The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.     

Insecticide induced disruptions in functioning of developing brain of Rana cyanophlictis.

Synthetic pyrethroid insecticide permethrin significantly decreased the levels of regulatory proteins (S-100 and calmodulin) in the developing CNS of tadpoles of R. cyanophlictis. Remarkable inhibition of enzymes acetylcholinesterase and choline acetylase and significant accumulation of neurotransmitter acetylcholine were observed in permethrin treated animals. Permethrin exposure significantly decreased the activity of phosphodiesterase. The results support molecular disruptions occurring due to permethrin induced toxicity. This in turn may bring about neuronal inefficiency in the treated tadpoles.     

Gallic acid based steroidal phenstatin analogues for selective targeting of breast cancer cells through inhibiting tubulin polymerization

Phenstatin analogues were synthesized on steroidal framework, for selective targeting of breast cancer cells. These analogues were evaluated for anticancer efficacy against breast cancer cell lines. Analogues 12 and 19 exhibited significant anticancer activity against MCF-7, hormone dependent breast cancer cell line. While analogues 10-14 exhibited significant anticancer activity against MDA-MB-231, hormone independent breast cancer cell line. Compound 10 showed significant oestrogen antagonistic activities with low agonistic activity in in vivo rat model. These analogues also retain tubulin polymerization inhibition activity. The most active analogue 10 was found to be non-toxic in Swiss albino mice up to 300 mg/kg dose. Gallic acid based phenstatin analogues may further be optimized as selective anti-breast cancer agents.     

Microbial fuel cells as an alternative energy source: current status

Microbial fuel cell (MFC) technology is an emerging area for alternative renewable energy generation and it offers additional opportunities for environmental bioremediation. Recent scientific studies have focused on MFC reactor design as well as reactor operations to increase energy output. The advancement in alternative MFC models and their performance in recent years reflect the interests of scientific community to exploit this technology for wider practical applications and environmental benefit. This is reflected in the diversity of the substrates available for use in MFCs at an economically viable level. This review provides an overview of the commonly used MFC designs and materials along with the basic operating parameters that have been developed in recent years. Still, many limitations and challenges exist for MFC development that needs to be further addressed to make them economically feasible for general use. These include continued improvements in fuel cell design and efficiency as well scale-up with economically practical applications tailored to local needs.     

An Endogenous Inhibitor of Ca++-ATPase from Human Placenta

Intracellular free calcium is regulated by Ca⁺⁺-ATPase, one form present on the plasma membrane (PM Ca⁺⁺-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca⁺⁺-ATPase). An endogenous inhibitor of SR Ca⁺⁺-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca⁺⁺-ATPase by this inhibitor was concentration-and time-dependent. The inhibitor neither bound to DEAE-nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15min up to 55°C and completely destroyed at 80°C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca⁺⁺-ATPase in the placenta during pregnancy.     

Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.The majority of proteins function as part of multiprotein complexes and not as isolated polypeptides. These protein complexes range from simple homodimers to large structures composed of many different polypeptides. Protein complexes vary in their size and shape from small globular dimers, such as 14-3-3 proteins, to large elongated filaments of variable length, such as microtubules. The wide variety of possible protein–protein interactions within multiprotein complexes contributes to the diversity of functions that are involved in cellular processes and regulatory mechanisms.Another important source of functional diversity and regulation is the large number of protein isoforms that may be generated from each gene. Functionally and structurally distinct isoforms can arise via multiple mechanisms, including alternative splicing, post-translational modification (PTM),¹ and proteolytic cleavage. Distinct isoforms can exhibit radically different properties. For example, including or excluding individual exons can either create or remove protein–protein interaction interfaces for binding specific interaction partners. Similarly, phosphorylation, and other PTMs, can either create or remove binding sites for interacting proteins, substrates, or ligands. PTMs can also promote structural changes in proteins and affect catalytic activity.The association of protein isoforms and post-translationally modified factors in multiprotein complexes can influence their subcellular location, activity, and substrate specificity. This can be dynamically regulated to modulate protein complex composition, and hence localization and function, to allow cells to respond to spatial and temporal stimuli. It is therefore important to characterize protein complexes at the level of the protein isoforms and post-translationally modified forms they contain in order to fully decipher the network of signaling and regulatory pathways within cells.Although many types of protein complexes have been studied in detail, in-depth analysis of the composition, dynamics, and isoform association of protein complexes formed in either human cells or model organisms is still not well documented at a system-wide level. The CORUM database, compiled using a variety of information from the literature describing protein interactions and assemblies, currently provides the largest public dataset of protein complexes (1). CORUM contains information relating to ∼1,970 protein complexes identified in human cells. However, these complexes are formed from proteins encoded by only ∼16% of the known human protein-coding genes, indicating that many forms of protein complexes still remain to be identified and characterized (1). Furthermore, the CORUM database does not describe how the protein compositions of the complexes may vary, either dynamically or in different subcellular locations, or how this relates to protein isoforms and PTMs. This illustrates that there is still a major deficit in our knowledge of the structure and functions of cellular protein complexes and how they contribute to biological regulatory mechanisms.The technique that is now most widely used to identify the components of protein complexes is affinity purification of an individual “bait” protein and subsequent analysis of the co-isolated proteins, usually via mass spectrometry (2). Affinity purification can use antibodies specific for an endogenous target protein (3, 4), if available, or, alternatively, can utilize a genetically constructed, epitope-tagged bait protein. The latter procedure is now widely used and is advantageous in that many different complexes can be compared using an identical antibody, or other affinity-purification method, targeted to the tag on the bait (for examples, see Refs. 5–7; for reviews, see Refs. 8 and 9). In contrast, it is harder to directly compare the results from immunoprecipitation of different endogenous protein complexes because each specific antibody that is used has different affinities and properties. Nonetheless, although epitope-tag “pull-down” techniques are now commonly used, they also have limitations. Not least, the addition of epitope tags to the bait can affect protein function and interactions (10, 11).To help determine whether co-purifying proteins detected using pull-down strategies represent specific partner proteins in bona fide complexes or are nonspecific contaminants, we and others have developed quantitative approaches, for example, based on variations of the stable isotope labeling of amino acids in cell culture (12–16). Additional data analysis procedures, including the use of a “super experiment” database that predicts the likelihood of nonspecific protein interactions based on the frequency with which any given protein is co-purified across many separate experiments, can also help to define the composition of protein complexes (17). Nonetheless, affinity purification strategies have a limited ability to distinguish multiple related complexes that may differ with respect to isoforms and PTMs. They are also costly and difficult to implement for large-scale studies to survey cellular complexes, and thus not well suited to study variations in complexes under different cellular growth conditions and responses.For system-wide studies of the composition and dynamics of protein complexes, alternative methods, in addition to immune-affinity purification, are required for convenient separation, characterization, and comparison of cellular protein complexes. To address this, a number of studies have utilized various forms of either column chromatography or native gel electrophoresis in combination with mass-spectrometry-based proteomics. For example, protein complexes have been separated using techniques including blue native polyacrylamide gel electrophoresis (18, 19), ion-exchange chromatography (20), and size-exclusion chromatography (21, 22) prior to MS analysis of proteins in the fractionated complexes. Size-exclusion chromatography (SEC) is a well-established technique used to separate proteins and protein complexes in solution on the basis of their shape/size (rotational cross-section) (23). SEC has been extensively used as an intermediate step in conventional multistep biochemical protein purification strategies. In contrast, SEC has been less commonly combined with mass-spectrometry-based proteomics for the high-throughput characterization of protein complexes. However, this has been demonstrated in previous studies that analyzed native protein complexes in plant chloroplasts (22) or large cytosolic complexes in mammalian cells (21).In this study, we combined native SEC with high-throughput mass-spectrometry-based proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma cells. Herein we demonstrate the utility and reproducibility of this approach for the system-wide characterization of endogenous, untagged protein complexes and show how it can be used to identify specific protein isoforms and PTMs associated with distinct protein complexes. The resulting data are available to the community in a convenient format in the Encyclopedia of Proteome Dynamics (EPD) (www.peptracker.com/encyclopediaInformation/), a user-friendly, searchable online database.     

Inhibition of 5-α-Reductase (TYPE-II) Expression by Antisense 3′-Deoxy-(2′-5′) Oligonucleotide Chimeras

Abstract

ABSTRACT: 3′-Deoxy-(2′-5′) oligonucleotides bind selectively to complementary RNA but not to DNA. 3′-Deoxy-(2′-5′) phosphorothioate ODN chimeras embedded with a short stretch of 3′-5′ phosphorothioate cassette are potent inhibitors of steroid 5-α-reductasc expression with significantly less non-specific interactions in cell culture.     

The Synthesis of Two Novel Ring-Expanded Xanthine Nucleosides Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The synthesis of two regioisomeric nucleosides, 4,5,7,8-tetrahydro-6H-3-(β-D-ribofuranosyl)imidazo[4,5-e][1,4]diazepin-5,8-dione (2) and its 1-glycosyl analogue (3) is reported. They were prepared by ribosydation of the heterocyclic aglycon 4 which in turn was synthesized in three steps from 4(5)-nitroimidazole-5(4)-carboxylic acid (5). Distinction between the two isomers was achieved by an unequivocal synthesis of 2.     

A Next-Generation Cleaved,Soluble HIV-1 Env Trimer,BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41_ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.     

Ethnic Variation in Inflammatory Profile in Tuberculosis

Distinct phylogenetic lineages of Mycobacterium tuberculosis (MTB) cause disease in patients of particular genetic ancestry, and elicit different patterns of cytokine and chemokine secretion when cultured with human macrophages in vitro. Circulating and antigen-stimulated concentrations of these inflammatory mediators might therefore be expected to vary significantly between tuberculosis patients of different ethnic origin. Studies to characterise such variation, and to determine whether it relates to host or bacillary factors, have not been conducted. We therefore compared circulating and antigen-stimulated concentrations of 43 inflammatory mediators and 14 haematological parameters (inflammatory profile) in 45 pulmonary tuberculosis patients of African ancestry vs. 83 patients of Eurasian ancestry in London, UK, and investigated the influence of bacillary and host genotype on these profiles. Despite having similar demographic and clinical characteristics, patients of differing ancestry exhibited distinct inflammatory profiles at presentation: those of African ancestry had lower neutrophil counts, lower serum concentrations of CCL2, CCL11 and vitamin D binding protein (DBP) but higher serum CCL5 concentrations and higher antigen-stimulated IL-1 receptor antagonist and IL-12 secretion. These differences associated with ethnic variation in host DBP genotype, but not with ethnic variation in MTB strain. Ethnic differences in inflammatory profile became more marked following initiation of antimicrobial therapy, and immunological correlates of speed of elimination of MTB from the sputum differed between patients of African vs. Eurasian ancestry. Our study demonstrates a hitherto unappreciated degree of ethnic heterogeneity in inflammatory profile in tuberculosis patients that associates primarily with ethnic variation in host, rather than bacillary, genotype. Candidate immunodiagnostics and immunological biomarkers of response to antimicrobial therapy should be derived and validated in tuberculosis patients of different ethnic origin.