Legume Lectins: I. Immunological Cross-Reactions between the Enzymic Lectin from Mung Beans and other Well Characterized Legume Lectins

A number of well characterized legume lectins including the enzymic lectin from Vigna radiata were examined for immunological relatedness. The immunological cross-reactions observed indicate that most of the legume lectins, including Vigna lectin, are evolutionarily closely related proteins. The possibility that these proteins are homologs with enzymic functions is discussed.     

Heterogeneous ethoxylation of cellulose: influence of alkali and available-water concentrations on substituent distributions

O-(2-Hydroxyethyl)cellulose (1) as formed by the alkali-catalyzed addition of ethylene oxide to cellulose flock in a slurry process is not uniformly substituted. Most of the ethylene oxide adds to HO-6 in a chain-fashion, so that ～20% of the d-glucose residues remain totally unsubstituted at 2.0 molar substitution. Consequently, an aqueous solution thickened by 1 is highly susceptible to enzymic degradation. Stepwise decrease in concentration of alkali during etherification gives improved stability to enzymic degradation associated with a more-uniform distribution of hydroxyethyl groups between the three hydroxyl groups of the glucose residues in cellulose. The relative reactivities of hydroxyl groups and patterns of substitution were established by matching the distribution patterns from a stochastic computer-model with the distribution of substituents as determined by chemical analyses [namely hydrolysis with sulfuric acid to determine the percentage of unsubstituted glucose residues (u-2) and with periodate oxidation for determining the percentage of unsubstituted 2,3-vicinal diol groups per residue]. The reactivities of the three hydroxyl groups at various alkali concentrations in a heterogeneous, slurry-addition process approximate those observed under homogeneous conditions, wherein the reactivities have been determined by tedious chromatographic analyses. In the variable-alkali procedure for addition of ethylene oxide, the amount of water available to the cellulose matrix in the low-alkali (m) sequence is important both for the stability to enzymic degradation and for obtaining gel-free, thickened, aqueous solutions. Optimal stabilities and gel-free solutions are observed at intermediate water: cellulose ratios of 1.10–1.23. At a ratio of 1.23, the stability to enzymic degradation is less sensitive to percentage variations of u-2 than in 1 prepared at higher or lower water: cellulose ratios. Although the initial degree of degradation between 1 of high molar substitution prepared at 6.8m alkali concentration and a similar product prepared under variable alkali conditions may be related to percentage differences of u-2, the rate and final degree of degradation do not relate to percentage differences of u-2. An adequate interpretation, utilizing known cellulase turnover-rates, is found in stochastic-model projections of the distribution of consecutive 2 residues not substituted at HO-2. The results indicate that (1) more-uniform substitution through equalization of hydroxyl reactivities is achieved by lowering the alkali concentration, and (2) more-uniform substitution of 2 of the many cellulose-chains being substituted is achieved by employing an optimal amount of “available” water during etherification.     

Three new Brazilian species of chamaecrista moench (Leguminosae: Caesalpinioideae)

Chamaecrista (sect.Apoucouita) aspidiifolia Irwin & Barneby from Bahia,Ch. (sect.Absus) glischrodes Irwin & Barneby from Minas Gerais andCh. (sect.Absus)didyma Irwin & Barneby from Brazilian Amazonia are described, figured, and discussed in terms of affinity. Several incidental new combinations inChamaecrista are proposed.     

Modified colorimetric ninhydrin methods for peptidase assay

Four colorimetric procedures suitable for the determination of peptidase activity on peptides having a free α- or ε-amino group are described. Two of the methods (A and B) are modifications of the conventional ninhydrin method described by S. Moore and W. H. Stein ((1948) J. Biol. Chem.176, 367–388; (1954) Ibid.211, 907–913); the heating time is shortened to 5 min at 100°C and the pH of the buffer in the reagent is lowered to 4.0. Method A differs from method B in buffer concentration. The other two methods (C and D) are modifications of the Cd-ninhydrin method described by A. P. Tsarichenko ((1966) Nauch. Tr. Krasnodar. Gos. Pedagog. Inst.70, 86–88, as cited in Chem. Abs.67, 79479c); the water content in the reagent is reduced to $\text{1}\text{20}$ of the original reagent and the sample is heated for 5 min at 84°C. Method C differs from method D in the ratio of sample to reagent. In contrast to the free amino acids which are sufficiently colored, various peptides and amino acid derivatives except for the glycylpeptides give only a faint color with these methods. These four methods are not only useful for the determination of peptidase activity on peptides (e.g., Leu-Gly and tert-butyloxycarbonyl-glycyl-lysyl-leucine), but are also useful for the determinations of amidase activity on amino acid amides (e.g., Leu-NH₃) and esterase activity on amino acid esters (e.g., tyrosine ethyl ester).     

Location of a proton-donating group at the re-face of a β-d-galactosidase-bound,diastereotopic substrate

(Z)-3,7-Anhydro-1,2-dideoxy-2-deuterio-d-galacto-oct-2-enitol (1) was used as a diastereotopic probe, in order to elucidate the stereochemistry of protonation by β-d-galactosidase. Compound 1 can be converted by the enzyme into 1,2-dideoxy-2-deuterio-d-galacto-3-octulopyranose (2), which was submitted to periodate degradation. Propanoic acid derived from C-1, 2, and 3 of 2 has the (S) configuration, which proved the enzymic protonation of 1 to have taken place exclusively from the re-face.     

Sodium/calcium exchanger as main effector of endogenous neuroprotection elicited by ischemic tolerance

The ischemic tolerance (IT) paradigm represents a fundamental cell response to certain types or injury able to render an organ more “tolerant” to a subsequent, stronger, insult. During the 16th century, the toxicologist Paracelsus described for the first time the possibility that a noxious event might determine a state of tolerance. This finding was summarized in one of his most important mentions: “The dose makes the poison”. In more recent years, ischemic tolerance in the brain was first described in 1991, when it was demonstrated by Kirino and collaborators that two minutes of subthreshold brain ischemia in gerbils produced tolerance against global brain ischemia.Based on the time in which the conditioning stimulus is applied, it is possible to define preconditioning, perconditioning and postconditioning, when the subthreshold insult is applied before, during or after the ischemic event, respectively. Furthermore, depending on the temporal delay from the ischemic event, two different modalities are distinguished: rapid or delayed preconditioning and postconditioning. Finally, the circumstance in which the conditioning stimulus is applied on an organ distant from the brain is referred as remote conditioning.Over the years the “conditioning” paradigm has been applied to several brain disorders and a number of molecular mechanisms taking part to these protective processes have been described. The mechanisms are usually classified in three distinct categories identified as triggers, mediators and effectors. As concerns the putative effectors, it has been hypothesized that brain cells appear to have the ability to adapt to hypoxia by reducing their energy demand through modulation of ion channels and transporters, which delays anoxic depolarization. The purpose of the present review is to summarize the role played by plasmamembrane proteins able to control ionic homeostasis in mediating protection elicited by brain conditioning, particular attention will be deserved to the role played by Na⁺/Ca²⁺ exchanger.     

Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.     

Fluctuations morphologiques et hétérochronies chez Toxaster (échinides,Crétacé inférieur)

Morphological fluctuations and heterochronies of Toxaster (echinoid, Lower Cretaceous). Several steps of the diversification of the spatangoid Toxaster are described with an analysis of six species distributed over the north-west Tethyan margin, and ranging from the Lower Valanginian to the Barremian. Morphological variation was investigated with the analysis of two parameters (angles), which allowed recognition of three modules on the test. Comparison of ontogenetic trajectories of two reference species (T. granosus kiliani and T. retusus) suggests a peramorphic heterochronic pattern. However, no peramorphocline can be attested when comparisons are extended to adults of the six species. Evolution among these species is partially driven by allometric changes in one module, indicating an important influence of size. On the temporal and spatial scale of the study, the evolution of Toxaster appears globally quite conservative. To cite this article: É. François et al., C. R. Palevol 2 (2003).     

Enzymic isoprenylation of tryptophanyl cyclic dipeptides by Aspergillus amstelodami

Cyclo-l-prolyl-l-tryptophanyl, a component of several metabolites of Aspergillus ustus and cyclo-l-alanyl-l-tryptophanyl were compared as co-substrates with 3-methyl-2-butenyl-[1-³H]-1-pyrophosphate for an enzyme from A. amstelodami which previously had been described to isoprenylate cyclo-l-alanyl-l-tryptophanyl. Both compounds were equally active as isoprene acceptors using the A. amstelodami enzyme. The mass spectrum of the isoprenylated cyclo-l-prolyl-l-tryptophanyl indicated that the product was a monoisoprenylated derivative of the starting cyclic dipeptide. The most probable structure for this enzymic product is cyclo-l-prolyl-2(1,1-dimethylallyl)-l-tryptophanyl. The cyclo-pentylidene analogue of 3-methyl-2-butenyl-1-pyrophosphate did not serve as an alkylating agent when cyclo-l-alanyl-l-tryptophanyl was used as co-substrate.     

A simple and sensitive apparatus for continuous monitoring of orthophosphate in the presence of acid-labile compounds

A new technique is described for obtaining reproducible transient-state isoelectric focusing patterns of human pancreatic secretory proteins. This method is excellent for preservation of the proteins for further analysis because of the avoidance of urea, the short running times, and low temperatures. The protein patterns were analyzed on a point-by-point basis by a computer using an extension of our previously published method (Allan, B. J., Kirk, J., and White, T. T., 1978, Biochem. Biophys. Res. Commun., 85, 1239–1246). It was possible with these techniques to subtract various types of backgrounds and to construct average densitometric tracings for statistical analysis.     

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (R_f) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (R_f × 100) was found to be directly proportional to the pK′₂ of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl-alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.     

Identification of proteins according to biological activity following separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis: analysis of human exocrine pancreatic proteins

A novel procedure is described whereby proteins can be identified according to their biological activity after their separation in two dimensions using isoelectric focusing and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (G. Scheele, 1975, J. Biol. Chem., 250, 5375–5385). This procedure includes an optimal staining method for the visualization of two-dimensional gel spots, which avoids the use of chemical fixatives, and a one-step method for elution and renaturation of proteins. Fifteen out of the twenty discrete proteins separated from human pancreatic juice by the two-dimensional gel method were successfully identified by this procedure.     

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a–d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.     

Two new species of Antispila Hübner (Lepidoptera: Adeloidea: Heliozelidae) associated with Hydrangea (Hydrangeaceae) from China

The genus Antispila Hübner, [1825] is poorly recorded from China. Two new Antispila species, A. longcangensis, sp. nov. and A. emeishanensis, sp. nov. feeding on Hydrangeaceae, are described from Sichuan, China. The adult male and female, genitalia and venation of both species are illustrated. Diagnostic characters of these species are compared with other described Antispila species from China. DNA barcodes sequences of the two new species are also provided.     

Glycosylated Fibronectin and Inhibin are Lower and Anti-Müllerian Hormone is Higher in Umbilical Cord Blood when Mothers have Preeclampsia

Objective: Preeclampsia is a common disorder of pregnancy, causing significant morbidity and mortality for mothers and infants. Several molecules, including glycosylated fibronectin (GlyFn), the inhibin-related proteins, anti-müllerian hormone (AMH), and the insulin-like growth factor axis, are altered in maternal plasma in the setting of preeclampsia; however, these molecules have not been previously measured in cord blood of infants born to mothers with preeclampsia, which may represent changes in fetal physiology. We evaluated potential biomarkers of preeclampsia in umbilical cord blood to fill the gap in knowledge.Methods: This is a case-control study of 196 neonates born at a tertiary teaching hospital in Boston from 2010–2017. Forty-nine neonates born to mothers with preeclampsia were matched 1:3 by gestational age, sex, and birth weight z-score with 147 controls. Eleven analytes were measured in cord blood by enzyme-linked immunosorbent assay. Binary logistic regression analyses were performed to evaluate associations between preeclampsia and analytes.Results: Mean cord blood levels of GlyFn and total inhibin were significantly lower in neonates born to mothers with preeclampsia compared to controls, and AMH levels were significantly higher in males born to mothers with preeclampsia than male controls. Associations remained significant after controlling for maternal and neonatal characteristics.Conclusion: Cord blood levels of GlyFn and inhibin are decreased and AMH (male) levels are increased in infants of preeclamptic mothers, which is opposite the pattern these biomarkers show in serum of mothers with preeclampsia. These molecules may be important in the pathophysiology and long-term effects of preeclampsia on the developing fetus.Abbreviations: AMH = anti-müllerian hormone; ELISA = enzyme-linked immunosorbent assay; GlyFn = glycosylated fibronectin; IGF = insulin-like growth factor; IGFBP5 = insulin-like growth factor binding protein 5; LOD = limit of detection; PAPP-A = pregnancy-associated plasma protein A; PAPP-A2 = pregnancy-associated plasma protein A2     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

A new bicyclic sesquiterpenoid from Merremia yunnanensis

A new sesquiterpenoid, 1α,4β,8β,9β-eudesmane-tetrol-1-O-β-D-glucopyranoside (1), together with nine known compounds (2–10), were isolated from Merremia yunnanensis. The structures of these compounds were elucidated by spectroscopic methods and compared to data in the literature. All these compounds (1–10) were firstly isolated from this plant, and compounds 3, 5, 7, and 10 were reported from the Merremia genus for the first time. The significance of the chemotaxonomy for these compounds is described herein.     

Cytotoxic terpenoids from Nardophyllum bryoides

The investigation of the ethanol extract of fresh aerial parts of the Patagonian shrub Nardophyllum bryoides collected in the province of Chubut, Argentina, yielded eleven terpenoids. These include: three seco-ent-halimane diterpenoids (1–3), two ent-halimanes (4–5) and six pentacyclic oleanane and ursane triterpenoids (6–11). Four of these compounds (2, 6, 8 and 11) are hitherto unknown, while two others (1 and 4) have been previously reported but only as synthetic products. Several of these compounds showed moderate cytotoxicity against a human pancreatic adenocarcinoma cell line while compounds 4 and 5 were active at micromolar concentrations. The main component, seco-chiliolidic acid (1), could be isolated from this extract in large amounts, turning N. bryoides into a sustainable source of this bioactive compound.     

Bacterial production of hydroxylated and amidated metabolites of flurbiprofen

Several Streptomyces and Bacillus strains were examined for their ability to transform the anti-inflammatory drug flurbiprofen 1 to the hydroxylated metabolites that are found in humans after ingestion of this compound. Of the seven Streptomyces spp. examined, all but one transformed flurbiprofen to the main mammalian metabolite 4′-hydroxyflurbiprofen 2, and the majority also produced 3′,4′-dihydroxyflurbiprofen 3. Three strains, Streptomyces griseus DSM40236 and ATCC13273, and Streptomyces subrutilis DSM40445, also elaborated 3′-methoxy, 4′-hydroxy-flurbiprofen 4. None of the Bacillus spp. examined yielded these metabolites. Examination of the extracted supernatants of Streptomyces lavenduligriseus and Streptomyces rimosus by fluorine-19 nuclear magnetic resonance (¹⁹F NMR), indicated new resonances and these new fluorometabolites were purified by HPLC and revealed to be flurbiprofenamide 5 and 7-hydroxyflurbiprofenamide 6 after MS and NMR analyses. Subsequent re-examination of the culture supernatants from Bacillus subtilis IM7, Bacillus megaterium NCIMB8291 and B. megaterium ATTC14581 showed that these strains also produced 5 and 6. Resting cell investigations suggested that the amidation reaction employed nitrogen from an as yet unidentified amino acid.