Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P₂). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

A flow cytometric assay for screening improved heterologous protein secretion in yeast

Selection of highly productive hosts for protein expression is a significant component of bioprocess design. As an alternative to traditional plate, halo, and suppression-based screens, we describe a high-throughput, flow cytometric assay, the Cell Surface Secretion Assay (CeSSA), that can be used to select for improved heterologous protein secretion from a population of S. cerevisiae mutants. A ligand is covalently attached to the cell surface via a PEG linker, and as cells secrete a protein that binds the tethered ligand, the protein is captured on the surface where it can be labeled and the cells sorted using flow cytometry. This report describes three different protein/ligand interactions that have been demonstrated with this system. Single-pass sorting enrichments from 23- to 54-fold have been validated in the separation of a 3-fold higher secretor from a background population of wild-type secretors making this system applicable to large library screening (10(8) clones). A mathematical model was developed to improve the parameters of the assay further. The model was validated with time course data and predicts an optimal screening window. The model also predicts a 60-fold enrichment rate for the validation experiment described above. With the development of this selection system, limitations presented by traditional, particularly plate-based, secretion assays can be overcome so that a larger search space can be examined under conditions closer to the growth physiology experienced by cells in fermentors.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Glycosaminoglycan-mimetic biomaterials. 3. Glycopolymers prepared from alkene-derivatized mono- and disaccharide-based glycomonomers

Mono- and disaccharide-containing glycopolymers were synthesized by two different free-radical processes, and their ability to act as heparan sulfate glycomimetics in promoting the binding of Fibroblast Growth Factor-2 (FGF-2) to its receptor (FGFR-1) was evaluated using an in vitro cell-based assay. Cyanoxyl (*OC triple bond N)-mediated polymerization of acrylamide with alkene-derivatized mono- and disaccharides including sulfated or nonsulfated N-acetyl-D-glucosamine is described. The results of this approach are compared to those obtained via the classical ammonium peroxodisulfate (APS)/N,N,N',N'-tetramethylethylenediamine (TMEDA) initiating system and confirm the capacity of cyanoxyl-mediated polymerization to generate a variety of glycopolymers with high saccharide contents and low polydispersity indexes. In vitro assays demonstrate that specific glycopolymers can potentiate FGF-2/FGFR-1 binding interactions.     

Glycosaminoglycan mimetic biomaterials. 4. Synthesis of sulfated lactose-based glycopolymers that exhibit anticoagulant activity

Cyanoxyl persistent radicals can be used as chain-growth moderators of the statistical copolymerization of a variety of monomers. We report herein the preparation of fully sulfated lactose-based glycopolymers by cyanoxyl (.OC[triple bond]N)-mediated free-radical polymerization of acrylamide derivatized glycomonomers in good yield (60-80%) and low polydispersity (1.1 < M(w)/M(n) < 1.6). Prolongation of the activated partial thromboplastin time (aPTT) was observed, and structure-activity relationships were defined. Specifically, the anticoagulant effect varied in response to both polymer molecular weight and the density of pendant sulfated lactose units. Nonetheless, measured thrombin times were only modestly prolonged suggesting that the observed anticoagulant effect is not primarily related to direct thrombin inhibition.     

Regulation of aromatase, 5α- and 5β-reductase in primary cell cultures of developing zebra finch telencephalon

Sex steroids act on the developing and adult telencephalon of songbirds to organize and activate the neural circuits required for the learning and production of song. Presumably, the availability of active androgens and estrogens to steroid-sensitive neural circuits controlling song is modulated by the local expression of androgen-metabolizing enzymes. Two enzymes, 5α- and 5β-reductase, are expressed widely in the songbird telencephalon, as they are in the telencephalons of other avian species. These enzymes convert circulating testosterone (T) into the active and inactive metabolites, 5α- and 5β-dihydrotestosterone (DHT), respectively. A third enzyme, aromatase, converts T into estradiol (E₂) and is expressed at unusually high levels in several regions of the songbird telencephalon. In many tissues, including the brain, the regulation of expression of one or more of these enzymes can be a critical feature of their ability to control the production of active sex steroids. We have used primary cell cultures to examine factors that might regulate the expression of these enzymes in developing zebra finch telencephalon. Cultures were treated for 0-72 h with sex steroids (T, E₂, 5α-DHT, and 5β-DHT) or with dibutyryl cAMP. Afterward, activities of aromatase, 5α-, and 5β-reductase were determined or total RNA was extracted for Northern analysis. Treatments with cAMP increased both aromatase activity and aromatase mRNA levels by 220%. E₂ significantly reduced aromatase activity by an average of 65%, whereas 5α- and 5β-DHT had no effect on aromatase activity. Compared to untreated controls, E₂ treatment decreased aromatase mRNA levels by 56%. None of these treatments consistently affected either 5α- or 5β-reductase activities. These results suggest that telencephalic E₂ may regulate its own synthesis by repression of aromatase expression, whereas factors that upregulate cAMP in the telencephalon can increase the local concentrations of E₂. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 30–40, 1998     

Ionizing radiation exposure results in up-regulation of Ku70 via a p53/ataxia-telangiectasia-mutated protein-dependent mechanism

Genome damaging events, such as gamma-irradiation exposure, result in the induction of pathways that activate DNA repair mechanisms, halt cell cycle progression, and/or trigger apoptosis. We have investigated the effects of gamma-irradiation on cellular levels of the Ku autoantigens. Ku70 and Ku80 have been shown to form a heterodimeric complex that can bind tightly to free DNA ends and activate the protein kinase DNA-PKcs. We have found that irradiation results in an up-regulation of cellular levels of Ku70, but not Ku80, and that this enhanced level of Ku70 accumulates within the nucleus. Further, we uncovered that the postirradiation up-regulation of Ku70 utilizes a mechanism that is dependent on both p53 and damage response protein kinase ATM (ataxia-telangiectasia-mutated); however, the activation of DNA-PK does not require Ku70 up-regulation. These findings suggest that Ku70 up-regulation provides the cell with a means of assuring either proper DNA repair or an appropriate response to DNA damage independent of DNA-PKcs activation.     

Sub chronic toxicity studies of lactulose in rats

Lactulose has profound health benefits by way of increasing bifidobacterial flora in the intestine of infants thereby protecting them against enteric infection, constipation and systemic encephalopathy. In the present study to assess the sub chronic toxicity of lactulose syrup, the rats were fed on a basal feed supplemented with lactulose syrup at 0.5, 1.0, 2.0 and 5.0% for a period of 21 weeks. Monitoring of food consumption, gain in body weight and physical observations did not reveal any treatment-related toxicity in any of the group of rats. Terminal autopsy also did not reveal any signs of toxicity. Further, no significant alterations in relative organ weight, serum biochemistry and urinalysis were observed up to 1% lactulose supplementation level. The results suggest that supplementation of lactulose in the diet does not produce any toxicity at the doses tested.     

A landscape-level assessment of Asian elephant habitat,its population and elephant–human conflict in the Anamalai hill ranges of southern Western Ghats,India

Spatial information at the landscape scale is extremely important for conservation planning, especially in the case of long-ranging vertebrates. The biodiversity-rich Anamalai hill ranges in the Western Ghats of southern India hold a viable population for the long-term conservation of the Asian elephant. Through rapid but extensive field surveys we mapped elephant habitat, corridors, vegetation and land-use patterns, estimated the elephant population density and structure, and assessed elephant–human conflict across this landscape. GIS and remote sensing analyses indicate that elephants are distributed among three blocks over a total area of about 4600 km². Approximately 92% remains contiguous because of four corridors; however, under 4000 km² of this area may be effectively used by elephants. Nine landscape elements were identified, including five natural vegetation types, of which tropical moist deciduous forest is dominant. Population density assessed through the dung count method using line transects covering 275 km of walk across the effective elephant habitat of the landscape yielded a mean density of 1.1 (95% CI = 0.99–1.2) elephant/km². Population structure from direct sighting of elephants showed that adult male elephants constitute just 2.9% and adult females 42.3% of the population with the rest being sub-adults (27.4%), juveniles (16%) and calves (11.4%). Sex ratios show an increasing skew toward females from juvenile (1:1.8) to sub-adult (1:2.4) and adult (1:14.7) indicating higher mortality of sub-adult and adult males that is most likely due to historical poaching for ivory. A rapid questionnaire survey and secondary data on elephant–human conflict from forest department records reveals that villages in and around the forest divisions on the eastern side of landscape experience higher levels of elephant–human conflict than those on the western side; this seems to relate to a greater degree of habitat fragmentation and percentage farmers cultivating annual crops in the east. We provide several recommendations that could help maintain population viability and reduce elephant–human conflict of the Anamalai elephant landscape.