A partial characterization of DNA fragments protected from nuclease degradation in histone depleted metaphase chromosomes of the Chinese hamster.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.     

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target structures which trigger killing activity of NK cells are determined by the phenotype of the target cell and are dependent on its MHC class I expression disregarding the haplotype of the cell.     

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.     

Structure-activity relationships of dimeric PPAR agonists

A series of dimeric PPAR agonists were designed and tested for PPAR activity in vitro. The SAR showed that dimeric ligands with a common group or full dimeric ligands had retained or even increased PPARgamma potency. The dimeric agonist concept can be used to fine tune the subtype selectivity of PPAR agonists. The PPARgamma potency could, at least partly, be explained using molecular modeling.     

Heterochromatin,HP1 and methylation at lysine 9 of histone H3 in animals

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.     

Functional ecology and palaeolimnology: using cladoceran remains to reconstruct anthropogenic impact

The field of lake palaeoecology has undergone significant changes. Powerful quantitative techniques have been developed to investigate anthropogenic impacts on lakes. Inclusion of zooplankton and benthic chydorid cladocerans has provided previously unavailable information on the historical development of planktivorous fish populations, submerged macrophytes and lake production, and has been used to document exotic species introductions, rapid genetic evolution and human disturbance of lakes. In particular, new techniques now allow a more complete evaluation of changes in past and present trophic structure to be made, and provide insights on the rapid evolutionary responses of aquatic invertebrate communities to anthropogenic perturbation of lakes.     

Landlocked Arctic charr (Salvelinus alpinus) population structure and lake morphometry in Greenland – is there a connection?

Landlocked Arctic charr (Salvelinus alpinus) populations in sub-Arctic and Arctic Greenland lakes were sampled with multi-mesh-sized survey gillnets. The study covered a range of small shallow lakes (0.01 km², maximum depth <3.3 m) to large deep lakes (43 km², maximum depth >200 m). Arctic charr were found in one to three different forms in lakes with maximum depths >3 m. A dwarf form occurred in all lakes inhabited by Arctic charr and was the only form in lakes with maximum depths <8 m. In deeper lakes with maximum depths >20 m and a surface area <0.5 km², larger charr were found, although in low numbers, the length-frequency distribution being unimodal with a tail towards large sizes. In lakes with a maximum depth >20 m, large-sized charr were more abundant, and the length-frequency distribution of the population was bimodal, with a first mode around 10–12 cm and a second mode around 26–37 cm. In a single large and deep lake, a distinct medium-sized pelagic zooplankton-eating charr form occurred. Maximum size of individual charr was significantly positively correlated with lake maximum depth and volume, and the mean size of large-sized charr was significantly positively correlated with lake volume. Our study indicates that the charr population structure became more complex with increasing lake size. Moreover, the population structure seemed to be influenced by lake-water transparency and the presence or absence of three-spined stickleback (Gasterosteus aculeatus). Accepted: 31 January 2000     

Rapid Ecological Shift Following Piscivorous Fish Introduction to Increasingly Eutrophic and Warmer Lake Furnas (Azores Archipelago,Portugal): A Paleoecological Approach

Lake ecosystems are nowadays often subjected to multi-stressors, such as eutrophication, climate change, and fish manipulations, the effects of which can be difficult to disentangle, not least from the usual short-term limnological time-series that are available. However, multi-proxy paleoecological approaches may offer such opportunities, especially in the study of remote island lakes characterized by being species poor and buffered somewhat against the recent climate change. We used a paleoecological multi-proxy approach to determine the relative importance of nutrient loading, meteorological forcing, and fish species introduction for recent lake ecosystem development in Lake Furnas on the island of São Miguel, the Azores. The lake was stocked with cyprinids in the late nineteenth century and recently also with piscivorous fish, and has been affected by increasing agricultural activities in its catchment. We analyzed marker pigments, cladoceran remains, and subfossil diatoms in a 46-cm core representing the last 40 years. Remains of large-bodied cladocerans were virtually absent until the introductions of piscivorous pike and pikeperch in 1980 and 1982, respectively, after which the zooplankton community composition changed abruptly. First Ceriodaphnia sp. appeared (ca. 1980), followed by Daphnia a few years later. Carotenoids from cyanobacteria (myxoxanthophyll, aphanizophyll) were regularly present in the lake sediment with a major shift occurring around 1994, from N₂-fixing to non-fixing groups. This shift coincided with the onset of anoxia in the lake water, evidenced by the presence of pigments from Chlorobiaceae (BChl-e homologues and isorenieratene), and with a rapid decrease in benthic-tychoplanktonic diatoms and an increase in cyanobacteria, chlorophytes, dinoflagellates, and cryptophytes. The composition of microbial and algal assemblages changed rapidly after Daphnia appearance, and the covariance between fish stocking, nutrient loading, and enhanced temperatures captured most of the variability in algae accumulation, and thus likely in lake primary production as well. Thus, lake production apparently did not respond strongly to specific changes in temperature, food-web structure, or nutrient input, but rather to the combined effects of all the three forcing mechanisms, emphasizing the role of multi-stressors in lake ecosystem functioning. Our study demonstrates the sensitivity of these remote species-poor lakes to increased nutrient loading, introduction of non-native species, such as fish and climate change.