Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer

Matrix metalloproteinases (MMPs) are suggested to play a critical role in extracellular matrix degradation and remodeling during inflammation and wound healing processes. However, the role of MMPs in indomethacin-induced gastric ulcer and its healing process are not clearly understood. This study is aimed at determining the regulation of MMP-9 and -2 activities in indomethacin-induced acute gastric ulceration and healing. Indomethacin-ulcerated stomach extracts exhibit significant up-regulation of pro-MMP-9 (92 kDa) activity and moderate reduction of MMP-2 activity, which strongly correlate with indomethacin dose and severity of ulcer. The anti-inflammatory and antioxidant properties of curcumin, an active component of turmeric, suggest that curcumin may exert antiulcer activity through scavenging reactive oxygen species, by regulating MMP activity, or both. To test these possibilities, the effect of curcumin in indomethacin-induced gastric ulcer is examined by biochemical and histological methods. The results show that curcumin exhibits potent antiulcer activity in acute ulcer in rat model by preventing glutathione depletion, lipid peroxidation, and protein oxidation. Denudation of epithelial cells during damage of gastric lumen is reversed by curcumin through re-epithelialization. Furthermore, both oral and intraperitoneal administration of curcumin blocks gastric ulceration in a dose-dependent manner. It accelerates the healing process and protects gastric ulcer through attenuation of MMP-9 activity and amelioration of MMP-2 activity. Omeprazole, an established antiulcer drug does not inhibit MMP-9 while protecting indomethacin-induced gastric ulcer. We conclude that antiulcer activity of curcumin is primarily attributed to MMP-9 inhibition, one of the major path-ways of ulcer healing.     

N-terminal region of the large subunit of Leishmania donovani bisubunit topoisomerase I is involved in DNA relaxation and interaction with the smaller subunit

Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (lacking amino acids 1-99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1-39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1-39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.     

Molecular basis for the origin of differential spectral and binding profiles of dansylamide with human carbonic anhydrase I and II

Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy. Arguments are presented that the detailed structural-functional investigations of enzyme-ligand interactions may provide insights into designing the isozyme-specific inhibitors of CAs.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.     

Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells

Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells.     

Rheology and composition of a multi-utility exopolymer from a desert borne cyanobacterium Anabaena variabilis

The desert cyanobacterium Anabaena variabilis produces an exopolymer during the stationary growth phase in batch culture. Optimal polymer production was observed at pH?10 under phosphorus limitation. Chemical analysis showed it to be composed of 49% carbohydrate and 19% protein. Monosaccharide analysis revealed a heteropolysaccharidic nature with glucose, mannose, and galactose as the main neutral sugars. Infrared (IR) spectrum of the exopolymer showed absorption bands at 1,645 and 1,421?cm^?1 characteristic of C=O in the carboxylate group. Strong band was observed at 1,072?cm^?1 due to C–O–C or C–O–P stretching vibrations. A band at 2,363?cm^?1 corresponding to C–H stretch of protein was also observed. IR spectrum suggested that the exopolymer is nonsulfated. Rheological properties of the polymer showed marked shear thinning non-Newtonian behavior in the concentration range of 0.1–0.4%. However, it appeared to undergo change in the internal structure on shearing thereby exhibiting thixotropic behavior. The polymer possessed 75% flocculating ability vis a vis alum, 71% emulsification of hexadecane, and good thermal stability making it a potent candidate for multiple industrial applications. The exopolymer bound 156?g H₂O g^?1 and exhibited antibacterial activity against Staphylococcus aureus suggesting a potential for application in wound management as well.     

Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G

In the present investigation, steady‐state and time‐resolved fluorescence with the combination of circular dichroism (CD) spectroscopic techniques were applied to study the interactions of the well‐known dye rhodamine 6 G (R6G) with the haem protein human myoglobin (Mb). From the analysis of the results it appears that the static type of fluorescence quenching mechanism is primarily involved, due to ground‐state interactions. Although considerable overlapping of fluorescence emission of the dye R6G with the absorption of Mb in the Q‐band region exists, the possibility of occurrences of the excitational singlet–singlet non‐radiative energy transfer process from R6G to Mb appears to be unlikely, according to time‐resolved fluorescence measurements. From the determinations of the thermodynamic parameters, it was apparent that the combined effect of van der Waals' interactions and hydrogen bonding plays a vital role in Mb–R6G interactions. Induced circular dichroism (ICD) studies demonstrate the possibility of interactions between R6G and Mb. The binding constants, number of binding sites and thermodynamic parameters have been computed. From CD measurements it is apparent that the binding of the dye R6G with the haem protein Mb induces negligible conformational changes in the protein and Mb retains its secondary structure and helicity when it interacts with R6G. The present detailed studies on the interactions with Mb should be helpful in further advancement of medical diagnostics and biotechnology. Copyright © 2011 John Wiley & Sons, Ltd.     

Overexpression of a delayed early gene hlg1 of temperate mycobacteriophage L1 is lethal to both M. smegmatis and E. coli

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growth-retardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.     

Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.     

Immunomodulatory effect of Tylophora indica on Con A induced lymphoproliferation.

Our preliminary studies with tylophora alkaloids had shown that they inhibit cellular immune responses like contact sensitivity to dinitro-flurobenzene and delayed hypersensitivity to sheep red blood cells, in vivo. Investigations were hence carried out to determine the cellular targets of tylophora alkaloids in in vitro systems. Con A induced proliferation of splenocytes was used as a model system to study the effect of the alkaloids on cellular immune responses. The alkaloid mixture was found to inhibit proliferation of splenocytes at higher concentrations and augment the same at lower concentrations. Both macrophages and T cells were found to be vulnerable to tylophora alkaloids. The alkaloid mixture suppressed IL-2 production in Con A stimulated splenocytes at the inhibitory or higher concentrations and enhanced production at the lower concentrations. IL-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentrations of tylophora. These studies indicate that tylophora alkaloids have a concentration dependent biphasic effect on Con A induced mitogenesis. At lower concentrations they augment Con A induced lymphoproliferation by enhancing IL-2 production. Inhibition of proliferation at higher concentrations of the alkaloid is due to inhibition of IL-2 production and activation of macrophages, which have a cytostatic effect.