Meloxicam, an inhibitor of cyclooxygenase-2, increases the level of serum G-CSF and might be usable as an auxiliary means in G-CSF therapy

Hematopoiesis-modulating action of meloxicam, a cyclooxyge-nase-2 inhibitor, has been evaluated in mice. Increased serum level of granulocyte colony-stimulating factor (G-CSF) after meloxicam administration has been found in sublethally gamma-irradiated animals. In further experiments hematopoiesis-stimulating effects of meloxicam and G-CSF given alone or in combination have been investigated. Granulocyte/macrophage progenitor cells counts were used to monitor these effects. Meloxicam and exogenous G-CSF did not act synergistically when given in combination, but could be mutually substituted during their repeated administration. The results suggest a promising possibility of using meloxicam as an auxiliary drug reducing the high costs of G-CSF therapy of myelosuppression.     

Prenylated flavanones and flavanonols as chemical markers in Glycosmis species (Rutaceae)

Fifteen prenylated or geranylated flavanones and flavanonols were isolated from the leaf extracts of different Glycosmis species collected in Thailand and Malaysia. All structures were elucidated by spectroscopic methods, especially 1D and 2D NMR. Six compounds were described for the first time and two were only known so far as synthetic products. The chemotaxonomic significance of flavanoid accumulation within the genus Glycosmis is highlighted.     

Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Neurofibromin Homologs Ira1 and Ira2 Affect Glycerophosphoinositol Production and Transport in Saccharomyces cerevisiae

Saccharomyces cerevisiae produces extracellular glycerophosphoinositol through phospholipase-mediated turnover of phosphatidylinositol and transports glycerophosphoinositol into the cell upon nutrient limitation. A screening identified the RAS GTPase-activating proteins Ira1 and Ira2 as required for utilization of glycerophosphoinositol as the sole phosphate source, but the RAS/cyclic AMP pathway does not appear to be involved in the growth phenotype. Ira1 and Ira2 affect both the production and transport of glycerophosphoinositol.Membrane phospholipids are continually synthesized and degraded as cells grow and respond to environmental conditions. A major pathway of phosphatidylinositol (PI) turnover in Saccharomyces cerevisiae is its deacylation to produce extracellular glycerophosphoinositol (GroPIns) (3). Plb3, an enzyme with phospholipase B (PLB)/lysophospholipase activity, is thought to be primarily responsible for the production of extracellular GroPIns, with Plb1 playing a lesser role (11, 12, 13). GroPIns is transported into the cell by the Git1 permease (17). GIT1 expression is upregulated by phosphate limitation and inositol limitation. In fact, GroPIns can act as the cell''s sole source of both inositol (17) and phosphate (1).A screening for gene products involved in the process by which GroPIns enters the cellular metabolism identified Ira1 and Ira2, yeast homologs of the mammalian protein neurofibromin. Alterations in NF1, the gene encoding neurofibromin, are associated with the pathogenesis of neurofibromatosis type 1, an autosomal dominant genetic disease (4, 5, 25). Ira1 and Ira2 and neurofibromin function as RAS GTPase-activating proteins (RAS GAPs). S. cerevisiae Ras1 and Ras2 activate adenylate cyclase to modulate cyclic AMP (cAMP) levels. The binding of cAMP to the regulatory subunits of protein kinase A (Bcy1) results in dissociation and activation of the catalytic subunits (Tpk1 to Tpk3). Ira1 and Ira2 inactivate RAS and thereby downregulate the pathway (18, 19). Hydrolysis of cAMP by the phosphodiesterases encoded by PDE1 and PDE2 also downregulate the pathway (7, 20, 23). The RAS/cAMP pathway responds to nutrient signals to modulate fundamental cellular processes, including stress resistance, metabolism, and cell proliferation (7, 20, 21).     

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

A new floating sensor array to detect electric near fields of beating heart preparations

A new flexible sensor for in vitro experiments was developed to measure the surface potential, Φ, and its gradient, E (electric near field), at given sites of the heart. During depolarisation, E describes a vector loop from which direction and magnitude of local conduction velocity θ can be computed. Four recording silver electrodes (14 μm × 14 μm) separated by 50 μm, conducting leads, and solderable pads were patterned on a 50 μm thick polyimide film. The conductive structures, except the electrodes, were isolated with polyimide, and electrodes were chlorided. Spacer pillars mounted on the tip fulfil two functions: they keep the electrodes 70 μm from the tissue allowing non-contact recording of Φ and prevent lateral slipping. The low mass (9.1 mg) and flexibility (6.33 N/m) of the sensor let it easily follow the movement of the beating heart without notable displacement. We examined the electrodes on criteria like rms-noise of Φ, signal-to-noise ratio of Φ and E, maximum peak-slope recording dΦ/dt, and deviation of local activation time (LAT) from a common signal and obtained values of 24–28 μV, 46 and 41 dB, 497–561 V/s and no differences, respectively. With appropriate data acquisition (sampling rate 100 kHz, 24-bit), we were able to record Φ and to monitor E and θ on-line from beat-to-beat even at heart rates of 600 beats/min. Moreover, this technique can discriminate between uncoupled cardiac activations (as occur in fibrotic tissue) separated by less than 1 mm and 1 ms.     

Lifelong learning: ocular dominance plasticity in mouse visual cortex

Ocular dominance plasticity has long served as a successful model for examining how cortical circuits are shaped by experience. In this paradigm, altered retinal activity caused by unilateral eye-lid closure leads to dramatic shifts in the binocular response properties of neurons in the visual cortex. Much of the recent progress in identifying the cellular and molecular mechanisms underlying ocular dominance plasticity has been achieved by using the mouse as a model system. In this species, monocular deprivation initiated in adulthood also causes robust ocular dominance shifts. Research on ocular dominance plasticity in the mouse is starting to provide insight into which factors mediate and influence cortical plasticity in juvenile and adult animals.     

Modelling the Species Distribution of Flat-Headed Cats (Prionailurus planiceps), an Endangered South-East Asian Small Felid

Background

The flat-headed cat (Prionailurus planiceps) is one of the world''s least known, highly threatened felids with a distribution restricted to tropical lowland rainforests in Peninsular Thailand/Malaysia, Borneo and Sumatra. Throughout its geographic range large-scale anthropogenic transformation processes, including the pollution of fresh-water river systems and landscape fragmentation, raise concerns regarding its conservation status. Despite an increasing number of camera-trapping field surveys for carnivores in South-East Asia during the past two decades, few of these studies recorded the flat-headed cat.

Methodology/Principal Findings

In this study, we designed a predictive species distribution model using the Maximum Entropy (MaxEnt) algorithm to reassess the potential current distribution and conservation status of the flat-headed cat. Eighty-eight independent species occurrence records were gathered from field surveys, literature records, and museum collections. These current and historical records were analysed in relation to bioclimatic variables (WorldClim), altitude (SRTM) and minimum distance to larger water resources (Digital Chart of the World). Distance to water was identified as the key predictor for the occurrence of flat-headed cats (>50% explanation). In addition, we used different land cover maps (GLC2000, GlobCover and SarVision LLC for Borneo), information on protected areas and regional human population density data to extract suitable habitats from the potential distribution predicted by the MaxEnt model. Between 54% and 68% of suitable habitat has already been converted to unsuitable land cover types (e.g. croplands, plantations), and only between 10% and 20% of suitable land cover is categorised as fully protected according to the IUCN criteria. The remaining habitats are highly fragmented and only a few larger forest patches remain.

Conclusion/Significance

Based on our findings, we recommend that future conservation efforts for the flat-headed cat should focus on the identified remaining key localities and be implemented through a continuous dialogue between local stakeholders, conservationists and scientists to ensure its long-term survival. The flat-headed cat can serve as a flagship species for the protection of several other endangered species associated with the threatened tropical lowland forests and surface fresh-water sources in this region.     

Molecular dynamics simulation of mammalian 15S-lipoxygenase with AMBER force field

A molecular dynamics simulation study of mononuclear iron 15S-lipoxygenase (15S-LOX) from rabbit reticulocytes was performed to investigate its structure and dynamics; newly developed AMBER force field parameters were employed for the first coordination sphere of the catalytic iron (II). The results obtained from this study demonstrate that the structural features of the catalytic iron coordination site are in good agreement with available data obtained from experiments. The motional flexibility of the N-terminal β-barrel domain is greater than the C-terminal catalytic domain; flexibility was assessed in terms of B-factors and secondary structure calculations. The significant features obtained for the relative motional flexibility of these two domains of 15S-LOX in solution as well as the isolated C-terminal domain were analyzed in terms of radius of gyration and maximum diameter, which correlated well with the structural flexibility of 15-lipoxygenase-1 in solution as probed by small-angle X-ray scattering. The motional flexibility indicates interdomain motion between the N-terminal β-barrel and the C-terminal catalytic domain; this was further verified by the evaluation of central bending in the solvated LOX molecule, which identified an unstructured stretch of amino acids as the interdomain linker. The average bending angle confirmed significant central bending between these two domains, which was linked to the high degree of motional freedom of the N-terminal β-barrel domain in aqueous solutions. This can be considered to have biological relevance for membrane binding as well as for regulating the catalytic domain.