Isoasparagine as a marker amino acid in the taxonomy of Characeae

Fourteen species of Characeae were analyzed for their free amino acids. This study, in combination with previous results (Sakanoet al., 1984) showed that all the species that belong to the generaChara (6 species),Lamprothamnium (1 species),Nitellopsis (1 species),Lychnothammus (1 species) and 5Nitella species of the members of the subsectionAnarthrodactylae contained a large amount of isoasparagine. In contrast, no isoasparagine was found in the species belonging toTolypella (3 species) and otherNitella (7 species). Presence of isoasparagine in some species of Characeae (N. flexillis andNitellopsis obtusa) was found to be independent of their localities (Japan, Canada and England). Species lacking isoasparagine (N. oligogyra and N. axilliformis) did not produce isoasparagine even under the condition that induced a great increase of this amino acid in the species that contained it (C. corallina andN. flexilis). These results indicate that isoasparagine is a distinct taxonomic marker and suggest that theNitella species of the subsectionAnarthrodactylae are the most primitive group inNitelleae in that they share synthesis and accumulation of isoasparagine withChareae and, hence, support the view (Kasaki, 1964) that the subsection may be treated as an independent genus.     

Biochemical analysis of four species ofCassia L. pollen

The present paper describes the comparative biochemical studies in terms of quantitative analyses of carbohydrates, lipids, proteins, free amino acids, nucleic acids, minerals, ash and moisture as well as the identification of free amino acids of pollen of four species ofCassia L. (C. alata L,C. fistula L,C. occidentalis L andC. siamea Lam.). A significant variation in the chemical constituents was observed among the four species.C. occidentalis showed the highest levels of carbohydrate (15.15%) and protein (22.45%), andC. siamea had the lowest levels of carbohydrate (7.15%), lipid (6.2%) and protein (13.85%).C. alata andC. fistula showed intermediate results. However,C. alata showed the highest amount of free amino acids (3.8%) and the least of 1.42% was found inC. fistula. Thin layer chromatography (TLC) of free amino acids of the four species showed some homology in their amino acid content, of which proline, glutamic acid, methionine and phenyl-alanine were the most dominant. The level of nucleic acids and minerals was found to be comparatively low.C. siamea andC. alata showed an exceptionally high level of ash content (8.6 and 8.8%, respectively) while moisture content varied from 8 to 11%.     

Purification of cadmium-binding proteins from related species of terrestrial helicidae (gastropoda,mollusca): A comparative study

Summary Three species of terrestrial Helicidae (Helix pomatia, Cepaea hortensis andArianta arbustorum) were fed cadmium-rich diet in the laboratory. The snails accumulated high amounts of the metal in their hepatopancreas. Most cadmium and some zinc were found, after centrifugation, in the soluble fractions from which a cadmium-binding protein was isolated for each species by ion exchange and gel chromatography. The proteins contained different amounts of cadmium, but little or no zinc, and showed high absorption at 254 nm indicating the presence of cadmium-mercaptide bonds. After gel filtration, a molecular weight of 12000 was found for cadmium-binding proteins fromHelix pomatia andArianta arbustorum, whereas a molecular weight of 10 000 was found for a cadmium-binding protein fromCepaea hortensis. SDS-polyacrylamide gel electrophoresis showed one single band for each protein fromHelix pomatia andArianta arbustorum and suggested a molecular weight of 11000 for both species. Amino acid analysis revealed, for each protein, high amounts of cysteine (12–20%), glycine (15–19%), and serine (12–14%), and moderately elevated contents of lysine (9–13%) and alanine (4–8%), but no methionine and only traces, if any, of aromatic amino acids. The ratios of cadmium to cysteine were 1:5, 1:10 and 1:3 in the proteins fromHelix pomatia,Cepaea hortensis andArianta arbustorum, respectively. Some features of the isolated proteins resembled mammalian metallothioneins. Most characteristics, however, differed from true metallothioneins and were similar to cadmium-binding proteins found in some marine molluscs.     

Chemical and cytological changes during the autolysis of yeasts

Summary Cell suspensions ofSacharomyces cerevisiae, Kloeckera apiculata andCandida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25–35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3–7%; organic acids 3–6%; protein 12–13%; free amino acids 8–12%; nucleic acid products 3–5%; and lipids 1–12%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85–90% of cellular RNA and 25–40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neutral lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization withinS. cerevisiae andC. stellata. There were differences in the autolytic behavior ofK. apiculata compared withS. cerevisiae andC. stellata.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Aminoacid profiles in the study of phylogenetic relationships in the genusOryza

Summary The amino acid profiles in seeds of thirteen different species ofOryza, including two cultivated rices,O. glaberrima andO. sativa and the two major geographical racesindica andjaponica were studied using an automatic amino acid analyser to assess differences in the profiles of cultivated species and their wild progenitors. The polygon graphic method was employed to envision the species relationship. Essential amino acid profiles in different species were also compared with those of the Food and Agriculture Organization (FAO) standards. The results suggest a wide range of variability amongOryza species for lysine (up to 4.4% as against 3.5% in cultivated rices) and other essential amino acids. This will be of considerable interest to rice breeders, when after overcoming genetic barriers, the possible utilization of these species in rice breeding becomes feasible.     

-Amino acid contents of mitochondria and some purple bacteria

The endosymbiont most likely to have given rise to mitochondria is an aerobic bacterium belonging to the α subdivision of the so-called purple bacteria such as Rickettsia, Bradythizobium and Agrobacterium [1 and 2]. Contents of the -enantiomers of serine, alanine, proline, glutamate and aspartate in rat liver whole mitochondria, mitochondrial outer membranes, inner membranes and matrix, soluble proteins and free amino acids were detected. These values for -amino acid content were compared with those in soluble proteins and free amino acids from the purple bacteria Paracoccus denitrificans, Pseudomonas aeruginosa and Escherichia coli, members, respectively of the α, β, and γ subdivisions, to find any similarity between mitochondria and these purple bacteria. A similarity was observed in protein -amino acid contents which were low (<1.5%, D-type/D-type+L-type) both in the membrane and soluble protein fractions from mitochondria and in soluble protein from bacteria. Oddly, substantial amounts of free -serine and free -aspartate (around 2%) were found for the first time in mitochondria. The contents of -serine and -aspartate were higher than those of -alanine, -proline and -glutamate. In purple bacteria, the concentration of -serine (<2%) was the lowest of the five amino acids examined, and those of -alanine (27–32%) and -glutamate (7–26%) were high. Therefore, no similarity was shown in the free -amino acid content between mitochondria and any of the three purple bacteria.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

Inhibition ofAzorhizobium andRhizobium by seed leachates

During germination of seeds ofSesbania rostrata, S. aculeata andS. speciosa various constituents, including tannin, free amino acids, protein, nitrogen and sugars, were released. Tannin released byS. speciosa after 24 h was particularly high. The growth ofAzorhizobium andRhizobium were inhibited in the presence of these materials.     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Further flavonoid studies onAttalea species and some related cocosoid palms

In a flavonoid survey of direct and hydrolysed leaf extracts of sixteenAttalea, sevenScheelea and fourOrbignya species free tricin, tricin 7-glycosides, tricin 5-glucoside and flavone C-glycosides were the most frequent constituents; present in 100, 89, 70, and 81% of species, respectively. Luteolin, quercetin and isorhamnetin were each found in only 15% of the sample. The present results confirm the findings of a previous survey thatAttalea, Scheelea andOrbignya are chemically heterogeneous with as much variation between species as between genera. Furthermore, threeAttalea species,A. allenii, A. guaranitica andA. victoriana showed some infraspecific variation. On the other hand all three accessions ofA. ferruginea and six ofA. geraensis examined gave identical flavonoid profiles. The results support the view thatA. geraensis andA. guaranitica are closely related but do not support the suggested close relationship based on morphology betweenA. oleifera, A. burretiana andA. piassabossu.     

Surface wax, a new taxonomic feature in Plagiochilaceae

A scanning electron microscope study of 81 species of Plagiochilaceae revealed the presence of superficial waxes on the leaves and stems ofPlagiochilion mayebarae and 5 species ofPlagiochila. The waxes are not visible in the light microscope and were unknown in Plagiochilaceae.Plagiochila fuscolutea andP. longiramea (=P. sect.Fuscoluteae) are characterised by the predominant occurrence of membraneous wax platelets;Plagiochila aerea, P. rudischusteri andP. tabinensis(=P. sect.Bursatae) predominately form various types of wax rodlets. Our findings show for the first time the systematic usefulness of leaf surface waxes in the liverworts.P. tabinensis contains surface waxes in amounts of ca. 1.4% dry weight composing of steryl esters, triacylglycerols and free fatty acids.     

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Pollen amino acids—an additional diet for a nectar feeding butterfly?

The increase of the amino acid concentration over different time intervals in artificial nectar (i.e., a sucrose solution) due to pollen contamination was investigated in four Californian plant species (Aesculus californica, Amsinckia lunaris, Brodiaea pulchella, Carduus pycnocephalus), which are important nectar resources for a Californian colony of the butterflyBattus philenor as well as for other insects. The increase of the amino acid concentration in the medium is different in all four species and seems to be determined by a variety of factors including permeability of the pollen grain wall and presence or absence of pores. The results suggest a passive diffusion process of the free pollen amino acids into the medium rather than an active release. Implications from the experiments forBattus philenor and for other nectar feeding pollinators are discussed. A possible complementary effect of free pollen and nectar amino acids is proposed for plant species in which pollen is likely to be knocked into nectar by their flower visitors. A possible evolutionary pathway from nectar feeding butterflies such asBattus philenor to the complex derived pollen feeding habit in theHeliconius butterflies is proposed.     

Taxonomic separation ofKengyilia (Poaceae: Triticeae) in relation to nearest relatedRoegneria,Elymus, andAgropyron,based on some morphological characters

Based on 100 species representative of the four genera, we scored 290 herbarium specimens for a number of morphological characters. The data were subjected to canonical discriminant analysis using characters different from those in the identification key to these genera byBaum, Yen, andYang (1991). These characters collectively support the four groupsAgropyron, Kengyilia, Roegneria, andElymus as previously defined. The four groups are also supported by the linear discriminant function with an overall rate of 83% correct classification. Length of lemma awn was found to be an additional diagnostic character asAgropyron andKengyilia have lemma awns shorter than 5 mm, whereasRoegneria andElymus have longer lemma awns with very few exceptions. Length of glume awns is also a useful supplementary generic diagnostic.Agropyron andElymus have glume awns, whereas the majority of species ofKengyilia and more than half of the species ofRoegneria lack them. If a glume awn is present it is usually not longer than 1 mm.     

The chemotaxonomy ofGeranium (Geraniaceae)

Relationships amongGeranium species constituting sectt.Anemonifolia, Lucida (monotypic) andRuberta together with representatives of sect.Unguiculata were investigated by gas-chromatographic study of essential oils, electrophoretic comparison of seed proteins and chromatographic separation of nectar amino acids. — Essential oil study gave little information.G. macrorrhizum (sect.Unguiculata) had far greater quantities of essential oils in its foliage than other species and differed from them qualitatively. — Species of sectt.Anemonifolia andRuberta, together withG. cataractarum (sect.Unguiculata), between them yielded 19 seed protein bands; the distribution of these indicated close relationship among the species and was consistent with hypotheses for the origin of certain species by alloand autopolyploidy partly within the group. Involvement of an unknown species in the origin of two allopolyploids was implied. Separate origins for the two octoploid species in this set are also inferred. In two instances there was evidence for the transformation of one band into another subsequent to the separation of related species. The inference of allopolyploidy was supported by the occurrence of additivity of parental bands shown by an artificial hybrid between two of the species. A model for the evolutionary divergence of the seed protein patterns is presented. Two species outside the above set appeared less closely related; they wereG. lucidum andG. macrorrhizum (sect.Unguiculata) and between them they showed 6 additional bands, four of which were shared. — Of 18 nectar amino acids found, 4 to 15 occurred in any one species, with low numbers (4 and 8) occurring in the most extreme inbreeders. The spectra of nectar amino acids of two hybrids showed additivity of those of the respective parents. The results echo rather closely those provided by seed proteins, but in the absence of data from outside the group their taxonomic significance is uncertain. However, the divergence between the two octoploids was again evident.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Nectary structure and nectar characteristics in someBignoniaceae

The nectary structure and chemical nectar composition of 15 species belonging to 12 genera ofBignoniaceae are analyzed. All taxa bear a conspicuous nuptial nectary surrounding the ovary base. The secretory tissue is mostly supplied by phloem branches. The stomata are located in the middle and upper part of the nectary epidermis with an homogeneous distribution. The nuptial nectary is proportionally large in relation to the ovary (15–30%), disregarding the nectary volume. Most species have extranuptial nectaries in both inner and outer surfaces of the calyx. Both kinds of nectaries lack a vascular tissue that straightly supplies them. Nuptial nectar concentration (wt/wt) ranges from 19 to 68%. Sugars and amino acids are found in all species. Half of the species have hexose predominant nectars, the remaining sucrose predominant. Phenols are detected in only three species, whereas reducing acids exclusively inTecoma stans. Alkaloids and lipids were never detected. Extranuptial nectar chemical composition is analyzed in two species:Dolichandra cynanchoides andPodranea ricasoliana. Bees constitute the main flower visitors of the species studied whereas hummingbirds were seen visiting three species. A correlation analysis is performed with the data obtained. There are a few significant correlations which indicate a parallel increase of three parameters: the longer the flower length, the more voluminous the nectary and the higher stomata number, independently of the floral biotype. Phenograms are obtained using 24 floral characters including nectary and nectar data. The clusters obtained do not reflect taxonomic relationships but are useful in the understanding of animal-plant interactions when the flower biotype is considered.This paper is based on a chapter of a doctoral thesis presented at the University of Córdoba (Argentina).     

Changes of chemical and nutrient composition of porcine blood during fermentation by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

A solid-state fermentation (SSF) of a mixture of porcine blood and wheat bran with a ratio of 8:1 by Aspergillus oryzae was investigated. Water content, pH, crude protein, heme and total iron, free amino acids (FAA) and total fatty acids (TFA) of the fermented mixture were determined at 0, 40, 80 and 120 h, respectively, and protein hydrolysis were analyzed with SDS-PAGE accordingly. The results showed that, during the fermentation, water contents and pH decreased significantly (P < 0.05) from 68.33% to 59.94%, 7.18% to 4.48%, respectively. Heme iron content changed slightly (P > 0.05). With the degradation of large protein molecules, free amino acids in the mixture increased from 872.83 mg l⁻¹ to 11560.94 mg l⁻¹ (P < 0.05). Content of free isoleucine, methionine and cystine, deficient in fresh porcine blood increased (P < 0.05) after fermentation. Percentages of saturated fatty acids such as C14:0, C17:0 and C18:0 in the total fatty acids decreased significantly (P < 0.05), and those of polyunsaturated fatty acids C18:1, C18:2, C18:3, C20:1 and C20:2 increased significantly from 27.06% to 47.90% (P < 0.05). The results indicated that Aspergillus oryzae could ferment porcine blood and bran mixture and change its chemical and nutrient composition.     

Leaching of zinc from an industrial filter dust withPenicillium,Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

Summary Heterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.