Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Focused antithrombotic therapy: Novel anti-platelet salicylates with reduced ulcerogenic potential and higher first-pass detoxification than aspirin in rats

The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicyclic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane Ag production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.

Factors affecting biosynthesis by Xanthomonas albilineans of albicidin antibiotics and phytotoxins

Albicidins are important factors in systemic pathogenesis by Xanthomonas albilineans, which causes the devastating leaf scald disease of sugar cane. They ale also of substantial interest as antibiotics that selectively block prokaryote DNA replication. Albicidin biosynthesis is highly sensitive to medium composition. An optimized, chemically defined medium (SMG3) yielded 30-fold more albicidin from half the accumulated biomass, relative to sucrose peptone (SP) medium. Phosphate starvation stimulated albicidin production in SMG3 and SP media. Addition of other amino acids, ammonium ions or peptones to the defined medium increased the growth rate of X albilineans XA3, but differentially inhibited albicidin biosynthesis. Knowledge of these factors indicates new approaches to understanding mechanisms of pathogenesis and resistance to sugar cane leaf scald disease, and to strain improvement for production of albicidin antibiotics.

Nuapapuin A and sigmosceptrellins D and E: New norterpene cyclic peroxides from a southern Australian marine sponge, Sigmosceptrella sp.

A Sigmosceptrella sp. from the Great Australian Eight, Australia, has yielded the new norditerpene cyclic peroxide, nuapapuin A (2a), and the norsesterterpene cyclic peroxide sigmosceptrellin D (3a), characterized as the corresponding methyl esters 2b and 3b. The crude methylated sponge extract also yielded the new norsesterterpene cyclic peroxide sigmosceptrellin E methyl ester (4). Relative stereochemistry about C2, C3, and C6 was assigned by established empirical rules and absolute stereochemistry by the advanced Mosher procedure. A plausible biosynthetic pathway has been proposed that rationalizes key transformations in the biosynthesis of all known norterpene cyclic peroxides and related norterpene ketones, dienes and sigmosceptrins.

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide

An increased degree of utilization of the potential N-glycosylation site In the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with, the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

First record in Australia of Myriogenospora atramentosa on lemongrass and sugarcane

Myriogenospora atramentosa has been found on lemongrass (Cymbopogon citratus) and sugarcane (Saccharum interspecific hybrids) in Queensland. These are the first records of this fungus outside of the Americas.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Use of an Arrhenius model to predict rheological behaviour in some Australian honeys

Rheological properties of nine unprocessed unifloral Australian honeys (bloodwood, blue top iron bark, gum top, heath, narrow leafed iron bark, stringy bark, tea tree yapunyah and yellow box) were analysed over a range of temperatures (1-40 degreesC) The temperature effect on the viscosity follow ed an Arrhenius-type relationship and ail honey varieties exhibited Newtonian behaviour. if the Arrhenius equation constants (mu (0) and E-a) for a particular honey are known, the Arrhenius model can be used to calculate the viscosity of these honeys at specific temperatures, negating the need for tedious viscosity determination. (C) 2000 Academic Press.

A multi-sectorial committee in directing HIV/AIDS-specific interventions in the occupational setting: An example from South Africa

We present a descriptive analysis of a mechanism to coordinate and implement human immunodeficiency virus (HIV) prevention and care in the occupational setting. The mechanism we describe is a multidisciplinary committee composed of stakeholders in the occupational health environment including unions, management, medical researchers, and medical personnel. The site chosen for the analysis was a South African sugar mill in rural KwaZulu-Natal. The factory is situated in an area of high HIV seroprevalence and has a workforce of 400 employees. The committee was initiated to coordinate a combined prevention-care initiative. The issues that were important in the formation of the committee included confidentiality, trust, and the traditional roles of the stakeholder relationships. When these points were addressed through the focus on a common goal, the committee was able to function in its role as a coordinating body. Central to this success was the inclusion of all stakeholders in the process, including those with traditionally opposing, interests and legitimacy conferred by the stakeholders. This committee was functionally effective and demonstrated the benefit of a freestanding committee dedicated to addressing HIV/acquired immune deficiency syndrome (AIDS) issues. We describe the implementation and feasibility of a multisectoral committee in directing HIV/AIDS initiatives in the occupational setting in rural South Africa.

Th structure of the P-II-ATP complex

P-II is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of P-II was solved using a hexagonal crystal form (form I). The more physiologically relevant form of P-II is a complex with small molecule effecters. We describe the structure of P-II with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of P-II with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the P-II/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that P-II and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.

Equilibrating isomers: Bromoindoles and a seco-xanthine encountered during a study of nematocides from the southern Australian marine sponge Hymeniacidon sp.

Bioassay-directed fractionation of a Hymeniacidon sp. yielded as nematocidal agents the equilibrating E/Z bromoindole ethyl esters 1 and 2 and corresponding methyl esters 3 and 4. Also isolated for the first time as a natural product was an equilibrating mixture of seco-xanthine formamides, attributed the trivial name hymeniacidin (5). The structure for 5 was assigned on the basis of detailed spectroscopic analysis and total synthesis.

Incorporation of bovine dry blood plasma into biscuit flour for the production of pasta

Spray-dried blood plasma (DBP) (10.9 g/100 g [w/w] nitrogen) was added to medium-protein biscuit flour (1.4 g/100 g N) during pasta manufacture. High-protein durum semolina (2.0 g/100 g N) Was used to produce the control pasta. Sensory data indicated that the addition of DBP produced pasta with significantly better colour intensity and acceptability. aroma intensity, flaN our intensity. textural strength, texture acceptability, aftertaste intensity, aftertaste acceptability. and overall acceptability The DBP/biscuit flour formulation that gave the optimum balance between pasta protein content and organoleptic acceptability contained 2.2 g/100 g DBP. A higher content of DBP resulted in increased protein levels, but these pasta formulations, ere less acceptable organoleptically. (C) 2002 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.

Effect of storage of adzuki bean (Vigna angularis) on starch and protein properties

Differential scanning calorimetry was used to evaluate the effect of storage at 10degreesC, 20degreesC and 30degreesC, and 40% and 65% relative humidity (RH) on adzuki bean starch gelatinisation and protein denaturation temperatures. Storage for 6 months at an elevated storage temperature (30degreesC) caused increases in the starch gelatinisation onset temperature (T-o) and gelatinisation peak temperature (T-p) for both Bloodwood and Erimo varieties. Storage at 40% RH resulted in higher T-o and T-p values than storage at 65% RH. The T-o of starch from Bloodwood and Erimo beans stored for up to 1.5 months at 10degreesC and 65% were similar to those of fresh beans. The changes in the salt-soluble protein component were less clear cut than those of the starch. Nonetheless, protein extracted from beans stored at 40% RH exhibited significantly lower T-o and T-p values compared with those stored at 65% RH. This indicates some destabilisation of the protein at the higher RH. These results suggest that detrimental changes occur in starch and, to a lesser extent protein, of adzuki beans stored under unfavourable conditions. On the basis of these results, the best storage conditions to maintain the characteristics of fresh beans are low temperatures (e.g. 10degreesC) and high RH (e.g. 65%). (C) 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.