Present genetic structure revealed by microsatellites reflects recent history of the Finnish moose (Alces alces)

Genetic structures of Holarctic species are largely formed by Pleistocene colonisation history, dispersal capacity and interactions between biotic and abiotic factors, even though the human impact can also be significant. The Holarctic moose (Alces alces) arrived in Fennoscandia around 9,000–8,000 years ago, and it has been exploited by humans ever since. During the last 400 years, the Finnish moose population has suffered from several population declines, and even local and regional extirpations have occurred. The purpose of the present study is to describe the genetic variation and population structure of the Finnish moose in order to clarify how historical events and human exploitation have influenced the present-day genetic patterns. Altogether 130 moose individuals from seven sampling sites in Finland were analysed at ten microsatellite loci. A variety of population genetic and coalescent-based methods was applied. The Finnish moose population was found to be divided into southern and northern subpopulations with additional lower hierarchical genetic structure. The estimated time of divergence between these two subpopulations was about 96–238 years ago. In addition, an isolation-by-distance pattern was discovered.     

Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.     

Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.     

Concentration and composition of serum lipoproteins during a low-fat diet at two levels of polyunsaturated fat

A 12-week dietary intervention was carried out among 40 families from North Karelia, a county in Finland with an exceptionally high rate of coronary heart disease and high serum cholesterol values. The proportion of dietary energy derived from fat was reduced during the 12-week intervention period from about 39% to 23% in all families. The families were randomly allocated into two groups. Twenty families consumed a diet with a polyunsaturated to saturated fat (P/S) ratio of 0.9 (group I), while the other 20 families had a diet with a P/S ratio of 0.4 (group II). Total serum cholesterol decreased by 16% and 9% in men of groups I and II, respectively, and by 16% in women of both groups. These changes were due to a decrease in both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. LDL cholesterol and phospholipid reached minimum values after 6 weeks on both intervention diets, but LDL protein responded more slowly. Thus, after 6 weeks LDL had an altered composition containing less cholesterol and phospholipids and more protein and triglycerides than during the baseline diet. During the intervention, the linoleic acid content in the serum cholesteryl ester fraction increased, and the magnitude of this change correlated negatively with the changes in total and LDL cholesterol. The decrease in HDL cholesterol during the two intervention diets was due to a fall in the HDL2 cholesterol (29% and 24% in men, and 26% and 25% in women in groups I and II, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of histones and their synthesis by ammoniacal silver reaction in meristematic root tip cells of Allium cepa

Summary The ammoniacal silver reaction was used for localization of histones in meristematic root tip cells of Allium cepa. Electron microscopic observations showed that yellow or brown colour of interphase and prophase nuclei and brown nucleolar colour produced in the reaction coincides with the appearance of silver grains, about 400 Å in diameter, in the interphase and prophase chromatin and nucleoli. This together with the complete absence of staining reaction and silver grains in the cytoplasm could mean quite a specific reaction with histones and might suggest also that in these cells the site of histone synthesis is in the nucleolus.     

Characterization of type 1 pili of Salmonella typhimurium LT2.

Type 1 pili from Salmonella typhimurium LT2 were purified and characterized. The pilus filaments were 6 nm in diameter and over 1 microns long. Estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the pilin was 21,000. The isoelectric point of the filament was 4.1. Hydrophobic amino acids comprised 40.3% of the total amino acids of the pilin, which contained more proline, serine, and lysine than reported for the type 1 pilin of Escherichia coli. Purified pili agglutinated both horse and chicken erythrocytes and yeast cells but not bovine, sheep, or human erythrocytes. Horse erythrocyte agglutination was inhibited at lower concentrations by alpha-methyl-D-mannoside than by yeast mannane and D-fructose. Agglutination was not affected by D-galactose or sucrose. Results of the present study confirm the role of type 1 pili as Salmonella hemagglutinins and show chemical differences between the type 1 pili of S. typhimurium and E. coli.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Localization of the heparin-releasable lipase in situ in the rat liver.

Immunofluorescence and immuno-electron microscopy were used for the localization of the heparin-releasable lipase in situ in the rat liver. The lipase is located exclusively on the liver endothelial cells. No labelling could be detected on the parenchymal of Kupffer cells, or in the livers of heparin-pretreated animals. The physiological significance of the endothelial localization of the hepatic lipase is discussed.     

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.The expert technical assistance of M. Jarva, L. Ollitervo, S. Kangas, and R. Jokisalo is gratefully acknowledged. This work was supported in part by grants from the Finnish Academy of Science, the Swedish Cancer Foundation, the Novo Nordisk Foundation and EC contract QLG1-CT-2000-01131 (K.T.), the Finnish Dental Society Apollonia and the Northern Finland Cancer Foundation (M.P.), as well as the K. Albin Johansson Foundation and the Einar and Karin Stroems Foundation (E.P.)