Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

Two acyl sucroses from Petunia nyctaginiflora

Two new acyl sucroses were isolated from the epigeal parts of Petunia nyctaginiflora Juss. (Solanaceae). Their structures were determined to be 2, 3, 4-tri (5-methylhexanoyl)-alpha-D-glucopyranosyl-beta-D-fructofuranoside (2) and 2, 3, 4-tri (6-methylheptanoyl)-alpha-D-glucopyranosyl-beta-D-fructofuranoside (4) on the basis of chemical and spectroscopic evidence.     

Transgene integration,organization and interaction in plants

It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.     

Skin graft survival in genetically identical cloned pigs

Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipient's skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible.     

Cloning, expression, activity and folding studies of serine hydroxymethyltransferase: a target enzyme for cancer chemotherapy

All the members of pyridoxal-5'-phosphate-dependent enzymes are involved in the metabolism of amino acids. The sequence homology studies further divide this family into three distinct groups. A fine scrutiny of the reactions catalyzed by these enzymes shows their regio specificity; they have been considered as the largest group of enzymes having tendency to affect the valency of the same carbon atom that carries the amino group forming an amine linkage with the coenzyme. Thus, this group was named 'alpha-class of enzymes'. Serine hydroxymethyltransferase (SHMT) is a member of this alpha-class; it reversibly catalyses the conversion of serine into glycine while the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate. The resultant compound is the sole precursor of purine biosynthesis. Henceforth, this enzyme greatly affects nucleic acid biosynthesis in all the organisms. It is obvious that SHMT plays an indispensable role in nucleic acid biosynthesis; therefore, designing and developing a repressor/inhibitor of the SHMT gene/protein may resolve the problem of drug resistance to cancer chemotherapy. SHMT has been widely studied in many living systems (e.g. Escherichia coli, humans, sheep, rabbits, Trypanosoma, Arabidopsis, peas, tobacco) in terms of its structure, cloning, expression, purification and folding patterns. Such studies have enabled one to assess the pattern of overall kinetic and activity behaviour of the enzyme, which may further help in developing a suitable cancer therapeutic molecule.     

Effect of animal bedding on rat liver endosome acidification

Animal beddings, such as pine products, and environmental factors are known to induce liver drug-metabolizing cytochrome P450 enzymes. We observed that a change to pine-based rat bedding altered baseline and cAMP-stimulated rates of acidification in rat liver endosomes, apparently by decreasing ATP-dependent proton transport in the presence and absence of chloride. Although cAMP altered phosphorylation of protein kinase B and extracellular signal-regulated kinases 1 and 2 (ERK 1,2) and p38 mitogen-activated protein kinases, changes in housing conditions did not affect baseline or cAMP-stimulated values of these or other selected signaling molecules. We conclude that compounds in rat bedding may alter not only drug metabolism, but also aspects of endocytosis.     

The Evolution of Enamel Microstructure: How Important Is Amelogenin?

The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes' process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure.