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Persistent seed banks are a key plant regeneration strategy, buffering environmental variation to allow population and species persistence. Understanding seed bank functioning within herb layer dynamics is therefore important. However, rather than assessing emergence from the seed bank in herb layer gaps, most studies evaluate the seed bank functioning via a greenhouse census. We hypothesise that greenhouse data may not reflect seed bank-driven emergence in disturbance gaps due to methodological differences. Failure in detecting (specialist) species may then introduce methodological bias into the ecological interpretation of seed bank functions using greenhouse data. The persistent seed bank was surveyed in 40 semi-natural grassland plots across a fragmented landscape, quantifying seedling emergence in both the greenhouse and in disturbance gaps. Given the suspected interpretational bias, we tested whether each census uncovers similar seed bank responses to fragmentation. Seed bank characteristics were similar between censuses. Census type affected seed bank composition, with >25% of species retrieved better by either census type, dependent on functional traits including seed longevity, production and size. Habitat specialists emerged more in disturbance gaps than in the greenhouse, while the opposite was true for ruderal species. Both censuses uncovered fragmentation-induced seed bank patterns. Low surface area sampling, larger depth of sampling and germination conditions cause underrepresentation of the habitat-specialised part of the persistent seed bank flora during greenhouse censuses. Methodological bias introduced in the recorded seed bank data may consequently have significant implications for the ecological interpretation of seed bank community functions based on greenhouse data.

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In chronic diseases such as Alzheimer's disease (AD), the arsenal of biomarkers available to determine the effectiveness of symptomatic treatment is very limited. Interpretation of the results provided in literature is cumbersome and it becomes difficult to predict their standardization to a larger patient population. Indeed, cognitive assessment alone does not appear to have sufficient predictive value of drug efficacy in early clinical development of AD treatment. In recent years, research has contributed to the emergence of new tools to assess brain activity relying on innovative technologies of imaging and electrophysiology. However, the relevance of the use of these newer markers in treatment response assessment is waiting for validation. This review shows how the early clinical assessment of symptomatic drugs could benefit from the inclusion of suitable pharmacodynamic markers. This review also emphasizes the importance of re-evaluating a step-by-step strategy in drug development.

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Converging evidence shows that the non-human primate gray mouse lemur (Microcebus murinus) is ideal for the study of the aging process and for testing the effects of new therapies and dietary interventions on age-associated pathologies. One such dietary supplement is resveratrol (RSV), a dietary polyphenolic compound with several positive effects on metabolic functions and longevity. However, little is known about the effect of RSV on the lemur sleep-wake cycle, which reflects mammalian brain function and health. In the present study, the authors investigated this effect by comparing sleep-wake cycles in adult lemurs based on electroencephalographic (EEG) rhythms. The effect of short-term RSV supplementation on the sleep-wake cycle of mouse lemurs was evaluated in entrained conditions (long-day photoperiods, light:dark 14:10). After 3 wks of RSV supplementation, the animals exhibited a significantly increased proportion of active-wake time, occurring mainly during the resting phase of the sleep-wake cycle (+163%). The increase in active-wake time with RSV supplementation was accompanied by a significant reduction of both paradoxical sleep (-95%) and slow-wave sleep (-38%). These changes mainly occurred during the resting phase of the sleep-wake cycle (RSV supplementation induced negligible changes in active-wake time during the active phase of the sleep-wake cycle). The present data suggest that RSV may be a potent regulator of sleep-wake rhythms and could be of major interest in the study of sleep perturbations associated with aging and neuropathology.

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To date, no consensus has been reached concerning the age of the earliest onset of age-related cognitive deficits in rodents. Our aim was to develop a behavioral model allowing early and individual detection of age-related cognitive impairments. We tested young (3 months), middle-aged (10 months) and aged (17 months) C57Bl/6 mice in the starmaze, a task allowing precise analysis of the search pattern of mice via standardized calculation of two navigation indices. We performed mouse-per-mouse analyses and compared each mouse's performance to a threshold based on young mice's performances. Using this method we identified impaired mice from the age of 10 months old. Their deficits were independent of any sensorimotor dysfunctions and were associated with an alteration of the maintenance of the hippocampal CA1 late-LTP. This study develops reliable methodology for early detection of age-related memory disorders and provides evidence that memory can decline in some individuals as early as from the age of 10 months.

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Lyophilised wafers are being developed as topical drug delivery systems for the treatment of chronic wounds. This study describes the formulation of xanthan wafers containing a selective, insoluble MMP-3 inhibitor (UK-370,106) and a non-ionic surfactant, designed to release accurate doses of UK-370,106 directly to a suppurating wound bed. Stability of UK-370,106 in the wafer compared to a non-lyophilised gel suspension was investigated using a combination of light scattering, thermal and microscopic techniques. Particle size distributions in UK-370,106-loaded wafers were constant throughout an accelerated stability study (12 weeks, 40 degrees C) while the mean particle size in a non-lyophilised suspension increased by 15 microm in the same period. Thermal analysis of UK-370,106-loaded wafers highlighted an unexpected interaction between the drug and the surfactant that was further investigated using simple mixtures of each component. It was concluded that an in situ solvate of UK-370,106 and the non-ionic surfactant can form and that this may have implications towards the stability of UK-370,106 during the formulation process. Further concerns regarding high water contents (14%) in the wafer and its effect on product stability were unfounded and it was concluded that these novel delivery systems provided a viable alternative to gel suspensions.

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Lyophilised wafers are being developed as drug delivery systems that can be applied directly to the surface of suppurating wounds. They are produced by the freeze-drying of polymer solutions and gels. This study investigates the possibility of sterilising these glassy, solid dosage forms with gamma-irradiation and determining the rheological properties of rehydrated wafers post-irradiation. One series of wafers was formulated using sodium alginate (SA) modified with increasing amounts of methylcellulose (MC), the other being composed of xanthan gum (XG) and MC. Batches were divided into three lots, two of which were exposed to 25 and 40 kGrays (kGy) of Cobalt-60 gamma-irradiation, respectively, the third being retained as a non-irradiated control. Apparent viscosities of solutions/gels resulting from the volumetric addition of distilled water to individual wafers were determined using continuous shear, flow-rheometry. Flow behaviour on proprietary suppurating surfaces was also determined. Large reductions in viscosity were apparent for irradiated SA samples while those of XG appeared to be largely unaffected. In addition, an increase in the yield stress of xanthan formulations was observed. Xanthan wafers appeared to withstand large doses of irradiation with no detrimental effect on the rheology of reconstituted gels. This offers the possibility of manufacturing sterilisable delivery systems for wounds.

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Lyophilised wafers have potential as drug delivery systems for suppurating wounds. A dual series of wafers made from low molecular weight sodium alginate (SA) and xanthan gum (XG) respectively, modified with high molecular weight methylcellulose (MC) were produced. The swelling and flow properties of these wafers on model suppurating surfaces were both qualitatively and quantitatively investigated. The wafers instantaneously adhered to the surfaces, absorbing water and transforming from glassy, porous solids to highly viscous gels. The rate at which this occurred varied for the series studied with clear distinctions between the behaviour of SA and XG systems. For SA wafers there was a distinct relationship between the flow-rate and MC content. Increased amounts of MC decreased the rate at which the SA wafers flowed across a model gelatine surface. Flow rheometry was used to quantify the effect of increased MC content on both series of wafers and for the SA series, highlighted a substantial increase in apparent viscosity as a function of incremental increases in MC content. These results reflected those from the gelatine model. Observations of the reluctance of a swollen, unmodified XG wafer to flow compared with the relative ease of unmodified, low molecular weight SA was attributed to the yield stress characteristic of xanthan gels. XG is known to exhibit complex, loosely bound network structures in solution via the association of helical backbone structures. The inclusion of sodium fluorescein as a visible model for a soluble drug highlighted the potential of lyophilised wafers as useful drug delivery systems for suppurating wounds.

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PURPOSE: To investigate chemical reactivity in water soluble glasses. METHODS: Rates of bond cleavage reactions in freeze-dried and freeze-concentrated aqueous carbohydrate solutions were measured above and below the glass transition temperatures (Tg). The kinetics of two reactions have been determined in formulations containing di- and polysaccharides: (1) fission of the Asp-Pro peptide bond in Physalaemin and Hamburger peptide by following the release of proline, using a ninhydrin based reaction and (2) the unimolecular dissociation of 2-(4-nitrophenoxy) tetrahydropyran by following the release of the 4-nitrophenoxy anion. RESULTS: The results show clearly that reaction occurs below the glass transition temperature, albeit at very reduced rates. No significant enhancement of the temperature dependence of the rate constant was observed near Tg. Different water soluble glasses provide different degrees of stability. The order of stabilisation was sucrose>Ficoll (low mol. weight)>Byco A approximately equal to Ficoll (high mol. weight)>dextran. The density of the matrix, and therefore the degrees of freedom of mobility of the reactant, is thought to be responsible for these differences. CONCLUSIONS: The storage of therapeutic agents, such as proteins, in glassy matrices below Tg does not confer indefinite stability. When formulating products, notice should be taken of the differing stabilisation properties of excipients.

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Acylpeptide hydrolase is shown to catalyse the specific addition of a single amino acid to the N-terminus of a peptide. The stabilised Sepharose-coupled form of the enzyme is used to couple a carboxy-methylated N-formyl (or N-acetyl) amino acid to a short pre-existing peptide. The yield is improved by optimal timing of the reaction and the presence of moderate concentrations (5%) of N,N-dimethylformamide. Two tripeptides, Ac-Ala-Ala-Ala and fMet-Leu-Phe (f, formyl) were synthesized by this technique (in yields of 2% and 0.064% respectively). The products were characterised by HPLC, amino acid analysis, mass spectroscopy and protein sequencing. The synthetic fMet-Leu-Phe also had biological activity, in that it stimulated superoxide generation by granulocytes. Acylpeptide hydrolase could therefore be a very useful tool for the synthesis and modification of peptides.

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Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.

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The structure and expression of the pea chloroplast atpH gene, encoding ATP synthase CFo subunit III, have been investigated. The atpH gene is situated between the atpI and atpF genes for CFo subunits IV and I, and encodes a hydrophobic polypeptide of 81 amino acid residues which is very similar to subunit III from other species. Analysis of transcripts from the region of chloroplast DNA encoding ATP synthase subunits IV-III-I-alpha shows a complex pattern of transcription, with large transcripts potentially coding for several subunits and also smaller gene-specific transcripts. Two abundant transcripts of 660 nucleotides (nt) and 980 nt specific for atpH were identified. Primer extension and S1 nuclease protection mapping suggested that the 660-nt transcripts were produced by endonucleolytic processing at the sequence, 5'-UGGAAU.

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Amino acid sequence studies show that the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa PAC142 consists of a single polypeptide chain of 346 residues, giving an Mr of 38,400. The evidence from the amino acid studies is in complete agreement with that deduced from the DNA sequence of the amiE gene. Studies of the protein from Pseudomonas putida A87 show that it differs from the Ps. aeruginosa protein by about 30 amino acid substitutions. It now becomes possible to relate changes in the enzyme which result in altered specificity to structural changes in the protein.

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The gene for CF(0) subunit I of ATP synthase has been located in wheat chloroplast DNA, between the genes for CF(0) subunit III and alpha subunit of CF(1). Nucleotide sequencing and analysis of RNA-DNA hybrids indicated that the gene is interrupted by an 823-bp intron which has boundaries similar to those previously described for the introns in protein-coding chloroplast genes of Euglena gracilis. The deduced amino acid sequence of CF(0) subunit I indicates a polypeptide of 183 amino acid residues. However, N-terminal amino acid sequencing of the mature spinach CF(0) subunit I suggests that the protein is synthesised with a N-terminal extension of 17 amino acid residues and is processed to give a protein of mol. wt. 19 001 of 166 amino acids residues. The mature CF(0) subunit I shows similarities in primary and predicted secondary structure to F(0) subunit b of Escherichia coli ATP synthase. A major transcript of 3.3 kb containing sequences from the genes for CF(0) subunit III, subunit I and CF(1) subunit alpha has been observed by RNA-DNA hybridisation.

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Lectins are proteins with multivalent carbohydrate-binding sites, which confer the ability to agglutinate. The seeds of legumes are particularly rich in lectins, for example, concanavalin A (Con A) comprises up to 15% of the protein in the cotyledons of jack bean (Canavalia ensiformis) seeds. The amino acid sequences of Con A and several other legume lectins have been partially or fully determined, and comparison of these sequences from different species reveals a circular homology (Fig. 1A); rearrangements within the genome have been suggested to explain this. We report here that the circular homology displayed by Con A is due to a post-translational transposition and ligation within the initial polypeptide. This type of modification has not been reported previously for eukaryotes, although it has been suggested to occur in bacteriophage lambda.

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A transmembrane arrangement of cytochrome f in chloroplast thylakoid membranes, with the N-terminal heme-containing region in the intrathylakoid space and a 15 amino acid C-terminal sequence in the stroma, is suggested by the amino acid sequence deduced from the nucleotide sequence of the pea chloroplast gene. This topology has been confirmed by partial proteolysis of the polypeptide in intact and disrupted thylakoid membranes and in inside-out and right-side-out vesicles of chloroplast membranes.

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The proton-translocating subunit of wheat chloroplast ATP synthase is encoded by a chloroplast gene that has been accurately mapped and whose nucleotide sequence has been determined. The predicted sequence of 81 amino acids has been confirmed in part by determination of the sequence of the first 40 amino acids from the NH(2) terminus of the protein, and it shows 100% homology with the known amino acid sequence of the spinach protein but no more than 35% homology with the amino acid sequences of bacterial and mitochondrial proteins. The gene shows no deviation from the "universal" genetic code and is not split. A potential ribosome binding site is located 12 nucleotides upstream from the initiation codon, but sequences homologous to prokaryotic promotors and transcription terminators are not apparent.

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An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.

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A restriction enzyme map was constructed for 5.1-kb fragment of Pseudomonas aeruginosa DNA inserted into plasmid pBR322. Restriction enzyme sites were matched to the N-terminal amino acid sequence of amidase to obtain alignment of the amiE gene within the cloned fragment.

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A partial sequence of the manganese-containing superoxide dismutase from Bacillus stearothermophilus has been determined by mass spectrometry. A new fragmentation reaction at N-terminal proline is described and an 'in-chain' fragmentation, also occurring at proline, has been observed. There is some evidence that the latter cleavage may also occur at other residues. Some differences from the classically determined sequence are reported, and the validity of these observations is discussed.

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