INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH-CELL-DENSITY DIALYSIS CULTURE1

The consumption of inorganic macronutrients (NO₃^?+ NO₂^?, NH₄⁺, and PO₄^?3) and the composition of intra- and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous-reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 10⁷ cells · mL^?1 was obtained in stationary phase, which coincided with the NO₃^?+ NO₂^? diffusion limit (Nd_max) of the dialysis apparatus. The Nd_max for NH₄⁺ occurred much earlier, at the end of exponential growth, whereas Nd_max for PO₄^?3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β-GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N-conversion coefficient (Y_N). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.     

Effects of Removal of Successful Males in a Fallow Deer Lek

Removal of 5 of the 8 most successful males in a fallow deer lek between breeding seasons led to an increase in fighting rate of males and a decrease in mating rate of both sexes. The increase in fighting was likely due to disruption of the social hierarchy. The lower mating rate of females was not due to disruption of copulations by territorial males, nor did it appear to result from disturbance from fights. We suggest that difficulties in mate choice related to an unstable male hierarchy resulted in females either increasing their length of stay in the lek, visiting the lek more than once before mating, or leaving the lek without mating.     

Locomotor Activity in the Freshwater Crab Potamon fluviatile: The Analysis of Temporal Patterns by Radio-telemetry

Locomotor activity in a field population of the freshwater crab, Potamon fluviatile, was studied during the breeding season by means of radio-telemetry and by direct counts of active animals along a transect of the stream. The basic pattern of crabs' locomotor activity can be described as a sequence of short distance movements around the shelters (foraging movements), followed by rarer long distance displacements (wandering movements). Whilst direct counting showed that the crabs exhibit a broadly nocturnal rhythm in foraging movements, no daily periodicity in wandering activity was revealed by telemetry. There is sexual difference in these latter excursions: females move farther along the stream and into the surrounding terrestrial habitat than do males. This behaviour is in contrast to observations made during the non-breeding season and is probably related to the stage of female reproduction.     

Impact of cell disruption and polymer recycling upon aqueous two-phase processes for protein recovery

A practical study is presented of the influence of cell debris and polymer recycling upon the operation of two-stage acqueous two-phase systems (ATPS) for the recovery of yeast bulk protein, pyruvate kinase and fumarase. Brewers' yeast was disrupted using one of two types of high-pressure homogenisers or a bead mill. The different cell debris suspensions were partitioned in a single PEG-phosphate ATPS extraction and the efficiency of solid-liquid separation was examined. A continuously operated two-stage ATPS process, using spray columns, is presented and practical problems of polymer recycling are discussed. Conclusions are drawn concerning the generic implementation and operational stability of ATPS in practical protein recoveries.     

Facile reduction of peptide oxime endothelin antagonist during trialkylsilane/TFA cleavage after solid-phase synthesis

Summary We describe a new solid-phase strategy for the selective reduction of the C=N bond in peptide oximes using a trialkylsilane in trifluoroacetic acid. The reduction is performed directly on the resin-bound peptide, with concomitant cleavage of the peptide from the resin and deblocking of protected side chains.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

Sex differentiation in the European eel: histological analysis of the effects of sex steroids on the gonad

The effects of sex steroids on sex differentiation in the European eel were studied. The steroids, 17α-methyltestosterone (MT) and 17α-ethynylestradiol (EE), were given in the diet to 6–8 cm elvers and to 15–18 cm and 22–25 cm yellow eels. In our rearing conditions a very large percentage of the untreated eels developed as males. No masculinizing effect of MT could be demonstrated. The EE, administered at a dose of 10 mg kg^-1 of diet to 6–8 cm elvers and 15–18 cm eels, induced ovarian differentiation in about 90 and 65% of eels respectively, while in the control <5% of females was recorded. In 22–25 cm yellow eels a moderated feminizing effect was observed.
Histological analysis of the gonads of treated eels showed that sex steroids affect the gonadal structure. The androgen stimulates hypertrophy of compact connective tissue, early differentiation of Leydig cells, Sertoli cells and early formation of the spermatic duct. Oestrogen inhibits the differentiation of these structural components and stimulates the differentiation of follicular cells and an ovarian structure.
The involvement of gonadal structural components is discussed in relation to the effect of steroid treatment and to the peculiarities of sex differentiation in the European eel.