Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Identification of putative regulatory motifs in the upstream regions of co-expressed functional groups of genes in Plasmodium falciparum

Background: Regulation of gene expression in Plasmodium falciparum (Pf) remains poorly understood. While over half the genes are estimated to be regulated at the transcriptional level, few regulatory motifs and transcription regulators have been found. Results: The study seeks to identify putative regulatory motifs in the upstream regions of 13 functional groups of genes expressed in the intraerythrocytic developmental cycle of Pf. Three motif-discovery programs were used for the purpose, and motifs were searched for only on the gene coding strand. Four motifs – the 'G-rich', the 'C-rich', the 'TGTG' and the 'CACA' motifs – were identified, and zero to all four of these occur in the 13 sets of upstream regions. The 'CACA motif' was absent in functional groups expressed during the ring to early trophozoite transition. For functional groups expressed in each transition, the motifs tended to be similar. Upstream motifs in some functional groups showed 'positional conservation' by occurring at similar positions relative to the translational start site (TLS); this increases their significance as regulatory motifs. In the ribonucleotide synthesis, mitochondrial, proteasome and organellar translation machinery genes, G-rich, C-rich, CACA and TGTG motifs, respectively, occur with striking positional conservation. In the organellar translation machinery group, G-rich motifs occur close to the TLS. The same motifs were sometimes identified for multiple functional groups; differences in location and abundance of the motifs appear to ensure different modes of action. Conclusion: The identification of positionally conserved over-represented upstream motifs throws light on putative regulatory elements for transcription in Pf.

An algebraic theory of functional and multivalued dependencies in relational databases

Computation of the dependency basis is the fundamental step in solving the membership problem for functional dependencies (FDs) and multivalued dependencies (MVDs) in relational database theory. We examine this problem from an algebraic perspective. We introduce the notion of the inference basis of a set M of MVDs and show that it contains the maximum information about the logical consequences of M. We propose the notion of a dependency-lattice and develop an algebraic characterization of inference basis using simple notions from lattice theory. We also establish several interesting properties of dependency-lattices related to the implication problem. Founded on our characterization, we synthesize efficient algorithms for (a): computing the inference basis of a given set M of MVDs; (b): computing the dependency basis of a given attribute set w.r.t. M; and (c): solving the membership problem for MVDs. We also show that our results naturally extend to incorporate FDs also in a way that enables the solution of the membership problem for both FDs and MVDs put together. We finally show that our algorithms are more efficient than existing ones, when used to solve what we term the ‘generalized membership problem’.

The Implication Problem for Functional and Multivalued Dependencies

Computation of the dependency basis is the fundamental step in solving the implication problem for MVDs in relational database theory. We examine this problem from an algebraic perspective. We introduce the notion of the inference basis of a set M of MVDs and show that it contains the maximum information about the logical consequences of M. We propose the notion of an MVD-lattice and develop an algebraic characterization of the inference basis using simple notions from lattice theory. We also establish several properties of MVD-lattices related to the implication problem. Founded on our characterization, we synthesize efficient algorithms for (a) computing the inference basis of a given set M of MVDs; (b) computing the dependency basis of a given attribute set w.r.t. M; and (c) solving the implication problem for MVDs. Finally, we show that our results naturally extend to incorporate FDs also in a way that enables the solution of the implication problem for both FDs and MVDs put together.

Exploring methods of preparing functional cartilage-bone xenografts for joint repair

This thesis explores the feasibility of donor-receiver concept for joint replacement where cartilage-bone tissues can be taken from either human or other mammals and prepared scientifically for repairing focal joint defects in knees, hips and shoulders. The manufactured construct is immunologically inert and is capable of acting as a scaffold for engineering new cartilage-bone laminates when placed in the joint. Innovative manufacturing procedures and assessment techniques were developed for appraising this tissue-based scaffold. This research has demonstrated that tissue replacement technology can be applied in situations where blood vessels are absent such as in articular cartilage.

Representation of functional dependencies in relational databases using linear graphs

Functional dependencies in relational databases are investigated. Eight binary relations, viz., (1) dependency relation, (2) equipotence relation, (3) dissidence relation, (4) completion relation, and dual relations of each of them are described. Any one of these eight relations can be used to represent the functional dependencies in a database. Results from linear graph theory are found helpful in obtaining these representations. The dependency relation directly gives the functional dependencies. The equipotence relation specifies the dependencies in terms of attribute sets which functionally determine each other. The dissidence relation specifies the dependencies in terms of saturated sets in a very indirect way. Completion relation represents the functional dependencies as a function, the range of which turns out to be a lattice. Depletion relation which is the dual of the completion relation can also represent functional dependencies and similarly can the duals of dependency, equipotence, and dissidence relations. The class of depleted sets, which is the dual of saturated sets, is defined and used in the study of depletion relations.

The process for implementation of a Quality Management System within a multi-functional cereal laboratory

The intent of this study was to design, document and implement a Quality Management System (QMS) into a laboratory that incorporated both research and development (R&D) and routine analytical activities. In addition, it was necessary for the QMS to be easily and efficiently maintained to: (a) provide documented evidence that would validate the system's compliance with a certifiable standard, (b) fit the purpose of the laboratory, (c) accommodate prevailing government policies and standards, and (d) promote positive outcomes for the laboratory through documentation and verification of the procedures and methodologies implemented. Initially, a matrix was developed that documented the standards' requirements and the necessary steps to be made to meet those requirements. The matrix provided a check mechanism on the progression of the system's development. In addition, it was later utilised in the Quality Manual as a reference tool for the location of full procedures documented elsewhere in the system. The necessary documentation to build and monitor the system consisted of a series of manuals along with forms that provided auditable evidence of the workings of the QMS. Quality Management (QM), in one form or another, has been in existence since the early 1900's. However, the question still remains: is it a good thing or just a bugbear? Many of the older style systems failed because they were designed by non-users, fiercely regulatory, restrictive and generally deemed to be an imposition. It is now considered important to foster a sense of ownership of the system by the people who use the system. The system's design must be tailored to best fit the purpose of the operations of the facility if maximum benefits to the organisation are to be gained.

A computational algorithm for the verification of tautologies in propositional calculus

A computational algorithm (based on Smullyan's analytic tableau method) that varifies whether a given well-formed formula in propositional calculus is a tautology or not has been implemented on a DEC system 10. The stepwise refinement approch of program development used for this implementation forms the subject matter of this paper. The top-down design has resulted in a modular and reliable program package. This computational algoritlhm compares favourably with the algorithm based on the well-known resolution principle used in theorem provers.

Structural and functional characterization of dendritic arbors and GABAergic synaptic inputs on interneurons and principal cells in the rat basolateral amygdala

The basolateral amygdala (BLA) is a complex brain region associated with processing emotional states, such as fear, anxiety, and stress. Some aspects of these emotional states are driven by the network activity of synaptic connections, derived from both local circuitry and projections to the BLA from other regions. Although the synaptic physiology and general morphological characteristics are known for many individual cell types within the BLA, the combination of morphological, electrophysiological, and distribution of neurochemical GABAergic synapses in a three-dimensional neuronal arbor has not been reported for single neurons from this region. The aim of this study was to assess differences in morphological characteristics of BLA principal cells and interneurons, quantify the distribution of GABAergic neurochemical synapses within the entire neuronal arbor of each cell type, and determine whether GABAergic synaptic density correlates with electrophysiological recordings of inhibitory postsynaptic currents. We show that BLA principal neurons form complex dendritic arborizations, with proximal dendrites having fewer spines but higher densities of neurochemical GABAergic synapses compared with distal dendrites. Furthermore, we found that BLA interneurons exhibited reduced dendritic arbor lengths and spine densities but had significantly higher densities of putative GABAergic synapses compared with principal cells, which was correlated with an increased frequency of spontaneous inhibitory postsynaptic currents. The quantification of GABAergic connectivity, in combination with morphological and electrophysiological measurements of the BLA cell types, is the first step toward a greater understanding of how fear and stress lead to changes in morphology, local connectivity, and/or synaptic reorganization of the BLA.

Representation of functional dependencies in relational databases using linear graphs

Functional dependencies in relational databases are investigated. Eight binary relations, viz., (1) dependency relation, (2) equipotence relation, (3) dissidence relation, (4) completion relation, and dual relations of each of them are described. Any one of these eight relations can be used to represent the functional dependencies in a database. Results from linear graph theory are found helpful in obtaining these representations. The dependency relation directly gives the functional dependencies. The equipotence relation specifies the dependencies in terms of attribute sets which functionally determine each other. The dissidence relation specifies the dependencies in terms of saturated sets in a very indirect way. Completion relation represents the functional dependencies as a function, the range of which turns out to be a lattice. Depletion relation which is the dual of the completion relation can also represent functional dependencies and similarly can the duals of dependency, equipotence, and dissidence relations. The class of depleted sets, which is the dual of saturated sets, is defined and used in the study of depletion relations.

Functional dynamics of the nitrogen balance of sorghum. II. Grain filling period.

Maintenance of green leaf area during grain filling can increase grain yield of sorghum grown under terminal water limitation. This 'stay-green' trait has been related to the nitrogen (N) supply-demand balance during grain filling. This study quantifies the N demand of grain and N translocation rates from leaves and stem and explores effects of genotype and N stress on onset and rate of leaf senescence during the grain filling period. Three hybrids differing in potential height were grown at three levels of N supply under well-watered conditions. Vertical profiles of biomass, leaf area, and N% of leaves, stem and grain were measured at regular intervals. Weekly SPAD chlorophyll readings on main shoot leaves were correlated with observed specific leaf nitrogen (SLN) to derive seasonal patterns of leaf N content. For all hybrids, individual grain N demand was sink determined and was initially met through N translocation from the stem and rachis. Only if this was insufficient did leaf N translocation occur. Maximum N translocation rates from leaves and stem were dependent on their N status. However, the supply of N at canopy scale was also related to the amount of leaf area senescing at any one time. This supply-demand framework for N dynamics explained effects of N stress and genotype on the onset and rate of leaf senescence.

Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts.

Stay-green, an important trait for grain yield of sorghum grown under water limitation, has been associated with a high leaf nitrogen content at the start of grain filling. This study quantifies the N demand of leaves and stems and explores effects of N stress on the N balance of vegetative plant parts of three sorghum hybrids differing in potential crop height. The hybrids were grown under well-watered conditions at three levels of N supply. Vertical profiles of biomass and N% of leaves and stems, together with leaf size and number, and specific leaf nitrogen (SLN), were measured at regular intervals. The hybrids had similar minimum but different critical and maximum SLN, associated with differences in leaf size and N partitioning, the latter associated with differences in plant height. N demand of expanding new leaves was represented by critical SLN, and structural stem N demand by minimum stem N%. The fraction of N partitioned to leaf blades increased under N stress. A framework for N dynamics of leaves and stems is developed that captures effects of N stress and genotype on N partitioning and on critical and maximum SLN.

Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1 B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.