopene and the 9-cis-and 13-cis--carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.     

Helicobacter infection and phospholipase A2 enzymes: Effect of Helicobacter felis‐infection on the expression and activity of sPLA2 enzymes in mouse stomach

The murine gastric mucosa possesses very high secretory type phospholipase A₂ activity. Northern and Western blots indicated that the pancreatictype, sPLA₂IB represents the predominant form of sPLA₂ enzymes present in the gastric mucosa. Both sPLA₂IB mRNA and protein in the gastric mucosa exceeded levels found in the pancreas, and in contrast to the pancreatic enzyme it was present primarily in the active state. The sPLA₂IB gene is not expressed in the murine small intestine and colon. Infection by the gastritis-inducing bacteria, Helicobacter felis (H. felis) dramatically and time dependently decreased the PLA₂ activity in the glandular stomach of the mouse strain, C57BL/6, sensitive to the organism, which appeared to be related to a decrease in the percentage of sPLA₂IB present in the active form. This bacterial-induced reduction in PLA₂ activity was not observed in BALB/c mice that fail to develop gastritis in response to H. felis infection. C57BL/6 mice do not, while BALB/c mice express, the PLA₂II enzyme. The H. felisinduced reduction in sPLA₂IB activity may weaken the gastric barrier by reducing the local concentration of arachidonic and linoleic acid, liberated from membrane phospholipids, the major precursors of cytoprotective prostaglandins. Data presented here suggest that both sPLA₂IB and sPLA₂II enzymes may contribute to the gastric response to Helicobacter infection.     

Estimation of symbiotic dinitrogen fixation in alder forest by the method based on natural15N abundance

Annual N₂-fixation in virgin forest ecosystems has been measured using a¹⁵N natural abundance (¹⁵N) procedure. This method was compared to a¹⁵N labelled fertilizer isotopic dilution method. For young alders (5–6 years old), ¹⁵N of leaves gave results in good agreement with the isotopic dilution of fertilizer method. Since ¹⁵N variability was expected according to plant physiology, for alder trees, leaves were collected at various heights after the end of the growing season, and, to take account of isotopic variations coming from derived inputs, ¹⁵N of leaves of a large number of other plants in the same are were measured to give control values. Following this procedure, the ¹⁵N method gave reliable evaluation of the nitrogen supply, by through N₂-fixation, to alders, which were found to maintain high nitrogen fixing capacity in a sequence ranging from first stage of establishment of climactic formation. Moreover, the same method is reported to discriminate various origins ofAlnus glutinosa grown in natural conditions, possibly in relation to the genetic diversity of this species.     

Theoretical study of N<Subscript>4</Subscript>X (X = O,S, Se) systems

A series of N₄X (X = O, S, Se) compounds have been examined with ab initio and density functional theory (DFT) methods. To our knowledge, these compounds, except for the C_2v ring and the C_3v towerlike isomers of N₄O, are first reported here. The ring structures are the most energetically favored for N₄X (X = O and S) systems. For N₄Se, the cagelike structure is the most energetically favored. Several decomposition and isomerization pathways for the N₄X species have been investigated. The dissociation of C_2v ring N₄O and N₄S structures via ring breaking and the barrier height are only 1.1 and −0.2 kcal mol⁻¹ at the CCSD(T)/6-311+G*//MP2/6-311+G* level of theory. The dissociation of the cagelike N₄X species is at a cost of 12.1–16.2 kcal mol⁻¹. As for the towerlike and triangle bipyramidal isomers, their decomposition or isomerization barrier heights are all lower than 10.0 kcal mol⁻¹. Although the C_S cagelike N₄S isomer has a moderate isomerization barrier (18.3–29.1 kcal mol⁻¹), the low dissociation barrier (−1.0 kcal mol⁻¹) indicates that it will disappear when going to the higher CCSD(T) level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photosynthetic Pathway Types in Rangeland Plant Species from Inner Mongolia,North China

Photosynthetic pathway types, based on ¹³C measurements, were determined for 125 species in 95 genera and 32 families growing in rangelands from Inner Mongolia. Of the total species, 4 species from 3 genera and 2 families had C₄ photosynthesis (2 species in Gramineae and 2 in Chenopodiaceae) and 118 species from 90 genera and 31 families had C₃ photosynthesis. The number of C₄ species differed significantly among four rangeland sites, 4 species in desert, 3 species in steppe, but no C₄ species were identified in meadow and dune. Six species [e.g. Agriophyllum arenarium Bieb., Bassia dasyphylla O. Kuntze, Saussurea japonica (Thunb.) DC.] earlier identified as C₄ species using the enzyme ratio method were found as C₃ species using the carbon isotope ratios (¹³C). Hence the enzyme ratio method for C₃ and C₄ identification may not always be reliable. The ¹³C values of 3 species of Crassulaceae, which had been considered as CAM species, differed remarkably [–25.79 for Sedum aizoon L., –24.42 for Osostachys fimbriatus (Turcz.) Berger, and –16.97 for O. malacophyllus (Pall.) Fisch], suggesting that the use of ¹³C method as a diagnosis for CAM photosynthetic pathway type may not always be reliable and supplementary measurements are needed.     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Glutamate,acetylcholine, and γ-aminobutyric acid as transmitters in the pyloric system of the stomatogastric ganglion of a stomatopod,Squilla oratoria

The neurotransmitters mediating the synaptic interactions in the pyloric system of the stomatogastric ganglion of a stomatopod, Squilla oratoria, were examined. Putative transmitters were applied iontophoretically to the pyloric cells. Glutamate and GABA produced inhibitory responses in all motoneurons but acetylcholine did not. These inhibitory responses were due to increases in conductance to either K⁺ or Cl^– or both, and blocked by picrotoxin. The inhibitory postsynaptic potentials evoked by the constrictor and dilator neurons were different in their time courses, reversal potentials, ion selectivities, and picrotoxin sensitivities. Glutamate is a transmitter candidate for inhibitory synapses made among the pyloric cells as well as for their neuromuscular junctions. In some cells, glutamate and acetylcholine evoked excitatory responses which were blocked by joro spider toxin and by tubocurare, respectively. They mediated the extrinsic inputs to modulate the pyloric rhythm. The transmitter, glutamate, is conserved in the ganglion neurons between stomatopods and decapods during evolution. Use of two transmitters, glutamate and acetylcholine, may have evolved in decapods, while the ionic mechanism is preserved in both orders. The neuromodulators, acetylcholine and -aminobutyric acid, are conserved between both orders. Glutamate may be used as the neuromodulator in stomatopods.Abbreviations ACh acetylcholine - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - Glu glutamate - IC inferior cardiac - IPSP inhibitory postsynaptic potential - JSTX joro spider toxin - LP lateral pyloric - pcp posterior cardiac plate - PTX picrotoxin     

Characterization of a half-molecular fragment obtained by reduction of human α2-macroglobulin with dithiothreitol

A half-molecular fragment of ₂-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric ₂-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of halfmolecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 Å, respectively. The match point, obtained by neutron scattering from solutions with different H₂O/D₂O rations, is at 43% D₂O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm³/g at+5°C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent threeaxial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72×10⁶ ų, we obtain a water content equal to 0.38 g H₂O/g protein (dry weight).Abbreviations SANS small-angle neutron scattering - SAXS small-angle X-ray scattering - ₂ M ₂-macroglobulin - DTT dithiothreitol - Tris tris(hydroxymethyl)aminomethane     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Charybdotoxin-sensitive K(Ca) channel is not involved in glucose-induced electrical activity in pancreatic β-cells

Summary The effects of charybdotoxin (CTX) on single [Ca²⁺] _i -activated potassium channel (K _(Ca)) activity and whole-cell K⁺ currents were examined in rat and mouse pancreatic -cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured -cells and -cells in intact islets were compared. K_(Ca) activity was very infrequent at negative patch potentials (–70<V _m <0 mV), channel activity appearing at highly depolarizedV _m . K_(Ca) open probability at these depolarizedV _m values was insensitive to glucose (10 and 20mm) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K⁺ channels of smaller conductance.The venom fromLeiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K_(Ca) channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18nm) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K⁺ currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20nm). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet -cells in the presence of glucose were not affected by CTX.We conclude that the K_(Ca) does not participate in the steady-state glucose-induced electrical activity in rodent pancreatic islets.     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

Stability of Schiff bases of amino acids and pyridoxal-5′-phosphate

Summary Stability of Schiff bases from Pyridoxal-5-phosphate and- and non-amino acids and amines have been studied in a wide range of pH. Furthermore the transamination process for the PLP-serine Schiff base and the cyclization reaction of PLP-histidine Schiff base have also been studied.Results show that the-position on carboxyl group of amino acids plays an important role on the mechanism of hydrolysis of imine bond. Absence of ionic groups in the surroundings of that bond seems to be an important fact of stability.In the transamination reaction, the rate-determining step is the isomerization of the Schiff base to ketoimine, since the rate constants for disappearance of Schiff base coincide with the rate constants for PMP formation. This process is catalyzed by the OH^–/H₂O system and the monoprotonated amino acid.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Expressed resistance to black shank among tobacco callus cultures

Summary Quantitatively inherited resistance to the black shank pathogen (Phytophthora parasitica var. nicotianae) was expressed among callus tissue cultures of tobacco (Nicotiana). Tissue cultures of genotypes known to posses polygenic mechanisms for black shank resistance expressed that resistance in vitro when challenged by the viable pathogen. Callus of a susceptible cultivar was readily parasitized in culture. Furthermore, single gene resistance to the common pathogen race was also shown to operate in vitro. Nongenetic factors examined did not contribute significantly to the observed differences. Disease expression in vitro appeared to be highly correlated with its expression at the whole plant level.Screening for quantitative disease resistance can be complicated at the whole plant level by variable hostpathogen reactions and by significant genotype × environment interactions. Since quantitatively inherited mechanisms of black shank resistance are expressed in tobacco callus cultures, an in vitro host-pathogen system may be useful in screening tobacco lines for black shank resistance.The research reported in this paper (No. 82-3-6) is in connection with a project of the Kentucky Agr. Exp. Stn., and the paper is published with the approval of the director     

Mode of action of atracotoxin at central and peripheral synapses of insects

These studies characterized the paralytic and neurophysiological effects of an atracotoxin (ACTX), from the Australian funnel web spider, Hadronyche versuta, and compared it to the established P/Q-type calcium channel blocker, -agatoxin-IVA (-Aga-IVA). ACTX-induced paralysis was of a spastic form in housefly (Musca domestica) larvae, but it was inactive on neuromuscular junction of housefly and tobacco budworm (Heliothis virescens). On cockroach (Periplaneta americana) cercal nerve–giant fiber synapse preparations, both toxins were effective blockers with potencies in the nanomolar range, but some spontaneous, high frequency trains of action potentials were observed with ACTX. In Drosophila melanogaster central nervous preparations, blockage of nerve firing occurred within 20 min when the nerve sheath was intact, demonstrating that the barrier could be breached by ACTX in vitro. There was a potent (pM) excitatory response to ACTX in this tissue, prior to the onset of block at higher concentrations. In contrast, -Aga-IVA was a pure blocker in both cockroach and Drosophila preparations. These studies demonstrate that central synaptic calcium channels underlie the action of ACTX. ACTX-dependent neuroexcitation has a number of possible mechanisms that warrant further study.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

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.