Trimeric reassembly of the globular domain of human C1q

C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.     

Early events induced by chitosan on plant cells

Chitosan (a polymer of beta-1,4-glucosamine residues) is a deacetylated derivative of chitin which presents antifungal properties and acts as a potent elicitor of plant resistance against fungal pathogens. Attention was focused in this study on the chitosan-induced early events in the elicitation chain. Thus, it was shown that chitosan triggered in a dose-dependent manner rapid membrane transient depolarization of Mimosa pudica motor cells and, correlatively, a transient rise of pH in the incubation medium of pulvinar tissues. By using plasma membrane vesicles (PMVs), it was specified that a primary site of action of the compound is the plasma membrane H(+)-ATPase as shown by its inhibitory effect on the proton pumping and the catalytic activity of the enzyme up to 250 microg ml(-1). As a consequence, chitosan treatment modified H(+)-mediated processes, in particular it inhibited the uptake of the H(+)-substrate co-transported sucrose and valine, and inhibited the light-induced H(+)/K(+)-mediated turgor reaction of motor cells. The present data also allowed the limit of the cytotoxicity of the compound to be established close to a concentration of 100 microg ml(-1) at the plasma membrane level. As a consequence, chitosan could be preferably used in plant disease control as a powerful elicitor rather than a direct antifungal agent.     

Charophytes, indicateurs de paléobathymétrie du lac Tigalmamine (Moyen Atlas, Maroc)

A paleolimnological study can be particularly accurate in situations where the species found in the sediments have persisted in the same lake and can provide a direct modern analogue for interpretation. Lake Tigalmamine, thus, represents an exceptionally favourable context, compared to most paleolakes in North Africa, as this site allows comparing the fossil remains of the Characeae to those produced by the living plants. Tigalmamine is a hydrosystem composed of three karstic lakes located at 1626 m above sea level in the Middle Atlas of Morocco. The site had been the subject of a 16 m long core taken in the center of the central lake under 16 m of water (core C 86 in Lamb and Kaars, 1995 [The Holocene 5 (1995) 400-408]). The section represents the total of the Holocene from 10,200 BP to Present (El Hamouti et al., 1991 [Comptes Rendus de l’Académie des Sciences de Paris 2 (1991) 259-265]; Lamb et al., 1995 [Nature 373 (1995) 134-137]). The latter published a synthesis of the pluridisciplinary studies conducted on this core. Also, the numerous gyrogonites isolated from that core had been determined as Chara hispida (Soulié-Märsche in Benkaddour, 1993 [Changements hydrologiques et climatiques dans le Moyen-Atlas marocain : chronologie, minéralogie, géochimie isotopique et élémentaire des sédiments lacustres de Tigalmamine. PhD thesis, Université Paris-Sud, Orsay (inédit)]). Further fieldwork at Tigalmamine by the authors of the present paper in year 2000 showed that Chara hispida is still present in the lake where it forms a belt of submerged vegetation restricted to the range of 2 to 9 m water depth. This paper describes the morphological and ecological characteristics of the extant Chara hispida as a reference for the interpretation of the fossil remains. Based on these data, the frequency changes of the gyrogonites in the core sediments become significant in terms of bathymetry. The detailed analysis of the charophytes from the core, as a complement to the previous studies, confirms the existence of four Holocene phases with lake level low stand at Tigalmamine. The aim of the present study is to enhance the value of the charophytes as a useful complementary tool for paleolimnological reconstruction.     

Mycotoxin Fumonisin B1 Increases Intestinal Colonization by Pathogenic Escherichia coli in Pigs

Fumonisin B₁ (FB₁) is a mycotoxin that commonly occurs in maize. FB₁ causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans. In the present study, we examined the effect of dietary exposure to FB₁ on intestinal colonization by pathogenic Escherichia coli associated with extraintestinal infection. Three-week-old weaned pigs were given FB₁ by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days. On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenic E. coli strain. All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination. Ingestion of FB₁ had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile. However, colonization of the small and large intestines by an extraintestinal pathogenic E. coli strain was significantly increased. Our results show that FB₁ is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food.     

Food Intake and Dietary Overlap in Native Lemur catta and Propithecus verreauxi and Introduced Eulemur fulvus at Berenty,Southern Madagascar

The introduction of Eulemur fulvus in 1975 into the Berenty Reserve and their recent attainment of population densities comparable to those of Lemur catta led us to analyze food partitioning among the 3 large prosimian species in the gallery forest. We assessed the diets of ring-tailed lemurs (Lemur catta), brown lemurs (Eulemur fulvus) and sifakas (Propithecus verreauxi) from food intake measurements during 3 successive short-term studies. All species exhibited marked seasonal changes in their major food categories. Dietary overlap was very high between ring-tailed lemurs and brown lemurs during 2 of 3 seasons, including the middle of the dry season. During the latter period, Eulemur appeared to compensate for a low quality diet by increasing the amount of food eaten. In contrast, Lemur fed on lower amounts of food and seemed more efficient at coping with fibrous plant materials. There is low dietary overlap of Lemur catta and Eulemur fulvus versus Propithecus, which exhibit by far the highest dietary diversity of the 3 species. We discuss sustainable coexistence among them, based on respective dietary adaptations and potential for dietary flexibility.     

Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I

 The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH 7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amount of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and hemoglobin. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, respectively. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that observed for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b ₅. Fe(III)-mimochrome I is able to catalyze styrene epoxidation by using a [Fe(III)-mimochrome I]/[H₂O₂]/[stryrene] ratio of 1 : 10 : 2000 in phosphate buffer solution (pH 7.2) containing 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0  °C. Received: 26 May 1998 / Accepted: 20 August 1998     

Characterization and visualization of cholecystokinin receptors in rat brain using [3H]pentagastrin

[³H]Pentagastrin binds specifically to an apparent single class of CCK receptors on slide-mounted sections of rat brain (K_D=5.6 nM; B_max=36.6 fmol/mg protein). This specific binding is temperature-dependant and regulated by ions and nucleotides. The relative potencies of C-terminal fragments of CCK-8(SO₃H), benzotript and proglumide in inhibiting specific [³H]pentagastrin binding to CCK brain receptors reinforce the concept of different brain and pancreas CCK receptors. CCK receptors were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry. CCK receptors are highly concentrated in the cortex, dentate gyrus, granular and external plexiform layers of the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, claustrum, accumbens nucleus, some nuclei of the amygdala, thalamus and hypothalamus.     

Purification and properties of the three o-diphenol-O-methyltransferases of tobacco leaves

Three o-diphenol-O-methyltransferases (OMTs I, II and III) which catalyse the monomethylation of various o-diphenols using S-adenosyl-L-methionine as methyl donor were isolated and purified about 210-, 70-, and 70-fold, respectively, from leaves of Nicotiana tabacum cv Samsun NN. They had slightly different MWs (93 000, 90 000 and 100000 for OMTs 1, 11 and Ill respectively) and slightly different pls (5.21, 4.80 and 4.74). The activities of all three enzymes were very stable when stored at 0° but they had different sensitivities to ultrafiltration and to heat treatment (45°). In none of the enzymes was there any change in reaction rate when Mg²⁺ ions or EDTA were added. The three enzymes exhibited very high and similar affinities towards the substrate S-adenosylmethionine and the reaction product S-adenosylhomocysteine, but they differed markedly in specificities towards the various o-diphenolic substrates. Relative methylation efficiencies were estimated from the calculation of the V/K_m ratios that led to the following decreasing order of best substrates: 5-hydroxyferulic acid > caffeic acid > homo-catechol > esculetin > protocatechuic aldehyde > catechol > hydrocaffeic acid > chlorogenic acid, for OMT I, and: homocatechol > catechol > protocatechuic aldehyde > esculetin ≈ cafreic acid > 5-hydroxyferulic acid, for both OMTsIIandIII. Most of the o-diphenols assayed were methylated exclusively in the meta position, but all three tobacco OMTs showed both para and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and escultin. Since K_m values towards the two position of methylation were always found to be identical, we conclude that each enzyme bears only one catalytic site.     

Structural and ultrastructural characteristics of the vascular apparatus of the sensitive plant (Mimosa pudica L.)

Summary 1. In motor organs ofMimosa pudica xylem contains living fibriform elements limited by a thick lignified highly pitted wall, whereas in other parts of the plant (stem, petiole, rachis), xylem and protoxylem vessels are closely associated with parenchyma cells which possess wall ingrowths. These ingrowths, at the apex of which the plasmalemma and the tonoplast touch, are localized like those of transfer cells of C type described byGunning andPate. Nevertheless, xylem parenchyma cells differ from cells of C type in several characteristics. Moreover, in motor organs, phloem contains cells characterized by wall ingrowths, less abundant on the parts adjacent to the sieve tubes; these cells which are localized near collenchyma cells of primary phloem, look like transfer cells of A type defined byGunning andPate; they are absent from internodes, petioles and rachides. 2. In motor organs, three types of vascular cells (companion cells, living xylem fibriform elements and protoxylem parenchyma cells) are characterized by reduced vacuolar volumes and well developed membrane systems, as compared with homologuous cells belonging to other parts of the plant. 3. A symplastic continuity holds from the middle of motor organs to their cortex: it is provided by the presence, in xylem and phloem respectively, of living fibriform elements and collenchyma cells bearing numerous pit fields containing large numbers of plasmodesmata. Several ultrastructural features suggest that the vascular apparatus ofMimosa pudica would be the site of intensive lateral transfer at different levels, specially in motor organs. Possible functions of certain structures observed are discussed in relation to some hypotheses relative to excitatory conduction pathways.     

Statistical criteria for the identification of protein active sites using Theoretical Microscopic Titration Curves

Theoretical Microscopic Titration Curves (THEMATICS) may be used to identify chemically important residues in active sites of enzymes by characteristic deviations from the normal, sigmoidal Henderson-Hasselbalch titration behavior. Clusters of such deviant residues in physical proximity constitute reliable predictors of the location of the active site. Originally the residues with deviant predicted behavior were identified by human observation of the computed titration curves. However, it is preferable to select the unusual residues by mathematically well-defined criteria, in order to reduce the chance of error, eliminate any possible biases, and substantially speed up the selection process. Here we present some simple statistical tests that constitute such selection criteria. The first derivatives of the predicted titration curves resemble distribution functions and are normalized. The moments of these first derivative functions are computed. It is shown that the third and fourth moments, measures of asymmetry and kurtosis, respectively, are good measures of the deviations from normal behavior. Results are presented for 44 different enzymes. Detailed results are given for 4 enzymes with 4 different types of chemistry: arginine kinase from Limulus polyphemus (horseshoe crab); beta-lactamase from Escherichia coli; glutamate racemase from Aquifex pyrophilus; and 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans. The relationship between the statistical measures of nonsigmoidal behavior in the predicted titration curves and the catalytic activity of the residue is discussed.