Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells.

BackgroundMycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein.ResultsThe recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like β-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity.ConclusionsThe Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.

We have examined the A431 (human epidermoid carcinoma) and HT29 (human colorectal carcinoma) cellular responses evoked by lectins of dietary origin, Jacalin of Artocarpus integrifolia (native jacalin; nJacalin), peanut agglutinin (PNA) of Arachis hypogea, and recombinant single-chain jacalin (rJacalin), which has the same protein backbone but approximately 100-fold less affinity for carbohydrates than nJacalin. All three lectins (nJacalin, rJacalin, and PNA) are cycotoxic inhibitors of proliferation of A431 cells. However, cells recover once jacalin but not PNA have been removed from the growth medium. Treatment of nJacalin results in morphologically visible cell rounding while retaining the membrane integrity when treated at 40 microg ml(-1), but treatment with PNA did not induce such changes. The observed cell rounding was found to be due to stress as the phosphorylation of caveolin-1 (at tyr14), p38 but not c-Jun N-terminal kinase were up-regulated, while PNA did not up-regulate the phosphorylation of the same. Jacalin also down-regulated the phosphorylation of the epidermal growth factor receptor and extracellular signal regulated kinase in contrast to PNA, which failed to down-regulate the same. Confocal microscopic studies reveal that jacalin is not internalized, unlike the lectin of Agaricus bisporous. Analysis of the proteins that bind to an nJacalin-sepharose column revealed the binding of six to eight proteins, and significant among them is a protein at approximately 110 kDa, which appears to be oxygen-regulated protein 150 (ORP150) (endoplasmic reticulum chaperone) as identified by its isoelectric point, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This 110-kDa band is detectable with anti-Hsp70 antibody because ORP150 has homology with Hsp70. Confocal microscopic studies reveal the presence of Hsp70-like proteins on the surface of A431 cells as revealed by immunostaining with anti-Hsp70 antibody. Moreover, overexpression of ORP150 in A431 cells has resulted in a dramatic protection of A431 cells against jacalin-induced toxicity, confirming that the jacalin-induced cytotoxicity is mediated through ORP150, and impairment of ORP150 functions with the help of jacalin makes the cells more susceptible to death due to stress. Our studies suggest that the cellular responses, as a consequence of lectin binding, may not be exclusively mediated by carbohydrate binding property alone, but other factors such as protein-protein interactions may also contribute to the observed cellular responses.

Additional Information:• This article was accepted for publication in the journal, Environment SUMMARYMunicipal water supply in Karachi has become grossly inadequate with reference to the user's needs and expectations. As a consequence of this inadequacy, communities suffer from the poor level of service. Peri urban locations, especially low-income settlements have very limited access to municipal water supply. Communities in these locations have to evolve alternate arrangements for acquiring the minimum quantities of water for human survival. In some situations, public sector agencies extend support in service delivery. Benefits of such support are appropriately availed when the community members organise themselves and subsequently develop working relationships with the concerned service providers. Awami tanks represent an example where water supply service was developed as a cooperative arrangement, partnership, between informally developed community organizations and public sector agencies. This paper reviews the performance of awami tanks and explores the evolving partnerships between service providers, recipients of the service and other related stakeholders. Findings and analysis in this paper are based on documentation of 20 awami tanks in different locations in Orangi including Ghaziabad, Gulshan-e-Zia, Mansoor Nagar, Gulshan-e-Bihar and Raees Amrohi Colony. Structured feedback is obtained from concerned stakeholders such as tanker operators and owners, Karachi Water and Sewerage Board (KWSB) staff, Rangers personnel and municipal representatives. In addition, focused group meetings were held with tanker operators and area residents. Interviews were held with KWSB staff, Rangers 1 , elected councillors and political activists all of whom shared their views under the condition of anonymity. The findings of this paper reveal the evolution, development, operation and management of awami tanks. Analysis is done and conclusions drawn to assess the possibilities of sustainability and expansion of awami tank network.

Municipal water supply in Karachi has become grossly inadequate with reference to the user's needs and expectations. As a consequence of this inadequacy, communities suffer from the poor level of service. Peri urban locations, especially low-income settlements have very limited access to municipal water supply. Communities in these locations have to evolve alternate arrangements for acquiring the minimum quantities of water for human survival. In some situations, public sector agencies extend support in service delivery. Benefits of such support are appropriately availed when the community members organise themselves and subsequently develop working relationships with the concerned service providers. Awami tanks represent an example where water supply service was developed as a cooperative arrangement, partnership, between informally developed community organizations and public sector agencies. This paper reviews the performance of awami tanks and explores the evolving partnerships between service providers, recipients of the service and other related stakeholders. Findings and analysis in this paper are based on documentation of 20 awami tanks in different locations in Orangi including Ghaziabad, Gulshan-e-Zia, Mansoor Nagar, Gulshan-e-Bihar and Raees Amrohi Colony. Structured feedback is obtained from concerned stakeholders such as tanker operators and owners, Karachi Water and Sewerage Board (KWSB) staff, Rangers personnel and municipal representatives. In addition, focused group meetings were held with tanker operators and area residents. Interviews were held with KWSB staff, Rangers 1 , elected councillors and political activists all of whom shared their views under the condition of anonymity. The findings of this paper reveal the evolution, development, operation and management of awami tanks. Analysis is done and conclusions drawn to assess the possibilities of sustainability and expansion of awami tank network. 1. WATER SUPPLY IN KARACHI-A BACKGROUND

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Low-income communities residing in squatter settlements usually develop their own services through self-help efforts. Such schemes include solid waste disposal, with the aim of placing household and other kinds of waste outside the locality from where it can be removed by municipal authorities. This minimalist system ensures cleanliness and basic upkeep in the community area. The system normally utilises a sweeper and basic waste-collection tools such as a wheelbarrow improvised for waste collection, hand tools and collection bins used by households for routine collection. A community mobilisation effort is required for the efficient working of such a system. In two low-income communities in Karachi, this approach was applied by a local non-government organisation (NGO), the Association for Protection of Environment (APE), in 1996. After providing continuous professional support for three years with the objective of acting as a catalyst, APE trained several members of the local community-based organisation (CBO) to manage and run the project on an independent basis. This paper provides an account of the approach and the system that evolved from it. After withdrawal of the NGO in 2000, the project has been working successfully under the supervision of the local CBO and area youth. This paper presents the lessons learned from the process of empowering communities to develop their own internal service systems. It addresses the issues that are vital in ensuring the sustainability of such attempts in urban lower-income localities.

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