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.     

Biochemical analysis of the allergenic pollen ofMadhuca indica Gmel. andSpathodea campanulata Beauv

Summary The present paper deals with the biochemical analysis in the form of quantitative estimation of carbohydrates, proteins, nucleic acids, minerals, ash and moisture of the pollen of two allergenic plants, i.e.Manhuca indica andSpathodea campanulata. Carbohydrates constitute the major dry matter fractions in both the pollen types with highest amount inM. indica (25.37%). Soluble proteins were estimated along with lipid content.S. campanulata showed 12.74% protein andM. indica 11.58%. The estimation of nucleic acids revealed the presence of DNA from 0.36 to 0.72% and RNA from 0.28 to 1.03%. Minerals were found in very low amounts ranging from 0.14 to 0.19% for Calcium and 0.15 to 0.20% for Magnesium. The ash and moisture content were also found variable. The present findings revealed that these chemical constituents may be responsible for the control of the rate and type of allergic reaction caused by the pollen of these two plants.     

Distribution of isoasparagine among different characean species

Thirteen species of Characeae were analyzed for their free amino acid contents. Large amounts of isoasparagine, accounting for 10 to 50% of the total free amino acids, were found in extracts fromChara (5 species including one unidentified),Nitellopsis (1 species), andLamprothamnium (1 species). In contrast, no isoasparagine was detected inNitella (5 species) andTolypella (1 species), except forN. flexilis in which as much as 40% of the free amino acids was isoasparagine. Other major amino acids found in the tested materials were Ala, Asp, Glu and Gln.     

Life-history parameters of western flower thrips on susceptible and resistant cucumber genotypes

The life-history parameters reproduction rate, developmental time and age specific survival of the western flower thrips,Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], were determined on susceptible and resistant cucumber (Cucumis sativus L.) genotypes. Both newly emerged andF. occidentalis females of mixed ages showed a substantial reduction (36 to 50%) of the reproduction rate on all resistant genotypes, in particular after the second day. On the resistant genotypes 9127 and 9140,F. occidentalis had a prolonged developmental period. This was primarily due to a prolongation of the second larval stage. On all resistant genotypes,F. occidentalis suffered from high (82 to 97%) preadult mortality, predominantly at the second larval stage. It is conclude that the resistant genotypes do not cause an immediate intoxication of adult nor preadult thrips stages.     

A new species ofLiparis (Orchidaceae) from India

Liparis indiraii spec. nova from India is close toL. alata A. Rich. andL. atropurpurea Lindl.     

Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii

Analysis of total aromatic amino acid (free and bound) in some cucumber accessions selected previously for resistance to western flower thrips, Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], indicated that low concentrations of these essential nutrients, relative to total leaf protein, were correlated with a reduction in damage by the insect. Further analysis of samples of four important horticultural crops (lettuce, tomato, pepper and cucumber) with unknown levels of resistance to thrips showed a significant genotypic variation in the concentrations of total aromatic amino acids relative to the total leaf protein. Accessions from each crop with low or high concentrations of aromatic amino acids in proteins were exposed to thrips larvae. Regression analysis showed a highly significant positive correlation between aromatic amino acid concentration in leaf protein and thrips damage, regardless of crop species. It is concluded that higher concentrations of aromatic amino acids in plant proteins are important for successful thrips development. These results provide plant breeders with a promising tool for indirect selection without using undesirable insect bioassays.     

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.     

Biological control of thrips [Thysanoptera: Thripidae] on greenhouse cucumber byAmblyseius cucumeris

The predatory miteAmblyseius cucumeris (Oudemans) [Acarina: Phytoseiidae] was evaluated as a biological control forThrips tabaci Lindeman andFrankliniella occidentalis (Pergande) [Thysanoptera: Thripidae] on greenhouse grown seedless cucumber.A. cucumeris spread throughout the greenhouses and provided control of both species of thrips.A. cucumeris adults persisted on plants for 7 weeks in the virtual absence of prey, and increased numerically in response to increases in prey population. On the basis of these resultsA. cucumeris is recommended as a useful biological control forT. tabaci andF. occidentalis on greenhouse cucumber. Publication No. 361, Agassiz Research Station, Agriculture Canada, Agassiz, B.C. Canada VOM 1AO.     

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.     

Assessment of the nutritional value of Capsosiphon fulvescens (Chlorophyta): developing a new species of marine macroalgae for cultivation in Korea

The nutritive value of Capsosiphon fulvescens (C. Agardh) Setchell et Gardner, a new developing species for cultivation of marine macroalgae in Korea, was assessed by comparison with common edible green seaweed Ulva prolifera (Oeder) J. Agardh collected from Korea and Japan, based on analysis of its chemical composition. The contents of protein and of total, essential, and free amino acids of C. fulvescens were significantly higher than those of U. prolifera, whereas those of lipid, carbohydrate, and total dietary fibres were significantly lower. There were no significant differences in the moisture content between Capsosiphon and Ulva species. The main minerals of Capsosiphon and Ulva are Na, Mg, K, Ca, and Al, making up approximately 97–99 % of the total minerals. The contents of Na and V of Capsosiphon were significantly greater than those of Ulva, while those of Mn and Sr were significantly lower in Capsosiphon. The contents of retinal (27.8 μg g⁻¹ dry wt) and ascorbic acid (0.28 mg g⁻¹ dry wt) of Capsosiphon were significantly higher than those of Ulva, but the contents of cobalamin were lower. These results suggest that C. fulvescens has greater potential to be used as human food and as an ingredient in formulated food.     

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.     

Feeding by emerald ash borer larvae induces systemic changes in black ash foliar chemistry

The exotic wood-boring pest, emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), has been threatening North American ash (Fraxinus spp.) resources, this being recognized since its first detection in Michigan, USA and Ontario, Canada in 2002. Ash trees are killed by larval feeding in the cambial region, which results in disruption of photosynthate and nutrient translocation. In this study, changes in volatile and non-volatile foliar phytochemicals of potted 2-yr-old black ash, Fraxinus nigra Marshall, seedlings were observed in response to EAB larval feeding in the main stem. EAB larval feeding affected levels of six compounds [hexanal, (E)-2-hexenal, (Z)-3-hexenyl acetate, (E)-β-ocimene, methyl salicylate, and (Z,E)-α-farnesene] with patterns of interaction depending upon compounds of interest and time of observation. Increased methyl salicylate emission suggests similarity in responses induced by EAB larval feeding and other phloem-feeding herbivores. Overall, EAB larval feeding suppressed (Z)-3-hexenyl acetate emission, elevated (E)-β-ocimene emission in the first 30 days, but emissions leveled off thereafter, and generally increased the emission of (Z,E)-α-farnesene. Levels of carbohydrates and phenolics increased overall, while levels of proteins and most amino acids decreased in response to larval feeding. Twenty-three amino acids were consistently detected in the foliage of black ash. The three most abundant amino acids were aspartic acid, glutamic acid, glutamine, while the four least abundant were α-aminobutyric acid, β-aminoisobutyric acid, methionine, and sarcosine. Most (16) foliar free amino acids and 6 of the 9 detected essential amino acids decreased with EAB larval feeding. The ecological consequences of these dynamic phytochemical changes on herbivores harbored by ash trees and potential natural enemies of these herbivores are discussed.     

Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of thegad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.     

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.     

Seasonal patterns of nucleic acid concentrations and amino acid profiles of Parapenaeus longirostris (Crustacea, Decapoda): relation to growth and nutritional condition

The present work describes the seasonal changes in nucleic acid concentrations and amino acid profiles in the muscle of juvenile Parapenaeus longirostris and their relation to growth and nutritional condition. RNA content varied significantly between seasons, being the highest values attained in spring and the lowest in winter (p < 0.05). Similar results were obtained with RNA:protein and RNA:DNA ratios. In respect to total amino acid content (TAA), a significant increase from winter to spring was observed (p < 0.05) and the major essential amino acids (EAA) were arginine, histidine and leucine. Within non-essential amino acids (NEAA) glutamic acid, aspartic acid, glycine and proline were dominant. From winter to spring, a significant variation in NEAA content occurred (26.8; p < 0.05), mainly due to the significant increase of glutamic acid (79.1) and serine (66.7) (p < 0.05). EAA content did not vary significantly between seasons (p > 0.05). In opposition, during this period a significant decrease in the free amino acid content (FAA) was observed (p < 0.05); a higher percentage of decrease was attained in free non-essential (FNEAA – 42.9) in comparison to free essential amino acids (FEAA – 40.2). The significant increase in RNA and TAA contents from winter to spring may be related with protein synthesis. On the other hand, the lowest values obtained in winter may be due to a reduction in feeding activity; in this period the muscle protein must be progressively hydrolysed, which is evident with the higher FAA content. The liberated amino acids enter FAA pool and become available for energy production. In conclusion, it was evident that the seasonal cycle in activities such as feeding and growth with nucleic acids and amino acid analyses was noticed.     

Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.     

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.     

A seasonal study of the red seaweeds Solieria tenera and three species of Gracilaria from Jamaica

Three species of Gracilaria viz G. cervicornis, G. domingensis, G. verrucosa and Solieria tenera (Gigartinales, Rhodophyta) have been studied for their monthly variation in dry wt yields, ash, soluble carbohydrate, protein, lipid and insoluble carbohydrate contents for one year. The dry wt yields were higher in G. domingensis. Soluble carbohydrate and ash contents showed an inverse relationship in all the species. Protein content was lower, below 5% of the dry wt for all the species. A comparison of protein:carbohydrate ratios showed a similarity between morphologically similar G. domingensis and G. cervicornis in that there was less variation in the ratio. From this, it is assumed that flattened morphology probably is more efficient in maintaining nutrient balance to keep up the growth rates.     

Morphological and molecular characterization of a natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae)

A putative natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae) from southern Italy, formerly known asO. alata, was characterized both on morphological and molecular grounds in order to confirm its hybrid status and to trace its maternal lineage. The morphological characters of the putative hybrid showed intermediacy between those of the parent species, and restriction fragment length polymorphism (RFLP) analysis of a region of the nuclear ribosomal DNA confirmed its hybrid origin. Chloroplast DNA RFLP analysis indicated thatO. morio provided the maternal genome.     

RAPD characterisation of two neotropical hybrid legumes

An investigation of randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker distribution was made for two well-characterised hybrids and their parents,Leucaena leucocephala andL. esculenta andParkinsonia aculeata andCercidium praecox. Three chloroplast DNA (cpDNA) markers identified the maternal parent of eachL. leucocephala ×L. esculenta hybrid. Fifteen species-diagnostic RAPD markers (invariant in one taxon and absent from the other) were always present in theLeucaena hybrid and assumed to be of nuclear origin, whilst three RAPD markers showed expression patterns identical to the cpDNA markers and were assumed to be of organellar origin. No RAPD or PCR-RFLP taxon-diagnostic markers were discovered for eitherP. aculeata orC. praecox. However, 21 RAPD markers were species-specific (polymorphic within one taxon but absent from the other) and Southern analysis indicated that none of the markers were of organellar origin. Only 67% additivity of markers specific toP. aculeata andC. praecox was demonstrated in the hybrids between these two species, whilst inLeucaena 97% additivity was demonstrated. Differences between the two hybridising situations were related to the behaviour of the molecular markers and the biology of the species.