Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.     

Assessing the effectiveness of community managed forests for plant diversity conservation in Meghalaya,Northeast India

In the state Meghalaya, northeast India, >80% of the forest lands are owned by local communities and managed by traditional institutions. These forests are under severe threats due to a number of human disturbances. The present study was conducted to assess the plant diversity and identify the community forests for priority conservation in Khasi Hills of Meghalaya. Floristic explorations carried out in the 87 forests reveals the presence of 1300 plant species of which 400 are either rare, endemic or threatened. Of the different forest categories, reserve forests had the highest number of species (1190), followed by sacred forests (987 species) and village forests (786 species). Majority of the forests (56) had high-species richness, irreplaceability level (42 forests) and vulnerability level (54). In terms of area, 13.8% (1666.8 ha) fall under low risk while 1855 ha under high risk zone. High risk zone was mostly represented by village forests. An area of 7661.56 ha of community forests falls under high priority category and hence calls for immediate conservation actions. The conservation priority map generated in the present study will help to concentrate the protection strategy to the demarcated and adjoining areas and help conservationists and planners to evolve effective strategies for conservation of the community forests.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

Audit of a tuberculosis contact tracing clinic.

OBJECTIVE--To determine the efficiency of tuberculosis contact tracing in South Glamorgan 1987-9. DESIGN--Review of records of contact tracing clinic and of data from the Mycobacterium Reference Unit. The clinic''s practice was compared with 1983 British Thoracic Society''s recommendations. SETTING--Health authority tuberculosis control programme. MAIN OUTCOME MEASURES--Proportion of contacts screened, follow up attendance rates, number of secondary cases detected, and quality of record keeping. RESULTS--101 index patients and 611 contacts were identified. 596 (97.5%) contacts were screened, of whom 139 should not have been. Of 356 contacts requiring a Heaf test, 237 were tested, seven refused the test, and 112 had chest radiography without a Heaf test. 95 contacts were unnecessarily tested. 87 contacts had chest radiography unnecessarily and seven should have had radiography but did not. 34 contacts were given follow up appointments inappropriately and seven were overlooked for follow up. Tuberculosis was diagnosed in five asymptomatic contacts, all at initial screening and all close contacts of index patients with pulmonary disease. CONCLUSION--Inadequacy of data, non-adherence to contact tracing guidelines, and failure to define the term highly infectious index case resulted in many contacts being unnecessarily screened or followed up. IMPLICATIONS--The efficiency of tracing contacts would be improved by specifying smear results and ethnic origin of the index case on the notification form, clearly classifying contacts as close or causal, and clearly defining the term highly infectious.     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

An Efficient Algorithm for Some Highly Nonlinear Fractional PDEs in Mathematical Physics

In this paper, a fractional complex transform (FCT) is used to convert the given fractional partial differential equations (FPDEs) into corresponding partial differential equations (PDEs) and subsequently Reduced Differential Transform Method (RDTM) is applied on the transformed system of linear and nonlinear time-fractional PDEs. The results so obtained are re-stated by making use of inverse transformation which yields it in terms of original variables. It is observed that the proposed algorithm is highly efficient and appropriate for fractional PDEs and hence can be extended to other complex problems of diversified nonlinear nature.