Development of a method for the purification and culture of rodent astrocytes

The inability to purify and culture astrocytes has long?hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in?vivo and that the vast majority of astrocytes contact blood vessels, suggesting that?astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in?vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation.     

Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days.     

The institute for genomic research Osa1 rice genome annotation database

We have developed a rice (Oryza sativa) genome annotation database (Osa1) that provides structural and functional annotation for this emerging model species. Using the sequence of O. sativa subsp. japonica cv Nipponbare from the International Rice Genome Sequencing Project, pseudomolecules, or virtual contigs, of the 12 rice chromosomes were constructed. Our most recent release, version 3, represents our third build of the pseudomolecules and is composed of 98% finished sequence. Genes were identified using a series of computational methods developed for Arabidopsis (Arabidopsis thaliana) that were modified for use with the rice genome. In release 3 of our annotation, we identified 57,915 genes, of which 14,196 are related to transposable elements. Of these 43,719 non-transposable element-related genes, 18,545 (42.4%) were annotated with a putative function, 5,777 (13.2%) were annotated as encoding an expressed protein with no known function, and the remaining 19,397 (44.4%) were annotated as encoding a hypothetical protein. Multiple splice forms (5,873) were detected for 2,538 genes, resulting in a total of 61,250 gene models in the rice genome. We incorporated experimental evidence into 18,252 gene models to improve the quality of the structural annotation. A series of functional data types has been annotated for the rice genome that includes alignment with genetic markers, assignment of gene ontologies, identification of flanking sequence tags, alignment with homologs from related species, and syntenic mapping with other cereal species. All structural and functional annotation data are available through interactive search and display windows as well as through download of flat files. To integrate the data with other genome projects, the annotation data are available through a Distributed Annotation System and a Genome Browser. All data can be obtained through the project Web pages at http://rice.tigr.org.     

Cryptochrome 1 contributes to blue-light sensing in pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea.     

A novel anti-inflammatory role of simvastatin in a murine model of allergic asthma

Statins, the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, are effective serum cholesterol-lowering agents in clinical practice, and they may also have anti-inflammatory properties. Asthma is characterized by chronic eosinophilic inflammation in the airways, which is thought to be regulated by the activity of T lymphocytes. We therefore examined the anti-inflammatory activity of simvastatin in a murine model of allergic asthma. In mice previously sensitized to OVA, simvastatin treatment, either orally or i.p., reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid in response to inhaled OVA challenge. Simvastatin therapy i.p. was also associated with a reduction in IL-4 and IL-5 levels in bronchoalveolar lavage fluid and, at higher doses, a histological reduction in inflammatory infiltrates in the lungs. OVA-induced IL-4, IL-5, IL-6, and IFN-gamma secretion was reduced in thoracic lymph node cultures from simvastatin-treated mice. Simvastatin treatment did not alter serum total IgE or OVA-specific IgG1 and IgG2a levels. These data demonstrate the therapeutic potential of statin-sensitive pathways in allergic airways disease.     

Heme oxygenase-1 gene transfer inhibits angiotensin II-mediated rat cardiac myocyte apoptosis but not hypertrophy

Cardiac myocyte apoptosis underlies the pathophysiology of cardiomyopathy, and plays a critical role in the transition from myocardial hypertrophy to heart failure. Angiotensin II (Ang II) induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress; however, the mechanisms underlying the activation of both pathways and their interactions remain unclear. In the present study, we have investigated whether overexpression of the antioxidant protein heme oxygenase-1 (HO-1) protects against apoptosis and hypertrophy in cultured rat cardiac myocytes treated with Ang II. Our findings demonstrate that Ang II (100 nM, 24 h) alone upregulates HO-1 expression and induces both myocyte hypertrophy and apoptosis, assessed by measuring terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) staining, caspase-3 activity and mitochondrial membrane potential. Ang II elicited apoptosis was augmented in the presence of tin protoporphyrin, an inhibitor of HO activity, while HO-1 gene transfer to myocytes attenuated Ang II-mediated apoptosis but not hypertrophy. Adenoviral overexpression of HO-1 was accompanied by a significant increase in Ang II induced phosphorylation of Akt, however, Ang II-mediated p38 mitogen activated protein kinase (MAPK) phosphorylation was attenuated. Inhibition of phosphotidylinositol-3-kinase enhanced myocyte apoptosis elicited by Ang II, however, p38MAPK inhibition had no effect, suggesting that overexpression of HO-1 protects myocytes via augmented Akt activation and not through modulation of p38MAPK activation. Our findings identify the signalling pathways by which HO-1 gene transfer protects against apoptosis and suggest that overexpression of HO-1 in cardiomyopathies may delay the transition from myocyte hypertrophy to heart failure.