Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.     

Role of Soil Organisms in the Maintenance of Species-Rich Seminatural Grasslands through Mowing

To preserve species-rich grasslands, management practices such as mowing are often required. Mowing is known to promote aboveground conditions that help to maintain plant species richness, but whether belowground effects are important as well is not known. We hypothesized that if mowing decreases belowground carbon transfer by reducing root mass, this will reduce the abundance and activity of soil decomposers and lead to diminished nutrient availability in soil. In grasslands, this would provide a means to mitigate the negative effects of nitrogen enrichment on plant species richness. We established experimental plots on grassland with one-third of plots growing untouched, one-third mowed once a summer, and one-third mowed twice a summer for three growing seasons. Root and soil attributes were measured at each plot 1 month after each mowing. Mowing decreased root mass but had few effects on belowground biota and soil NH₄-N, NO₃-N, and PO₄-P concentrations. Mowing did not affect root N concentrations. Our results show that despite reducing root mass, mowing may have few effects on those soil biota that control nutrient supply and, as a result, have no clear-cut effects on nutrient availability in soil. This suggests that the effects of mowing on soil decomposers and soil nutrient availability may not provide an effective means to mitigate N enrichment and enhance plant species richness in grasslands in a timescale of a few years. Whether such effects could, however, be achieved with longer lasting mowing remains open.     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Decline in cardiovascular mortality in North Karelia and other parts of Finland.

The trends in mortality from ischaemic heart disease, cerebrovascular stroke, and all cardiovascular diseases were analysed for the province of North Karelia and for the rest of Finland. Linear trends in mortality were computed for the population aged 35 to 64 for the period from 1969 to 1982, and changes in mortality between the three year means of 1969-71 and 1980-2 were calculated. In North Karelia, where a community based preventive programme has been carried out since 1972, the annual decline in mortality from ischaemic heart disease in men was on average 2.9%, whereas in the rest of Finland it was 2.0%. For women the respective average annual declines in mortality were 4.9% and 3.0%. The net decline from 1969-71 to 1980-2 in North Karelia was 100 deaths/100,000 men. The annual mortality from all cardiovascular disease in men decreased by 2.9% in North Karelia and by 2.6% in the rest of Finland; in women the decreases were 6.0% and 5.0% a year, respectively. The net decline in North Karelia was 71 deaths/100,000 men. The decline in mortality from all causes was also appreciable in both sexes in North Karelia, but it did not differ significantly from national trends.     

Escherichia coli plasmid production in fermenter

Escherichia coli harboring a recombinant plasmid was grown in a fermenter to study the effects of selected process parameters on the growth of the microbe and on plasmid amplification with chloramphenicol treatment. Eighteen fermentations were carried out according to a statistical experimental design in which the fermentation temperature, pH, and turbidity of culture at the onset of plasmid amplification were the selected independent process variables. Static regression models describing the process were derived from the experimental results. It turned out that recombinant plasmid copy numbers could be influenced by controlling fermentation temperature and pH. The maximal copy number during bacterial growth phase and the optimal plasmid production were found to require fermentation conditions different from those needed for optimal bacterial growth and cell division. The conditions also differed significantly from those routinely used in research laboratories for plasmid preparation. The chloramphenicol treatment increased the plasmid copy number compared with chromosome numbers up to fivefold. Some of the data suggest that under certain conditions the number of chromosome molecules in E. coli cells may rise during the plasmid amplification stage. Statistical experimental design, a nucleic acid sandwich hybridization technique for plasmid quantification, and regression models proved to be useful in this study.     

Gene therapy in epilepsy: the focus on NPY

Gene therapy represents an innovative and promising alternative for the treatment of epileptic patients who are resistant to conventional antiepileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant viral vectors have been most widely used so far. Several gene targets could be used to correct the compromized balance between inhibitory and excitatory transmission in epilepsy. Transduction of neuropeptide genes such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. In particular, the long-lasting NPY over-expression obtained in the rat hippocampus using intracerebral application of recombinant adeno-associated viral (AAV) vectors reduced the generalization of seizures from their site of onset, delayed acquisition of fully kindled seizures and afforded neuroprotection. These results establish a proof-of-principle for the applicability of AAV-NPY vectors for the inhibition of seizures in epilepsy. Additional investigations are required to demonstrate a therapeutic role of gene therapy in chronic models of seizures and to address in more detail safety concerns and possible side-effects.     

Depletion of alphaV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis.

Integrin heterodimers sharing the common alphaV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alphaV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alphaVbeta3 and alphaVbeta5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alphaV integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor alphaV integrin subunit was not affected. However, the maturation of alphaV integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of alphaV integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, alphaV integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation.     

Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal,population based study

Objective To examine the relation of midlife raised blood pressure and serum cholesterol concentrations to Alzheimer''s disease in later life.Design Prospective, population based study.SettingPopulations of Kuopio and Joensuu, eastern Finland.ParticipantsParticipants were derived from random, population based samples previously studied in a survey carried out in 1972, 1977, 1982, or 1987. After an average of 21 years'' follow up, a total of 1449 (73%) participants aged 65-79 took part in the re-examination in 1998.Results People with raised systolic blood pressure (⩾160 mm Hg) or high serum cholesterol concentration (⩾6.5 mmol/l) in midlife had a significantly higher risk of Alzheimer''s disease in later life, even after adjustment for age, body mass index, education, vascular events, smoking status, and alcohol consumption, than those with normal systolic blood pressure (odds ratio 2.3, 95% confidence interval 1.0 to 5.5) or serum cholesterol (odds ratio 2.1, 1.0 to 4.4). Participants with both of these risk factors in midlife had a significantly higher risk of developing Alzheimer''s disease than those with either of the risk factors alone (odds ratio 3.5, 1.6 to 7.9). Diastolic blood pressure in midlife had no significant effect on the risk of Alzheimer''s disease.Conclusion Raised systolic blood pressure and high serum cholesterol concentration, and in particular the combination of these risks, in midlife increase the risk of Alzheimer''s disease in later life.

What is already known on this topic

Vascular risk factors may play an important part as risk factors for Alzheimer''s diseaseNo population based studies have evaluated prospectively the impact of both midlife blood pressure and cholesterol concentration in both men and women on the subsequent development of Alzheimer''s disease

What this study adds

Raised systolic blood pressure and high serum cholesterol concentration, and in particular the combination of these risks, in midlife increased the risk of Alzheimer''s disease in later lifeRaised systolic blood pressure and hypercholesterolaemia may have a role in the pathogenesis of Alzheimer''s disease; more emphasis should be placed on identification and appropriate treatment of these conditions