In vivo analysis reveals different apoptotic pathways in pre- and postmigratory cerebellar granule cells of rabbit

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation.     

Intercalation of daunomycin into stacked DNA base pairs. DFT study of an anticancer drug

We have computationally studied the intercalation of the antitumor drug daunomycin into six stacks of Watson-Crick DNA base pairs (i.e., AT-AT, AT-TA, GC-AT, CG-TA, GC-GC, GC-CG) using density functional theory (DFT). The proton affinity of the DNA intercalator daunomycin in water was computed to be 159.2 kcal/mol at BP86/TZ2P, which is in line with the experimental observation that daunomycin is protonated under physiological conditions. The intercalation interaction of protonated daunomycin with two stacked DNA base pairs was studied through a hybrid approach in which intercalation is treated at LDA/TZP while the molecular structure of daunomycin and hydrogen-bonded Watson-Crick pairs is computed at BP86/TZ2P. We find that the affinity of the drug for the six considered base pair dimers decreases in the order AT-AT > AT-TA > GC-AT > GC-TA > GC-CG > GC-GC, in excellent agreement with experimental data on the thermodynamics of the interaction between daunomycin and synthetic polynucleotides in aqueous solution. Our analyses show that the overall stability of the intercalation complexes comes mainly from pi-pi stacking but an important contribution to the computed and experimentally observed sequence specificity comes from hydrogen bonding between daunomycin and hetero atoms in the minor groove of AT base pairs.     

Morphogenesis and differentiation of Dictyostelium cells interacting with immobilized glucosides: dependence on DIF production

Previous work has shown that multicellular morphogenesis of submerged Dictyostelium cells is inhibited when they bind to glucosides covalently linked to polyacrylamide gels. The amoebae aggregate normally, but then the aggregates repeatedly disperse and reaggregate, whereas control cells go on to form tight aggregates. We have investigated the role of the stalk cell differentiation inducing factors (DIFs) in this process. In the presence of cyclic AMP, amoebae submerged at high cell density accumulate DIF and differentiate into stalk cells. We find that stalk cell differentiation is inhibited by interaction of the cells with glucoside gels in these conditions, but can be restored by the addition of exogenous DIF-1. Since the responsiveness of cells to DIF-1 is not altered, it appears likely that the effect of the glucoside gel is to block DIF-1 production. Further, the addition of DIF-1 or DIF-2 stimulates the formation of tight aggregates by cells developing on glucoside gels in the absence of cyclic AMP, thus preventing the rounds of aggregation and disaggregation otherwise seen. This suggests a role for DIF in morphogenesis as well as in controlling cell differentiation. We propose a model in which immobilized glucosides activate a specific receptor ("food sensor") which drives the amoebae toward the vegetative state and inhibits DIF accumulation. DIF, on the other hand, induces tight aggregate formation and so locks the amoebae into the developmental program.     

Molecular evolution of rodent insulins

Several trees of amino acid sequences of rodent insulins were derived with the maximum-parsimony procedure. Possible orthologous and paralogous relationships were investigated. Except for a recent gene duplication in the ancestor of rat and mouse, there are no strong arguments for other paralogous relationships. Therefore, a tree in agreement with other biological data is the most reasonable one. According to this tree, the capacity to form zinc-binding hexamers was lost once in the ancestor of the hystricomorph rodents, followed by moderately increased evolutionary rates in the lineages to African porcupine and chinchilla but highly increased rates in at least three independent lines to other taxa of this suborder: guinea pig, cuis, and Octodontoidea (coypu and casiragua).      

Ecology and management of mahogany (Swietenia macrophylla King) in the Chimanes Forest, Bed, Bolivia

Mahogany ( Swietenia macrophylla King) regenerates in areas of erosion on high terraces and in forest killed by flooding and deposition of alluvial sediments in the Chimanes Forest, Bolivia. These hydrological disturbances are patchy, and only one of five stands of mahogany that we inventoried was regenerating. Mahogany survives these disturbances significantly better than the common tree species. The long time between disturbances appears to favour late maturation. Mahogany trees allocate little photosynthates to reproduction until they are very large emergents, at least 80 cm in diameter. The episodic nature of the regeneration sites means that mahogany stands are composed of one or a few cohorts, which are vulnerable to overharvesting, particularly with the current use of a minimum cutting diameter to regulate harvest. The delayed onset of fecundity means that the small trees that escape harvest are not very fecund, resulting in minimal seed input to logged forest. Only 7–9% of the gaps created by logging contain natural regeneration after 20 + yr. A successful management plan for mahogany would entail a monocyclic harvest, with a rotation age of 100 + years, the estimated time that it takes for trees to achieve commercial size in natural forest. Since the number of seed trees that will be left is small, they should be concentrated in sites that are likely to be conducive to natural regeneration, such as near rivers and flood damaged forest. Seed production will be maximized for a given basal area (opportunity cost to loggers) if trees c. 110 cm dbh are selected as seed trees. The mahogany stocks in the Chimanes Forest are nearly exhausted, but the findings of this study could be used to help rebuild the mahogany populations, or to design management plans for the commercial species that have similar ecologies to mahogany.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.