Gene expression patterns to define stages of post-harvest senescence in Alstroemeria petals

Petal senescence in many species is regulated by ethylene but some flowers, such as those on the monocotyledonous plant Alstroemeria, var. Rebecca are ethylene insensitive. Changes in gene expression during the post-harvest senescence of Alstroemeria flowers were investigated using several different techniques. Suppressive subtractive hybridization (SSH) was used to obtain cDNA libraries enriched for genes expressed at selected stages of petal senescence. Sequencing of the EST clones obtained resulted in over 1000 sequences that represent approximately 500 different genes. Analysis of the potential functions of these genes provides a snapshot of the processes that are taking place during petal development. Both cell wall related genes and genes involved in metabolism were present at a higher proportion in the earlier stages. Genes encoding metal binding proteins (mostly metallothionein-like) were the major component of senescence enhanced libraries. This limited the diversity of genes identified showing differential expression at the later stages. Changes in the expression of all genes were analysed using microarray hybridization, and genes showing either up or down-regulation were identified. The expression pattern of a selection of genes was confirmed using Northern hybridization. Northern hybridization confirmed the up-regulation of metallothioneins after floral opening, however, this was not detected by the microarray analysis, indicating the importance of using a combination of methods to investigate gene expression patterns. Considerably more genes were up-regulated than down-regulated. This may reflect the need during Alstroemeria petal senescence for the expression of a whole new set of genes involved with degradation and mobilization. The potential uses of expression profiling to improve floral quality in breeding programmes or as a diagnostic tool are discussed.     

Novel cationic triazine dyes for protein purification

The effectiveness of a new immobilized cationic triazine dye was investigated alongside two new amphoteric triazine dyes and two well known anionic triazine dyes, Procion Red H-3B and Procion Blue H-B, as chromatographic media for binding four familiar proteases-trypsin, chymotrypsin, thrombin and carboxypeptidase-B-as well as a typical oxidoreductase, lactate dehydrogenase, and human serum albumin. The new affinity adsorbent, CL-Sepharose-immobilized Cationic Dye, specifically binds trypsin-like proteases such as trypsin, thrombin, and carboxypeptidase-B, but none of the other proteins tested. In contrast, the amphoteric and anionic immobilized dyes bind all the other proteins tested in a similar fashion. The specificity of the cationic dye was exploited in the resolution of trypsin and chymotrypsin from a crude activated bovine pancreatic extract. The procedure described here affords trypsin with specific activity of 7400 units/mg with a 79% overall yield in a single step. The immobilized cationic dye, unlike previously reported adsorbents for trypsin, is inexpensive, readily synthesized, and displays a workable capacity of 4000 trypsin units or 0.55 mg protein/g moist weight gel (1.2 mumol dye/g moist weight gel) from a crude bovine pancreatic extract and, thus, is potentially amenable to process-scale operations.     

The effect of hydroxycinnamic acids on the growth of wine-spoilage lactic acid bacteria

Hydroxycinnamic acids and their derivatives occur naturally in grape juice and wine. To assess their potential as natural preservatives the effect of caffeic, coumaric and ferulic acids on the growth of three wine-spoilage strains of Lactobacillus collinoides and one of Lact. brevis was studied in acid tomato broth containing 5% ethanol at pH 4.8. At concentrations of 500 and 1000 mg l^-1, all three compounds markedly inhibited growth; coumaric and ferulic acids were more effective than caffeic acid. At a concentration of 100 mg l^-1, all compounds stimulated growth. In general, the strains of Lact. collinoides were more susceptible both to inhibition and stimulation by the hydroxycinnamic acids than was the strain of Lact. brevis. The possible influence of hydroxycinnamic acids on the malolactic fermentation of wine is discussed.     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

The hippocampus as a cognitive graph

A theory of cognitive mapping is developed that depends only on accepted properties of hippocampal function, namely, long-term potentiation, the place cell phenomenon, and the associative or recurrent connections made among CA3 pyramidal cells. It is proposed that the distance between the firing fields of connected pairs of CA3 place cells is encoded as synaptic resistance (reciprocal synaptic strength). The encoding occurs because pairs of cells with coincident or overlapping fields will tend to fire together in time, thereby causing a decrease in synaptic resistance via long-term potentiation; in contrast, cells with widely separated fields will tend never to fire together, causing no change or perhaps (via long-term depression) an increase in synaptic resistance. A network whose connection pattern mimics that of CA3 and whose connection weights are proportional to synaptic resistance can be formally treated as a weighted, directed graph. In such a graph, a "node" is assigned to each CA3 cell and two nodes are connected by a "directed edge" if and only if the two corresponding cells are connected by a synapse. Weighted, directed graphs can be searched for an optimal path between any pair of nodes with standard algorithms. Here, we are interested in finding the path along which the sum of the synaptic resistances from one cell to another is minimal. Since each cell is a place cell, such a path also corresponds to a path in two-dimensional space. Our basic finding is that minimizing the sum of the synaptic resistances along a path in neural space yields the shortest (optimal) path in unobstructed two-dimensional space, so long as the connectivity of the network is great enough. In addition to being able to find geodesics in unobstructed space, the same network enables solutions to the "detour" and "shortcut" problems, in which it is necessary to find an optimal path around a newly introduced barrier and to take a shorter path through a hole opened up in a preexisting barrier, respectively. We argue that the ability to solve such problems qualifies the proposed hippocampal object as a cognitive map. Graph theory thus provides a sort of existence proof demonstrating that the hippocampus contains the necessary information to function as a map, in the sense postulated by others (O'Keefe, J., and L. Nadel. 1978. The Hippocampus as a Cognitive Map. Clarendon Press, Oxford, UK). It is also possible that the cognitive mapping functions of the hippocampus are carried out by parallel graph searching algorithms implemented as neural processes. This possibility has the great attraction that the hippocampus could then operate in much the same way to find paths in general problem space; it would only be necessary for pyramidal cells to exhibit a strong nonpositional firing correlate.     

Development of a toxicity test system using primary rat liver cells

Summary A model in vitro rat liver parenchymal cellular toxicity system employing cells obtained by the in situ collagenase perfusion technique has been developed to detect potential liver toxicants. The initial evaluation of this test system was accomplished using cadmium chloride, chromium chloride, cobalt chloride, mercuric chloride, nickelous chloride, sodium arsenite, sodium selenite, and ammonium vanadate. Linear regression analysis of the dose response curves was used to determine the effective concentration at which the viability was reduced to 50% (EC₅₀). The relative toxicity of the compounds was as follows: Cd>V=As>Se>Hg>Cr=Co>Ni. Since several of the compounds with very similar EC_50S had significantly different dose response slopes, an additional parameter, lowest effective concentration tested (LECT) was employed to assess the relative toxicity. The LECT was determined using the Williams test and the relative toxicity of the compounds was found to be Cd=Se>V>As=Hg>Co>Cr=Ni. The primary objective in developing this rat liver cellular toxicity test system was to employ an in vitro test system utilizing metabolically active primary cells from a potential target organ. This study demonstrates the utility of this test system in determining the relative liver cell toxicity of a series of inorganic agents of differing toxicity.     

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).     

Marine megafauna interactions with small-scale fisheries in the southwestern Indian Ocean: a review of status and challenges for research and management

In developing regions, coastal communities are particularly dependent on small-scale fisheries for food security and income. However, information on the scale and impacts of small-scale fisheries on coastal marine ecosystems are frequently lacking. Large marine vertebrates (marine mammals, sea turtles and chondrichthyans) are often among the first species to experience declines due to fisheries. This paper reviews the interactions between small-scale fisheries and vulnerable marine megafauna in the southwestern Indian Ocean. We highlight an urgent need for proper documentation, monitoring and assessment at the regional level of small-scale fisheries and the megafauna affected by them to inform evidence-based fisheries management. Catch and landings data are generally of poor quality and resolution with compositional data, where available, mostly anecdotal or heavily biased towards easily identifiable species. There is also limited understanding of fisheries effort, most of which relies on metrics unsuitable for proper assessment. Management strategies (where they exist) are often created without strong evidence bases or understanding of the reliance of fishers on resources. Consequently, it is not possible to effectively assess the current status and ensure the sustainability of these species groups; with indications of overexploitation in several areas. To address these issues, a regionally collaborative approach between government and non-governmental organisations, independent researchers and institutions, and small-scale fisheries stakeholders is required. In combination with good governance practices, appropriate and effective, evidence-based management can be formulated to sustain these resources, the marine ecosystems they are intrinsically linked to and the livelihoods of coastal communities that are tied to them.     

Abscission of flowers and floral parts

The abscission of inflorescences, flowers, petals, sepals, styles,and stamens is discussed, with emphasis on the anatomy and ultrastructureof the abscission zones, and the role of cell wall degradingenzymes and hormonal control. Shedding of these parts is usuallydue to cell wall dissolution, but abscission of petals, stamens,and styles in some species occurs due to the forces generatedby the growing fruit. Flower abscission is clearly regulatedby ethylene, whilst auxins apparently decrease the sensitivityto ethylene. Petal, style and stamen abscission also seems tobe controlled by endogenous ethylene. Auxin is apparently involvedin abscission of styles and stamens, but in petals its roleis at yet unclear. The ultrastructural data indicate high proteinsynthesis and high secretory activity of material toward cellwalls of abscission zone cells. The physiological evidence indicatesa role of both polygalacturonase and cellulase in cell walldissolution, whilst the role of other cell wall degrading enzymesis still unknown. The physiological processes occurring in thewalls of the separating cells should be distinguished from thoserelating to defence against microbial intrusion, such as depositionof lignin and suberin and tylose formation. Experimentationusing mutants and transgenic plants may aid in separating theseprocesses. Sequencing of the isoenzymes specific for the abscissionzone and a search for abscission zone-specific promoters seemsa requirement for the successful evaluation of the enzymes involvedin cell wall degradation. Key words: Abscission, anatomy, abscission zone, hormonal control, cell wall degrading enzymes, inflorescences