Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

No evidence of mineralization abnormalities in iliac bone of premenopausal women with type 2 diabetes mellitus

Objectives:Patients with type-2 diabetes mellitus (T2DM) have increased risk for bone fractures which points towards impaired bone quality.Methods:We measured bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics based on quantitative backscattered electron images of transiliac biopsy samples from n=26 premenopausal women with T2DM. Outcomes were compared to those from reference cohorts as well as between T2DM subgroups defined by clinical characteristics.Results:Comparison to references did not reveal any differences in BMDD (all p>0.05) but a lowered OLS-density in cancellous bone in T2DM (-14.9%, p<0.001). Neither BMDD nor OLS-characteristics differed in T2DM subgroups defined by HbA1c (<7% versus >7%). The average degree of bone mineralization (CaMean) was higher (0.44 wt%Ca in T2DM, 0.30 wt%Ca in reference) and consistently the calcium concentration between the tetracycline double labels (CaYoung) was higher (0.76 wt%Ca, all p<0.001) in cancellous versus cortical bone.Conclusions:Our findings suggest that bone matrix mineralization was neither affected by the presence nor by the glycemic control of T2DM in our study cohort. The intra-individual differences between cancellous and cortical bone mineralization gave evidence for differences in the time course of the early mineralization process in these compartments in general.     

Delta-sleep-inducing peptide (DSIP): An update

The isolation and characterization of delta-sleep-inducing peptide (DSIP) achieved from 1963 to 1977 were reviewed in 1984. The first reports describing sleep as well as extra-sleep effects of DSIP also were included in that work. Only two years later, much additional literature concerning DSIP has accumulated. Besides further sleep-inducing and/or-supporting effects of DSIP in animals, considerable work has been carried out to evaluate the potential use of the peptide for therapeutic purposes such as treatment of insomnia, pain, and withdrawal. Immunohistochemical as well as radioimmunochemical studies provided further insights into the natural occurrence of the nonapeptide and the distribution of DSIP-like material in the body, suggesting possible relations of the peptide to certain diseases. Various physiological functions of DSIP and a possible mechanism of action involving the modulation of adrenergic transmission remain to be established.     

Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.     

Short-term effects of temperature enhancement on growth and reproduction of alpine grassland species

In order to study the effects of temperature enhancement on alpine calcareous grassland species, a warming experiment was carried out in the Berchtesgaden National Park (Southeast Germany, Northern Calcareous Alps) between 2002 and 2004. The study was conducted in stands of the Carex sempervirens and the Carex firma communities; the two most widespread grassland types in the alpine zone of the Northern Calcareous Alps. The temperature of the vegetation stand and the upper soil was passively enhanced using open top chambers (OTCs). The construction of the OTCs was appropriate since temperature was clearly increased while water conditions (humidity, soil water content) were not changed.

By comparing manipulated (temperature enhancement) with non-manipulated plots, the effects of warming on growth and reproduction of selected key species were studied. To test if vegetation response to temperature enhancement is at least partly due to increases in nutrient availability, soil solution concentrations of nitrate and ammonium were analysed.

We found that most of the studied plant species are sensitive to temperature enhancement. Growth and/or reproduction of 12 of the 14 studied species were significantly stimulated by warming. Only two species showed no response; none of the species experienced decreases in growth or reproduction. Dwarf shrubs and graminoids showed a stronger response than herbaceous perennials. A significant effect of warming on nutrient availability could not be detected. The observed response of vegetation is therefore mainly caused by direct and not by indirect temperature effects.     

Positive feedback favors invasion by a submersed freshwater plant

The submersed macrophyte Utricularia inflata has invaded lakes in northern New York State, thereby threatening native isoetids such as Eriocaulon aquaticum. Isoetids often dominate and modify softwater lakes due to their capacity to oxidize sediment and thus influence solute mobilization. Greenhouse experiments tested the hypotheses that U. inflata invasion could result in higher porewater iron (Fe) concentrations and greater ammonium (NH₄ ⁺) and Fe release from the sediment into the water column, and that this mobilization would stimulate further U. inflata growth. In the first experiment, three levels of U. inflata impact on E. aquaticum were imposed using sediment cores overlain by lake water: E. aquaticum alone, E. aquaticum with a cover of U. inflata, and bare sediment—the latter to simulate local extirpation of the isoetid by the invasive. After 16 weeks, sediment porewater NH₄ ⁺ and total dissolved Fe concentrations were significantly higher (P < 0.05) for the U. inflata and bare sediment treatments. Water column concentrations of these solutes were five-fold higher (P < 0.05) for the bare sediment treatment than E. aquaticum alone, indicating that isoetid extirpation by U. inflata can compromise water quality. A second experiment demonstrated that U. inflata grew faster over bare sediment than over sediment with E. aquaticum (P < 0.05), likely due to greater solute mobilization in the absence of E. aquaticum. Where U. inflata causes a decline of native isoetids in Adirondack Mountain lakes, changes to lake sediment and water chemistry can create a positive feedback loop further escalating the impact of this invasive species.     

Association between CSN3 and BCO2 gene polymorphisms and milk performance traits in the Czech Fleckvieh cattle breed

Daily milk, fat and protein yield and amount of somatic cells in cow milk are very important factors that influence milk performance traits. An association between polymorphisms in the kappa casein (CSN3) gene and milk production, composition and technical properties has been previously reported; however, this type of information is not available for the bovine β-carotene oxygenase 2 (BCO2) gene--the BCO2 gene has relationship with milk color and meat fat color, which is dependent on content of β-carotene. We analyzed these two genes and their relationship with milk performance traits (daily milk, fat and protein yield, somatic cell count, SCC) in one cattle population, Czech Fleckvieh (N = 152). All animals were milked twice a day and kept in the same environmental conditions. The Fleckvieh is a typical Czech cattle breed farming for milk and meat production. It is the most common breed in the Czech Republic. DNA was isolated from milk or from hairs. Genes were analyzed using PCR-RFLP, frequencies of alleles and genotypes were calculated and association analysis was performed using a GLM Procedure in SAS. Statistical analysis established that the CSN3 gene has no statistically significant influence on daily milk, fat and protein yield and SCC. Compared to other references this result can be explained by, e.g., small group of animals and different cattle breed. The BCO2 gene (genotypes AA and AG) shows a statistically significant relationship (P = 0.05) with daily milk, protein yield and SCC.     

Pergolide Presynaptically Inhibits Calcium-Stimulated Release of γ-Aminobutyric Acid

The release of gamma-aminobutyric acid (GABA) in rat dorsolateral striatum was studied using in vivo microdialysis. Dialysis was conducted 2 days after probe implantation in awake, freely moving rats using a modified Ringer solution. Calcium induced a reversible increase in GABA release that was abolished by tetrodotoxin but was only slightly attenuated by a maximally effective dose of pergolide, a D2 receptor agonist. It was thus concluded that pergolide inhibits calcium-stimulated release of GABA presynaptically by a mechanism distinct from that of tetrodotoxin.