Metabolism and excretion of nitrogen during metamorphosis and egg production in the sawfly, Neodiprion sertifer

Measurements of whole-body dry matter, total nitrogen, water-soluble protein, amino acids, and uric acid were determined at successive stages during metamorphosis in Neodiprion sertifer. The major change was in the uric acid fraction: in females, it increased up to the non-pharate pupa and then decreased during the subsequent stages of adult development and egg production; in males, it continued to increase during adult development. The decline of uric acid could not be explained by the accumulation of allantoin, allantoic acid, urea, or uric acid riboside. Examination of amino acid levels in the gut revealed an accumulation in the pupa followed by a depletion at the onset of adult development. This was followed by an excretory phase marked by the progressive accumulation of large quantities of uric acid and small quantities of urea, ammonia, and amino acids during the formation of the meconium. Amino acid analysis of the meconium revealed the presence of large proportions of proline, hydroxyproline, and histidine in comparison with the other amino acids.     

Structural studies of the Vibrio cholerae o-antigen

The dominant part of the O-antigen of Vibrio cholerae is a homopolysaccharide composed of (1→2)-linked 4-amino-4,6-dideoxy-α-d-mannopyranosyl (perosaminyl) residues, the amino groups of which are acylated by 3-deoxy-l-glycero-tetronic acid. Most of the amino sugar is decomposed during acid hydrolysis. Treatment of the polymer with anhydrous hydrogen fluoride, which cleaves the glycosidic linkages but does not cause N-deacylation, followed by acid hydrolysis under mild conditions, produced the monomer in good yield. Treatment of the N-deacylated polysaccharide with nitrous acid caused deamination with concomitant rearrangements, typical of 4-amino-4-deoxyhexopyranosyl residues in which the amino group occupies an equatorial position.     

A new neuraminic acid derivative and three types of glycopeptides isolated from the Cuvierian tubules of the sea cucumber Holothuria forskali

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.     

Production and Partial Characterization of Stylet Exudate from Adult Females of Meloidogyne incognita

Adult females of Meloidogyne incognita were excised from tomato roots and incubated in 0.04 M phosphate buffered saline, pH 7.4 for 18-72 hours to allow accumulation of stylet exudate. Twenty-four percent of the females produced exudate during the initial 18-hour incubation period; 70% of those females producing exudate initially produced additional exudate during the subsequent 54-hour incubation period. Analysis of exudate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least nine major protein bands. Differential staining with silver and Coomassie Brilliant Blue G-250 stains indicated that three of the bands were glycoproteins. Upon acid hydrolysis, 14 amino acids were detected in the stylet exudate. The basic amino acids lysine, histidine, and arginine comprised 21.8% of the total amino acids detected. No peroxidase activity was detected in the stylet exudates. Data presented extend and generally confirm prior work on the chemical composition of stylet exudate.     

Conjugated Amino Acids in the Non-Cationic Fraction of Pea Seedlings

Many amino acids were liberated by acid hydrolysis of the non-cationic fraction of pea seedling extract, suggesting the existence of ninhydrin-negative conjugated amino acids. The non-cationic fraction was subjected to cellulose column chromatography and gel-filtration with Sephadex G-15 and was separated into 20 fractions which gave ninhydrin color after alkaline hydrolysis. Alanine and glutamic acid, the most predominant amino acids in the hydrolyzate of the non-cationic fraction, occurred in many fractions. These results indicated that alanine and glutamic acid existed in various conjugated forms. In one of the fractions, there was evidence suggesting the presence of a peptide containing equimolar amounts of alanine and glutamic acid.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Synthesis of 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate by phosphorylation of methyl (methyl 3-deoxy-d-arabino-heptulopyranosid)onate

3-Deoxy-d-arabino-2-heptulosonic acid 7-phosphate (5), the first committed intermediate in aromatic amino acid biosynthesis, has been synthesized in good yield by treatment of methyl (methyl 3-deoxy-d-arabino-2-heptulopyranosid)onate with diphenylphosphoric chloride under mild conditions to give the 7-diphenyl phosphate. Catalytic removal of the phenyl residues, followed by base-catalyzed hydrolysis resulted in formation of (methyl 3-deoxy-d-arabino-2-heptulopyranosid)onic acid dihydrogen 7-phosphate (4), which yielded a crystalline tris-(cyclohexylammonium) salt. Acid-catalyzed hydrolysis of 4 afforded 5, which was used to purify 3-dehydroquinate synthase.     

Fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid,a constituent of glycoproteins of the cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje

Analysis of the sialic acid fraction obtained by mild acid hydrolysis of the Cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje revealed the presence of F-glycolylneuraminic acid and fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid. Furthermore, methylation analysis of a pronase-digest of the tubules demonstrated that all N-glycolylneuraminic acid residues are substituted at C-4, explaining the earlier reported resistance of sialic acid in tubules to cleavage by neuraminidase.     

An evaluation of the potential of Acacia dealbata as raw material for bioethanol production

In this work, the potential of Acacia dealbata as raw material for ethanol production was evaluated, as well as its composition with regard to cellulose, hemicelluloses, lignin, extractives and ash. The tree samples were subjected to several dilute acid pretreatments using a combined severity parameter ranging from 0.7 to 3.7. The highest ethanol concentration obtained was 10.31 g ethanol/L within 24 h by using a separate hydrolysis and fermentation of the water insoluble fraction after pretreatment at 180 °C with 0.8% of sulfuric acid for 15 min. With simultaneous saccharification and fermentation, results obtained for the washed solids of water insoluble fraction were better than those obtained with the whole slurry.     

A rapid procedure for assaying nicotinamide phosphoribosyltransferase

We examined the potential use of bovine enterokinase for the limited proteolysis of proteins containing sequences of one or more acidic residues preceding a basic residue. Proteolysis was followed by observing the appearance of fragments by sodium dodecyl sulfate-gel electrophoresis. The susceptible peptide bond was identified from a knowledge of the size of the fragment and the amino acid sequence of the protein. Bovine serum albumin was resistant to proteolysis in its native state, was somewhat susceptible as the S-carboxyamidomethyl derivative, and was highly susceptible as the S-carboxymethyl derivative. S-Alkylated soybean trypsin inhibitor and hen egg white lysozyme were both susceptible to limited hydrolysis, but only in the presence of deoxycholate. All susceptible bonds were either lysine or arginine. The preceding acidic residues could be either aspartic acid, glutamic acid, or carboxymethyl cysteine. If a single acidic residue immediately preceded the basic residue, the rate of hydrolysis was slow. The rate of hydrolysis was also slow if a carboxymethyl cysteine was introduced at the position following the basic residue. In addition to better defining the specificity of enterokinase, these results indicate that enterokinase may be useful in amino acid sequence studies for the production of large fragments. The enzyme may also be useful in DNA-recombinant studies in releasing the desired polypeptide chain from neighboring sequences.     

Pyroglutamyl N-termini of thermal polyamino acids

It has been established indirectly that the N-termini of the thermal polyamino acids are pyroglutamic acid. This was determined by trifluoroacetic acid hydrolysis of the lactam ring followed by Dansyl labelling. The polyamino acids contained Ala, Gly, Glu, Leu, Phe, and Pro. In the experiments described here, the presence of pyroglutamic acid at the N-terminus of a polyamino acid was determined directly by the use of pyrrolidone carboxylyl peptidase. The enzyme catalyzes the removal of pyroglutamyl residues at the N-terminus of polypeptide chains. The polyamino acids used in these studies contained glutamic acid, aspartic acid, alanine, glycine, isoleucine, proline and valine. Alkaline hydrolysis was also used to determine indirectly that the N-termini of these polyamino acids are pyroglutamic acid. Another interesting finding was that many of the amino acids in the polymerization mixture were found to occur penultimate to the N-terminal amino acid. This is interpreted to mean that the diffusible fraction contains many polyamino acids.     

The preparation of matrix-bound proteases and their use in the hydrolysis of proteins

Four proteases, crude acid protease from Aspergillus, pronase, amino-peptidase M, and prolidase, have been covalently attached to activated agarose and to amino propyl glass beads. The matrix-bound enzymes have been tested as catalysts for the complete hydrolysis of protein substrates, with the primary goal to isolate unstable amino acid derivatives present in the substrate protein. Under conditions used in the present work, the total amino acid release from the protease-catalyzed hydrolysis of four substrate proteins (pancreatic ribonuclease, egg white lysozyme, yeast enolase, and bovine insulin) was 95–103% of that observed in standard acid hydrolysis. Recovery of individual amino acids showed greater deviation from the theoretical values, but cystine was the only amino acid recovered in low yields (42–77%) from all four proteins. Derivatized amino acids, such as methionine sulfoxide, O-(butylcarbamoyl)-serine, and N-glycosyl asparagine have been obtained from chemically modified proteins or from unmodified glycoprotein in good yield, and normal amino acid constituents of proteins which cannot be quantified after acid hydrolysis (tryptophan, asparagine, and glutamine) have also been determined either directly after proteolysis or after proteolysis in conjunction with acid hydrolysis.     

A lichenase-like family 12 endo-(1-->4)-beta-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on beta-glucans in comparison with other glycoside hydrolases

Using anion-exchange chromatography on Source 15Q followed by hydrophobic interaction chromatography on Source 15 Isopropyl, a lichenase-like endo-(1→4)-β-glucanase (BG, 28 kDa, pI 4.1) was isolated from a culture filtrate of Aspergillus japonicus. The enzyme was highly active against barley β-glucan and lichenan (263 and 267 U/mg protein) and had much lower activity toward carboxymethylcellulose (3.9 U/mg). The mode of action of the BG on barley β-glucan and lichenan was studied in comparison with that of Bacillus subtilis lichenase and endo-(1→4)-β-glucanases (EG I, II, and III) of Trichoderma reesei. The BG behaved very similar to the bacterial lichenase, except the tri- and tetrasaccharides formed as the end products of β-glucan hydrolysis with the BG contained the β-(1→3)-glucoside linkage at the non-reducing end, while the lichenase-derived oligosaccharides had the β-(1→3)-linkage at the reducing end. The BG was characterized by a high amino acid sequence identity to the EG of Aspergillus kawachii (UniProt entry Q12679) from a family 12 of glycoside hydrolases (96% in 162 identified aa residues out of total 223 residues) and also showed lower sequence similarity to the EglA of Aspergillus niger (O74705).     

The identification of N-acetylglycine as a contaminant of glacial acetic acid.

This paper provides evidence by gas chromatography-mass spectrometry (GC-MS) that N-acetylglycine is present, in varying amounts, as a contaminant of all samples of analytical grade glacial acetic acid that have been examined in our laboratory. Supportive evidence for the GC-MS data was obtained by amino acid analyses of evaporated samples of acetic acid which were subjected to acid hydrolysis and then analyzed by ion-exchange chromatography.Although the identification of N-acetylglycine has been established with certainty, small quantities of other amino acid derivatives which have not yet been identified are also present in glacial acetic acid. These additional amino acids have been identified after acid hydrolysis. It should be pointed out that although amino acids are of chief interest here, they comprise approximately 1% or less of the total organic contamination.A very marked reduction of the concentration of N-acetylglycine and all other contaminants was accomplished by slow distillation of the glacial acetic acid through a column of packed Raschig rings.     

Applications of the hexanic fraction of Agave sisalana Perrine ex Engelm (Asparagaceae): control of inflammation and pain screening

The present study evaluated the anti-inflammatory and analgesic properties of Agave sisalana Perrine in classic models of inflammation and pain. The hexanic fraction of A. sisalana (HFAS) was obtained by acid hydrolysis followed by hexanic reflux. Anti-inflammatory properties were examined in three acute mouse models (xylene ear oedema, hind paw oedema and pleurisy) and a chronic mouse model (granuloma cotton pellet). The antinociceptive potential was evaluated in chemical (acetic-acid) and thermal (tail-flick and hot-plate test) models of pain. When given orally, HFAS (5, 10, 25 and 50 mg/kg) reduced ear oedema (p < 0.0001; 52%, 71%, 62% and 42%, respectively). HFAS also reduced hind paw oedema at doses of 10 mg/kg and 25 mg/kg (p < 0.05; 42% and 58%, respectively) and pleurisy at doses of 10 mg/kg and 25 mg/kg (41% and 50%, respectively). In a chronic model, HFAS reduced inflammation by 46% and 58% at doses of 10 mg/kg and 25 mg/kg, respectively. Moreover, this fraction showed analgesic properties against the abdominal writhing in an acetic acid model (at doses of 5-25 mg/kg) with inhibitory rates of 24%, 54% and 48%. The HFAS also showed an increased latency time in the hot-plate (23% and 28%) and tail-flick tests (61% and 66%) for the 25 mg/kg and 50 mg/kg doses, respectively. These results suggest that HFAS has anti-inflammatory and analgesic properties.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.     

Incorporation of 3H-Gibberellin A20 in roots of G2 peas

Following application of ³H-Gibberellin A₂₀ (GA₂₀) to roots of G2 pea seedlings and homogenization of the roots, about 3% of the radioactivity in the tissue could be precipitated from a 30,000 × g supernatant with trichloroacetic acid (TCA) (soluble fraction) while about 5% of the radioactivity pelleted at 30,000 × g (particulate fraction). The radioactivity in the particulate fraction was soluble in sodium dodecyl sulfate (SDS), but was not dialyzable and was insoluble in ethanol. Electrophoresis of the soluble fraction gave only one band of radioactivity, while that of the particulate fraction gave multiple bands. Acid hydrolysis of the soluble fraction released radioactivity that ran coincident with acid-treated GA₂₀ on silicic-acid column chromatography. The particulate fraction gave numerous radioactive peaks following acid hydrolysis, two of which were coincident with GA₂₀ and GA₂₉ (hydroxylation product of GA₂₀) on silicic acid chromatography. Treatment of the particulate and soluble fractions with RNase, DNase, and proteases showed a significant solubilization of radioactivity only with the proteases, suggesting that the GA is bound to a proteinaceous macromolecule. Complete proteolytic hydrolyis followed by thin layer chromatography showed 65% of the radioactivity from the soluble fraction running separately from free GAs or the individual amino acids; the particulate fraction gave mainly (60%) free GAs on enzymatic hydrolysis and much smaller amounts (17%) in a position separate from that of the GAs or amino acids. Binding of ³H-GA to protease-sensitive material was obtained with biologically active ³H-GA₂₀ and ³H-GA₁.