Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure

Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal (n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs (n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.     

Mast cell-mediated apoptosis of endothelial cells in vitro: a paracrine mechanism involving TNF-alpha-mediated down-regulation of bcl-2 expression

Degranulated mast cells are present in the subendothelial space of eroded (de-endothelialized) coronary atheromas. Upon degranulation, mast cells secrete into the surrounding tissue an array of preformed and newly synthesized mediators, including proapoptotic molecules, such as chymase and TNF-alpha. In a co-culture system involving rat serosal mast cells and rat cardiac (microvascular) endothelial cells, we could show, by means of competitive RT-PCR, immunoblotting, immunocytochemistry, annexin staining, flow cytometry, and DNA-laddering, that stimulation of mast cells with ensuing degranulation rapidly (within 30 min) down-regulated the expression of both bcl-2 mRNA and protein, with subsequent induction of apoptosis in the endothelial cells. The major effect of bcl-2 down-regulation resided in the exocytosed granule remnants, a minor effect also being present in the granule remnant-free supernatant. No significant changes were observed in the expression levels of the pro-apoptotic protein, bax. The mast cell-mediated apoptotic effect was partially (70%) dependent on the presence of TNF-alpha and involved the translocation of cytochrome C from mitochondria into cytoplasm. These results are the first to show that one of the cell types present in the atherosclerotic plaques, namely the mast cell, by releasing both granule-remnant-bound and soluble TNF-alpha, may contribute to the erosion of atherosclerotic plaques by inducing apoptosis in adjacent endothelial cells. Published 2003 Wiley-Liss, Inc.     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

The good bad wolf—wolf evaluation reveals the roots of the Finnish wolf conflict

This article focuses on the roots of the Finnish wolf conflict by using stakeholder evaluations of the wolf as a tool. The recent growth of the wolf population has highlighted stakeholders’ contradictory objectives and revealed a conflict between the two main stakeholders, conservationists and hunters, in wolf management. The question of hunting emerges as the core of the conflict. The negative evaluation of the wolf by hunters reflects a competitive situation, which is typical of the historical development of wolf management in Finland. In areas with the most abundant wolf populations, hunters view the wolf most negatively. This study clearly demonstrates that the Finnish wolf conflict is rooted in the values of modern society and carries a long historical, practical and ecological background in which humans and wolves compete over resources, mainly the moose. The conflict between hunters and conservationists in wolf management is connected to the appreciation of moose as game and stems from competition between humans and wolves over their prey and the historical presence or absence of the wolf.     

Bottom-up and cascading top-down control of macroalgae along a depth gradient

On marine rocky shores, macroalgal herbivory is often intense, such that the cascading effects of fish predation may contribute to the control of algal communities. To estimate the magnitudes of top-down and bottom-up control on a macroalgal community, we manipulated the access of carnivorous fish to macroalgal colonization substrates, as well as nutrient availability, at two sub-littoral depths. There were three levels of fish manipulation: natural fish community, no fish and the enclosure of one common species, the perch, Perca fluviatilis. We found a clear cascade effect of fish predation on both the total density and several individual species of macroalgae, which was more pronounced in deep than shallow water. The density of the dominant grazers, i.e. snails, increased in nutrient-enriched conditions; perch were inefficient in controlling herbivores, and had therefore no cascading effect on algal densities under such conditions. Although nutrients enhanced the growth of opportunistic algae, herbivores, in the absence of fish, inhibited this response. While algal diversity was higher in shallow than in deep water, the enrichment effect was opposite at the two depths with lowered diversity in the shallows and increased at depth. Our results indicate that fish predation is an efficient regulator of meso-herbivores and that its effect thereby cascades onto the producer trophic level such that both perennial and opportunistic algae benefit from the presence of fish. This cascade effect is probably stronger at depth where predation efficiency is less disturbed by wave motion.     

Regional and local spatial genetic structure of Siberian primrose populations in Northern Europe

We have investigated the local and regional scale genetic structure of Siberian primrose (Primula nutans) populations in Northern Europe. The genetic diversity and structure of fifteen populations sampled from the Bothnian Bay in Finland, the Barents Sea in Norway and the White Sea in Russia were assessed using eleven microsatellite markers. We investigated the distribution of genetic variation within and between populations, and studied the local genetic structure using spatial autocorrelation analysis. We found very low genetic and allelic diversity in the Bothnian Bay and Barents Sea populations, and only slightly higher in the White Sea population. The level of genetic differentiation between the regions was very high, whereas differentiation between the populations within the regions was moderate. We found no spatial structuring of populations in any region suggesting efficient dispersal on a local scale. Clonal reproduction seemed to have no effect on genetic structure.     

Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.     

Further characterisation of the translational termination-reinitiation signal of the influenza B virus segment 7 RNA

Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAAUG and ～10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAAUG, known as the 'termination upstream ribosome binding site' (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAAUG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.