Biodegradable and biocompatible multi-arm star amphiphilic block copolymer as a carrier for hydrophobic drug delivery

Multi-arm star amphiphilic block copolymers (SABCs) with approximately 32 arms were synthesized and characterized for drug delivery applications. A hyperbranched polyester, boltorn^® H40 (H40), was used as the macroinitiator for the ring-opening polymerization of ?-caprolactone (?-CL). The resulting multi-arm H40-poly(?-caprolactone) (H40-PCL-OH) was further reacted with carboxyl terminated methoxy poly(ethylene glycol) (MPEG-COOH) to form H40-PCL-b-MPEG copolymers. The resulting SABCs were characterized by ¹H NMR spectroscopy and gel permeation chromatography (GPC). The critical aggregation concentration (CAC) of H40-PCL-b-MPEG was 3.8 mg/L as determined by fluorescence spectrophotometry. Below the CAC, stable unimolecular micelles were formed with an average diameter of 18 nm as measured by TEM. Above the CAC, unimolecular micelles exhibited agglomeration with an average diameter of 98 nm. The hydrodynamic diameter of these agglomerates was found to be 122 nm, as measured by dynamic light scattering (DLS). The drug loading efficacy of the H40-PCL-b-MPEG micelles was 26 wt%. Drug release study showed an initial burst followed by a sustained release of the entrapped hydrophobic model drug, 5-fluorouracil, over a period of 9–140 h. These results indicate that the H40-PCL-b-MPEG micelles have great potential as hydrophobic drug delivery carriers.     

Development and evaluation of a rapid flow-through immuno filtration test using recombinant filarial antigen for diagnosis of brugian and bancroftian filariasis

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid-format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP-1. This test system employs colloidal gold-protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n = 70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non-reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil-transmitted helminthiases (n = 34) and protozoan infections (n = 33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow-through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.     

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.GroEL, an essential chaperonin, is known to form a ring-shaped structure for sequestering substrate proteins from the crowded cellular milieu and is responsible for the occurrence of various cellular processes, such as de novo folding, transport, and macromolecular assembly, within a biologically relevant time scale (7, 26, 48, 53). In Escherichia coli, GroEL, along with its cofactor GroES, assists the folding of about 10 to 30% of cytosolic proteins, among which some are known to be essential for cell viability (15, 26, 27, 31). GroEL was originally identified as the host factor responsible for phage λ and T4 capsid protein assembly and was subsequently shown to be essential for cell viability (17, 20). E. coli groEL is found in an operonic arrangement with groES (groESL), and its expression is regulated by multiple promoter elements.GroEL function has been shown to be a complex interplay between its interaction with and encapsulation of substrate proteins, with concomitant conformational changes induced by ATP binding, hydrolysis, and GroES binding (24, 56, 62). E. coli GroEL exists as a homotetradecamer forming two isologous rings of seven identical subunits each. Crystallographic analyses have delineated the three-domain architecture of GroEL monomers and the GroES-GroEL interactions (4, 63). The central region of the GroEL polypeptide, spanning amino acid residues 191 to 376, constitutes the GroES and substrate polypeptide-binding apical domain. The equatorial ATPase domain spanning two extremities of the GroEL polypeptide, that is, residues 6 to 133 and 409 to 523, is responsible for the ATPase activity and the bulk of intersubunit interactions. The hinge-forming intermediate domain, spanning two regions on the polypeptide, namely, residues 134 to 190 and 377 to 408, connects the said two domains in the tertiary structure. The conformational changes resulting from ATP binding and hydrolysis at the equatorial domain are coupled to those occurring at the apical domain via this hinge region (4, 63).The usual size limit for the substrate proteins, as shown by both in vitro and in vivo studies, is around 57 kDa, although the cis cavity is reported to theoretically accommodate larger proteins, on the order of 104 kDa (10, 27, 35, 46). Productive in vivo folding of the proteins larger than the usual size limit, such as the 86-kDa maltose binding protein fusion and 82-kDa mitochondrial aconitase, has also been reported (9, 29). Since such large substrates are difficult to accommodate in the central cavity, it has been suggested that their productive folding might occur outside the cis cavity. These studies therefore indicate that the substrate recognition patterns of GroEL may be more diverse than initially thought.Recent genome annotation studies of various bacteria have revealed that a few bacterial genomes possess multiple copies of groEL genes (2, 18, 30). The Mycobacterium tuberculosis genome bears two copies of groEL genes (groELs). One of these, groEL1, is arranged in an operon, with the cognate cochaperonin groES being the first gene, while the second copy, groEL2, exists separately on the genome (13). Recombinant mycobacterial GroELs were shown to possess biochemical features that deviated significantly from the trademark properties of E. coli GroEL. The most striking feature of M. tuberculosis GroELs, however, was their oligomeric state, where contrary to expectations, in vitro they did not form the canonical tetradecameric assembly when purified from E. coli. The proteins instead existed as lower oligomers (dimers) irrespective of the presence or absence of cofactors, such as the cognate GroES or ATP (40, 41). Furthermore, they displayed weak ATPase activities and GroES independence in preventing aggregation of the denatured polypeptides.Evolutionary studies of M. tuberculosis groEL sequences have suggested rapid evolution of the groEL1 gene, yet without turning these into pseudogenes (21). The other hypothesis suggests that M. tuberculosis, being an organism that grows slowly, might require GroEL function that does not utilize ATP rapidly but, rather, with a slow turnover rate. Alternately, additional mechanisms might exist in M. tuberculosis which could mediate regulated oligomerization of M. tuberculosis chaperonins. Such regulation might help in the controlled utilization of ATP in nutrient-deprived M. tuberculosis, as observed for other chaperones, such as small heat shock proteins (23).In the present study, we have exploited the unusual oligomeric status of the recombinant M. tuberculosis GroELs to study the significance of oligomer formation for GroEL''s function as a molecular chaperone. Furthermore, we have explored the possibility of the existence of regulated oligomerization for native M. tuberculosis GroELs in their natural setting. We first show that M. tuberculosis groEL genes are not capable of complementing a conditional allele of E. coli groEL, namely, groEL44. The results of phenotypic and biochemical analyses of GroEL variants obtained by gene shuffling and domain swapping suggest that the impaired chaperoning ability of recombinant M. tuberculosis GroELs is a consequence of their inability to form higher-order oligomers in E. coli and that oligomerization is the prelude to the formation of an active GroEL chaperonin. Further, by immunochemical and mass spectrometric (MS) analysis of native mycobacterial GroELs, we show that M. tuberculosis GroEL1 exists in multiple oligomeric forms, viz., monomeric, dimeric, heptameric (single ring), and tetradecameric (double ring) forms, and that the switch between single-ring and double-ring variants is operated by phosphorylation on a serine residue. These observations suggest that the determinants of oligomerization for M. tuberculosis GroEL1 are distinct from those of its E. coli counterpart and that it does oligomerize in M. tuberculosis (its native environment), whereas it loses its oligomerization capability when expressed in E. coli. It could thus be possible that M. tuberculosis GroEL1 requires a certain native M. tuberculosis protein, probably a eukaryotic-like Ser-Thr protein kinase, to oligomerize properly, though the precise reason cannot be discerned by these observations.     

Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui

Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRβ, is upregulated in response to TH. Similarly, EcTRβ, the E. coqui ortholog, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.     

P2-P4 macrocyclic inhibitors of hepatitis C virus NS3-4A serine protease

Synthesis and HCV NS3 serine protease inhibitory activity of 4-hydroxyproline derived macrocyclic inhibitors and SAR around this macrocyclic core is described in this communication. X-ray structure of inhibitor 38 bound to the protease is discussed.     

Effects of Chronic Arginase Inhibition with Norvaline on Tau Pathology and Brain Glucose Metabolism in Alzheimer's Disease Mice

Neurochemical Research - Alzheimer's disease (AD) is an insidious neurodegenerative disorder representing a serious continuously escalating medico-social problem. The AD-associated progressive...     

Association between Farming and Chronic Energy Deficiency in Rural South India

Objective

To examine factors associated with chronic energy deficiency (CED) and anaemia in disadvantaged Indian adults who are mostly involved in subsistence farming.

Design

A cross-sectional study in which we collected information on socio-demographic factors, physical activity, anthropometry, blood haemoglobin concentration, and daily household food intake. These data were used to calculate body mass index (BMI), basal metabolic rate (BMR), daily energy expenditure, and energy and nutrient intake. Multivariable backward stepwise logistic regression was used to assess socioeconomic and lifestyle factors associated with CED (defined as BMI<18 kg/m²) and anaemia.

Setting

The study was conducted in 12 villages, in the Rishi Valley, Andhra Pradesh, India.

Subjects

Individuals aged 18 years and above, residing in the 12 villages, were eligible to participate.

Results

Data were available for 1178 individuals (45% male, median age 36 years (inter quartile range (IQR 27–50)). The prevalence of CED (38%) and anaemia (25%) was high. Farming was associated with CED in women (2.20, 95% CI: 1.39–3.49) and men (1.71, 95% CI: (1.06–2.74). Low income was also significantly associated with CED, while not completing high school was positively associated with anaemia. Median iron intake was high: 35.7 mg/day (IQR 26–46) in women and 43.4 mg/day (IQR 34–55) in men.

Conclusions

Farming is an important risk factor associated with CED in this rural Indian population and low dietary iron is not the main cause of anaemia. Better farming practice may help to reduce CED in this population.     

A search tool for identification and analysis of conserved sequence patterns in Saccharomyces spp. orthologous promoter

We describe a web-based resource to identify, search and analyze sequence patterns conserved in the multiple sequence alignments of orthologous promoters from closely related / distant Saccharomyces spp. The webtool interfaces with a database where conserved sequence patterns (greater than 4 bp) have been previously extracted from genome-wide promoter alignments, allowing one to carry out user-defined genome-wide searches for conserved sequences to assist in the discovery of novel promoter elements based on comparative genomics. The web-based server can be accessed at http://www2.imtech.res.in/ anand/sacch_prom_pat.html.