Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

Manipulation of the bacteriochlorophyll c homolog distribution in the green sulfur bacterium Chlorobium tepidum

We have shown that the green sulfur bacterium Chlorobium tepidum can be grown in batch culture supplemented with potentially toxic fatty alcohols without a major effect on the growth rate if the concentration of the alcohols is kept low either by programmed addition or by adding the alcohol as an inclusion complex with -cyclodextrin. HPLC and GC analysis of pigment extracts from the supplemented cells showed that the fatty alcohols were incorporated into bacteriochlorophyll c as the esterifying alcohol. It was possible to change up to 43% of the naturally occurring farnesyl ester of bacteriochlorophyll c with the added alcohol. This change in the homolog composition had no effect on the spectral properties of the cells when farnesol was partially replaced by stearol, phytol or geranylgeraniol. However, with dodecanol we obtained a blue-shift of 6 nm of the Q_y band of the bacteriochlorophyll c and a concomitant change in the fluorescence emission was observed. The possible significance of these findings is discussed in the light of current ideas about bacteriochlorophyll organization in the chlorosomes.Abbreviations -CD -cyclodextrin - BChl bacteriochlorophyll - BChl c _H bacteriochlorophyllide c - [E,M] BChl c _F 8-ethyl, 12-methyl, farnesyl BChl c - [E,E] BChl c _F 8-ethyl, 12-ethyl, farnesyl BChl c - [P,E] BChl c _F 8-propyl, 12-ethyl, farnesyl BChl c - [I,E] BChl c _F 8-isobutyl, 12-ethyl, farnesyl BChl c - Car carotenoids     

Reduced programmed cell death in the retina and defects in lens and cornea of Tgfbeta2(-/-) Tgfbeta3(-/-) double-deficient mice

We have previously shown that immunoneutralization of transforming growth factor- (TGF-) in the chick embryo significantly reduces programmed cell death (PCD) in peripheral neurons, spinal cord, and retina. In order to validate these results we have begun to analyze PCD in mice with targeted ablations of the TGF-2 and TGF-3 genes. Recent analyses of mice lacking TGF-3 had failed to reveal an overt eye phenotype, while retinae of TGF-2-deficient mice showed retinal hypercellularity. We report now that eyes of Tgf2/Tgf3 double-deficient mice display severe alterations in the morphology of the retina, lens, and cornea. The inner neural retina—the region where TGF- receptor (TR) I and II immunoreactivities are most prominent—is significantly thickened, and numbers of TUNEL-positive cells are significantly reduced compared to wild-type littermates. In Tgf2^–/–Tgf3^–/– and Tgf2^–/–Tgf3^+/– littermates the retina was consistently detached from the underlying pigment epithelium. Cornea, corneal stroma, and lens epithelium were significantly thinner in these mutants. In contrast, retinal morphology in Tgf2^+/–Tgf3^–/– mutant littermates resembles the situation observed in wild-type retinae except for the retinal detachment. Thus, regression in the thickness of cornea and corneal stroma seems to be TGF- isoform and gene dose dependent. Our results substantiate the notion based on previous analyses of chick embryos with reduced levels of endogenous TGF- that TGF-, most notably TGF-2, is required to mediate PCD in developing retinal cells in vivo. Moreover, our data indicate that TGF-s play essential roles in cornea and lens development.This work was supported by grants from the Deutsche Forschungsgemeinschaft.     

Temperature sensitivity of vinblastine-induced tubulin polymerization in the presence of microtubule-associated proteins

The antitumor drug vinblastine has been a useful probe for examining the interaction of tubulin with the microtubule-associated proteins (MAPs), specifically with and MAP 2. Although and MAP 2 can stimulate microtubule assemblyin vitro, their specific interactions with tubulin are known to differ. For example, in the presence of vinblastine, both and MAP 2 cause tubulin to form spirals, but causes formation of clustered spirals of high turbidity, while MAP 2 causes formation of loose spirals of low turbidity [Ludueñaet al., J. Biol. Chem. 259, 12890–12898 (1984)]. Although cold temperatures can inhibit microtubule assembly, cold has no effect on vinblastine-induced tubulin spiral formation. Consequently, we used the vinblastine-tubulin system to examine the interactions of and MAP 2 with tubulin at low temperatures. We found that -tubulin-vinblastine complexes form about as well at 0°C as at 37°C. In contrast, MAP 2-tubulin-vinblastine complexes form much less well at 0°C than at 37°C. We find, however, that MAP 2, at 0°C, will strongly inhibit, and even reverse, formation of the -tubulin-vinblastine complex. This suggests that the temperature-sensitive factor is the MAP 2-stimulated tubulin-tubulin interaction rather than the MAP 2-tubulin interactionper se; this raises the possibility that the tubulin-tubulin interactions stimulated by differ in their temperature sensitivity from those stimulated by MAP 2.     

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a -1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa -1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic -1,3-glucanase and a basic 35 kDa -1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic -1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa -1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.     

Effect of “restricted ovulator” gene on uptake of yolk-precursor protein

Summary The bulk of the yolk proteins and lipoproteins constituting the yolks of mature oocytes in birds are synthesized by the liver and transported via the plasma to the oocytes where they are incorporated by micropinocytosis. Evidence is presented indicating that oocytes of hens possessing a mutation identified by Jones, Briles, and Schjeide as a restricted ovulator gene fail to incorporate normal amounts and proportions of low density lipoproteins, lipovitellin and possibly other proteins making up the bulk of the yolk material. Plasma albumin is taken into the yolks but the other proteins synthesized by the liver for deposition within the oocytes accumulate in the plasma, attaining very high levels. The possible nature of the lock preventing normal deposition of the excluded yolk proteins is discussed.     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

Phylogenetic Analysis of Vertebrate Fibrillar Collagen Locates the Position of Zebrafish α3(I) and Suggests an Evolutionary Link Between Collagen α Chains and Hox Clusters

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two 1 and one 2 chains. In some teleosts, a third chain has been identified by chromatography, suggesting that type I collagen should also exist as an 1(I)2(I)3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen 1(I), 2(I), and 3(I) chains. In this study on the evolution of fibrillar collagen chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the 3(I) chain is phylogenetically close to the 1(I) chain and support the hypothesis that the 3(I) chain arose from a duplication of the 1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the chains of fibrillar collagens of vertebrates could be related to Hox cluster history. Present address (Laure Bonnaud): Institut Jacques Monod, Tour 43, CNRS, Universités Paris 6 et 7, Equipe Evolution du Développement des Nématodes, 2 place Jussieu, 75005 Paris, France     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Photoactivatable Analogues of α-Conotoxins GI and MI and Their Interaction with Nicotinic Acetylcholine Receptor

Two photoactivatable analogues of -conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, -conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N of Gly1 or N of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native -conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [¹²⁵I] Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR crosslinking. All the AChR subunits were found to be crosslinked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the -conotoxin molecule.     

Cytoarchitectonic pattern of the hypothalamus in the cobra,Naja naja

Summary The distribution and cytoarchitectonic pattern of the magno- and parvocellular hypothalamic nuclei of the cobra, Naja naja, are described at the light-microscopic level. With respect to their tinctorial affinity to paraldehyde fuchsin (AF) as a representative of the Gomori-type of stains, the magnocellular neurons belong to the AF-positive and the parvocellular neurons to the AF-negative elements. In addition to the supraoptic and paraventricular nuclei proper, two accessory aggregations of magnocellular neurons, the nucleus retrochiasmaticus and nucleus circularis, can be identified. Although in a peculiar location, they may be regarded as subunits of the supraopticoparaventricular neurosecretory complex. As many as 22 AF-negative nuclear areas are identified in the hypothalamus of the cobra. The nucleus periventricularis hypothalami of earlier authors is subdivided into several circumscribed neuronal complexes. The nucleus arcuatus, nucleus hypothalamicus lateralis and nucleus lateralis recessus infundibuli are well developed. An attempt is made to interpret the significance of these nuclei on a comparative and phylogenetic basis.On leave from the Department of Zoology, Nagpur University, Nagpur, India     

Infertility in mice induced by the rhesus monkey chorionic gonadotropin β-subunit glycoprotein (rmCGβ) using DNA immunization

The recombinant eukaryotic expression vector pCMV4-rmCG, inserted full-length cDNA of the -subunit of rhesus monkey chorionic gonadotropin (rmCG), as DNA immuno-contraceptive against CG glycoprotein, has previously demonstrated the biological expression of rmCG in vitro and in vivo. The plasmid DNA of pCMV4-rmCG was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCG DNA delivery method for BALB/c mice, and the dose of 10 g should be enough to elicit immune response. With different doses from 10–50 g, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCG DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 g of pCMV4-rmCG DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCG DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of -subunit of mouse chorionic gonadotropin (muCG) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCG shares 99.6% homology with rmCG and 90.6% with hCG respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCG contraceptive should be further studied as a CG DNA contraceptive.     

Bounded water kinetic model of β-galactosidase in reverse micelles

In the present investigation, -galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (W_o) were collected. It was observed that the usual kinetic model used for -galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and W_o. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Tryptophanase in aqueous methanol: the solvent effects and a probable mechanism of the hydrophobic control of substrate specificity

To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K_i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K_i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K_D) and deprotonation to form a quinonoid intermediate (K_q) were determined for the reactions of tryptophanase with 2-oxindolyl- -alanine and -alanine by stopped-flow technique. For 2-oxindolyl- -alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the α-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably “closed”, conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, “open”, conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl- -alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid–base properties of the catalytic groups to ensure the movement of protons in the “correct” direction depending on the elementary stage of catalysis.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

Models for glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation

2-(4-Methoxybenzyl)-1-cyclohexyl--d-glucopyra nosides (1b and 2b) and 2-(4-methoxybenzyl)-1-cyclohexyl--d-galactopy ranosides (1c and 2c), models for glycosidic juvenogens, were synthesized using either D-glucose or D-galactose [in their natural form (3 and 5) or activated form (4 and 6)], and the respective racemic cis or trans isomers of 2-(4-methoxybenzyl)-1-cyclohexanol (1a and 2a) by either enzymic reverse hydrolysis or transglycosylation under both standard heating and microwave irradiation. Commercially available almond -glucosidase (EC 3.2.1.21) or -galactosidase (EC 3.2.1.23) from Escherichia coli were employed using different acetonitrile/water mixtures [9/1 (v/v) for the reverse hydrolysis, and 4/1 (v/v) for the transglycosylation].     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Wave separation versus bandpass filtering: a comparative non-linear analysis of brain α-EEG signals with and without psychotropic drug treatment

Attempts to demonstrate low-dimensional attractor behaviour in the analysis of electroencephalographic (EEG) signals meet with difficulties that in part stem from a departure from single-system dynamics. In order to address this problem, the -waves can be extracted by digital filtering or by wave separation; these two techniques are compared in order to specify the conditions in which finite impulse response (FIR) bandpass filters can be used. The comparison was made using 18 EEG records of 3 min duration under resting conditions (6 subjects, 3 records per subject: prior to apomorphine administration, then 90 min and 150 min post-treatment). No presence of low-dimensional dynamic episodes in -signals was observed without digital processing. Sixty 5 s sections showing attractor behaviour were found after filtering and twenty five 5 s sections after wave separation. The mean correlation dimension was calculated for each experimental condition and for 4 subjects, in order to observe the temporal profile of the drug. When attractors were found after wave separation, bandpass filtering then also showed attractor behaviour, with the same temporal profile. However, the reverse is not true: attractors were found after bandpass filtering that were not present after wave separation; in this case the results deserve confirmation, although the temporal profiles for all cases in which attractors were found after filtering remained comparable.