Fatty acids-carotenoid complex: An effective anti-TB agent from the chlorella growth factor-extracted spent biomass of Chlorella vulgaris.

ETHNOPHARMACOLOGICAL RELEVANCE: The multidrug-resistant (MDR) pathogen, Mycobacterium tuberculosis, still remains as one of the major threat to mankind, despite the availability of a live attenuated vaccine and effective antibiotics. Marine microalgae, at all times, act as a key resource for valuable therapeutic compounds with limited side effects. AIM OF THE STUDY: The present explorative attempt is to isolate the biomolecules of pharmacological importance from the marine microalgae, Chlorella vulgaris, and to evaluate its effect on the ever dreadful disease, Tuberculosis. The study is also aimed to develop an economically feasible methodology for by-products extraction from microalgae. MATERIALS AND METHODS: Fatty acids-carotenoid complexes (FACC), namely, FACC-1 (red oil) and FACC-2 (brown oil) were isolated, in addition to lipid and lutein from the Chlorella Growth Factor (CGF, a protein fraction enriched with vitamins, minerals and carbohydrates)-extracted spent biomass through column chromatography. RESULTS: FACC-1 is a complex of fatty acids such as oleic and linoleic acids, and carotenoids such as canthaxanthin and neoxanthin. FACC-2 is a complex of oleic, linoleic and linolenic acids and carotenoids (cryptoxanthin and echinenone). Initial screening for evaluation of minimum bactericidal concentration (MBC) of FACC-1 and -2 was performed against Mycobacterium tuberculosis strains such as H37Rv, SHRE sensitive clinical isolate and SHRE resistant clinical isolate. MBC was noted at 10 µg/mL by FACC-1 and at 5 µg/mL by FACC-2, determined using colony forming and Lucipherase Reporter Mycobacteriophages (LRP) assay. Testing in the PAN sensitive isolates indicated that the MBC was noted at 5 µg/mL by FACC-1 and at 2.5 µg/mL by FACC-2. Complete inhibition (100%) was observed at 100 µg/mL by FACC-1 and at 50 µg/mL by FACC-2. Testing of FACC-1 and FACC-2 individually as well as in combination on two different types of MDR strains confirmed the efficacy of the algal oils, wherein in MDR-strain 1, FACC-1 revealed 50% inhibition at 10 µg/mL, while FACC-2 exhibited the same at 5 µg/mL. Conversely, in the case of MDR strain-2, MBC of FACC-1 was at 500 µg/mL and MBCof FACC-2 to be at 250 µg/mL. No significant synergistic effect was observed on combining both the oils. CONCLUSION: The study signifies the development of a potent therapeutic agent comprising of a complex of anti-TB agent (fatty acids) and antioxidants (carotenoids) from the CGF-extracted spent biomass of C. vulgaris.

Granzyme B regulates antiviral CD8+ T cell responses.

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.

Bioactivity-guided isolation of mosquitocidal constituents from the rhizomes of Plumbago capensis Thunb.

A bioassay-guided fractionation and chemical examination of chloroform extract of Plumbago capensis roots resulted in isolation and characterization of two new napthaquinone derivatives (4, 8) along with six known compounds (1-3, 5-7). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested for their mosquito larvicidal activity against fourth instar larvae of Aedes aegypti, and compared with that of rotenone. Among the tested compounds, isoshinanolone (3) and plumbagin (1) showed excellent toxicity with LC(50) values of 1.26 and 5.43 microg/mL. New compound (8) displayed moderate toxicity against the tested mosquito species.

A new benzil derivative from Derris scandens: Structure-insecticidal activity study.

Bioactivity-directed investigation of root extract of Derris scandens has led to the isolation and characterization of a new benzil derivative (11), along with ten known compounds (1-10). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. The insect antifeedant activity and growth inhibitory studies of these compounds were investigated against castor semilooper pest, Achaea janata using a no-choice laboratory bioassay. Several of the isolates displayed potent feeding deterrence and were also toxic or caused developmental abnormalities following topical administration. The new compound, derrisdione A was moderately active with an antifeedant index of 58.6+/-1.7% at 10microg/cm(3) against A. janata.

CD25 deficiency causes an immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome, and defective IL-10 expression from CD4 lymphocytes.

BACKGROUND: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) results in systemic autoimmunity from birth and can be caused by mutations in the transcription factor forkhead box P3 (FOXP3). OBJECTIVE: To determine if Foxp3 is required for the generation of IL-10-expressing T regulatory cells. METHODS: CD4 lymphocytes were isolated from patients with IPEX-like syndromes and activated with antibodies to CD3 and CD46 to generate IL-10-expressing T regulatory cells. RESULTS: We describe a patient with clinical manifestations of IPEX that had a normal Foxp3 gene, but who had CD25 deficiency due to autosomal recessive mutations in this gene. This patient exhibited defective IL-10 expression from CD4 lymphocytes, whereas a Foxp3-deficient patient expressed normal levels of IL-10. CONCLUSION: These data show that CD25 deficiency results in an IPEX-like syndrome and suggests that although Foxp3 is not required for normal IL-10 expression by human CD4 lymphocytes, CD25 expression is important. CLINICAL IMPLICATIONS: Any patient with features of IPEX but with a normal Foxp3 gene should be screened for mutations in the IL-2 receptor subunit CD25.

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

A new method of synthesis and antibacterial activity evaluation of piper amides.

Several natural piper amides and their mimics were synthesized by developing a new strategy of amide formation. The piper amides were tested against both gram +ve and gram -ve bacterial strains and found that they are particularly more active against Staphylococcus aureus and Chromobacterium violaceum strains.

Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris.

A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene. The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography. Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase. To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man. The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site. Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain. In conclusion, the full-length CD-MPR produced in P. pastoris is structurally and functionally suitable for crystallization studies.

High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris.

Mannose 6-phosphate receptors (MPRs) form essential components of the lysosomal enzyme targeting system by binding newly synthesized acid hydrolases with high (nM) affinity. We report the use of Pichia pastoris as a host to efficiently express the extracytoplasmic ligand-binding domain of the cation-dependent mannose 6-phosphate receptor. A truncated and glycosylation-deficient form of the receptor AF-Asn(81)/Stop(155) was secreted into the culture medium, yielding approximately 28mg/L after purification, which is an improvement of 10-100-fold compared to expression in baculovirus-infected insect cells and mammalian cells, respectively. Enzymatic deglycosylation indicated high-mannose sugars at the single potential glycosylation site of Asn 81. The extent and heterogeneity of N-glycans were revealed by applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the case of AF-Asn(81)/Stop(155), the majority (75%) of the oligosaccharides contained chain lengths of Man(8-10)GlcNAc(2) while Man(11-12)GlcNAc(2) comprised the remaining (25%) N-linked sugars. A comparative MALDI-TOF spectra of Asn(81)/Stop(155) purified from insect cells indicated that Man(2-3)GlcNAc(2) and GlcNAcMan(2-3)GlcNAc(2) share the oligosaccharide pool. The receptor isolated from yeast was functional with respect to ligand binding and acid-dependent dissociation properties, as determined by pentamannosyl phosphate-agarose affinity chromatography. In addition, the protein was biochemically and functionally similar to Asn(81)/Stop(155) expressed in insect cells concerning its oligomeric state and binding affinity to the lysosomal enzyme, beta-glucuronidase (K(d)=1.4nM). These results demonstrate that P. pastoris is a convenient system for the production of large quantities of functional recombinant MPRs suitable for structure-function studies.

Insect antifeedant and growth regulating activities of quassinoids from Samadera indica.

Four quassinoids, indaquassin C (1), samaderins C (2), B (3) and A (4), isolated from the seeds and bark of Samadera indica, were tested for insect antifeedant and growth regulatory activities against the tobacco cutworm, Spodoptera litura. Indaquassin C was the most effective antifeedant. Samaderin C increased pupal duration and induced pupal mortality.