Parul Chakrabarti

Flavonoid constituents of Eupatorium odoratum

Abstract Isosakuranetin and a new chalcone, odoratin, have been isolated from the leaves of Eupatorium odoratum . The structure of odoratin has been shown to be 2′-hydroxy-4,4′,5′,6′-tetramethoxy chalcone.

Isolation and characterization of an extracellular lipase from the conidia of Neurospora crassa.

A triacylglycerol lipase (EC 3.1.1.3) from the conidia of Neurospora crassa was purified and characterized. The enzyme was purified by Sephadex G-100 column chromatography. Homogeneity was checked by PAGE, and isoelectric focusing gave a single band corresponding to a pI of 6.4. The enzyme had an apparent Mr 54000 +/- 1000 as determined by gel filtration. SDS-PAGE gave a single band of Mr 27000, suggesting the presence of two identical subunits. This lipase preferred triglycerides with C16- and C18-fatty acyl chains. It cleaved only the primary groups of triglycerides. The lipase also exhibited a marked preference for substrates containing endogenously occurring fatty acids and so may prove useful in detailed studies on the physiological relevance of fatty acyl specificity of lipases. The enzyme was not affected by detergents, or thiol-binding agents. Modification of free amino groups caused 90% inhibition, suggesting a role of these groups in the maintenance of lipase activity.

An oligopeptide transporter of Mycobacterium tuberculosis regulates cytokine release and apoptosis of infected macrophages.

Background The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppAMTB. Methodology/Principal Findings OppAMTB was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppAMTB confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1β, IL-6 and TNF-α was also compromised after inactivation of oppD. Conclusions Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

Purification and characterization of an extracellular lectin from Mycobacterium smegmatis

Lectin, extracelllular; Arabinogalactan; Mannan; (Mycobacterium smegmatis)

Purification and characterisation of a non-specific lipid transfer protein from goat liver

A non-specific lipid transfer protein has been purified from the pH 5.1 supernatant of goat liver by DEAE-cellulose, CM-cellulose and Sephadex G-75 column chromatography. The protein shows a single band on polyacrylamide gel electrophoresis and transfers 450 nmol of phosphatidylcholine per min per mg of protein under the present assay condition. This protein has a subunit molecular weight of 12,000 and an isoelectric point of 8.65. Amino acid analysis reveals the absence of methionine. Histidine has been identified as the only N-terminal amino acid. Besides phosphatidylcholine, the protein transfers phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and cholesterol. Chemical modification studies showed the involvement of free amino and thiol groups in the maintenance of the transfer activity of the goat liver protein.

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Abstract Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.

Mycotin: a lectin involved in the adherence of Mycobacteria to macrophages

Pathogenic Mycobacteria colonize host macrophages. Attachement of these organisms to macrophages is the preliminary step prior to invasion of the macrophages by the bacteria. Western blot confirmed that walls of Mycobacterium avium and Mycobacterium tuberculosis contain molecules which are immunologically related to mycotin, a lectin found in Mycobacterium smegmatis. We have demonstrated that the adherence of Mycobacteria to macrophages is significantly inhibited by anti‐mycotin antibody or the mycotin‐specific sugar, mannan. These observations suggest that prevention of the interaction of mycotin‐related molecules on the surfaces of Mycobacteria with mannose‐specific receptors on macrophages, offers an important approach for blocking attachment of pathogenic Mycobacteria to macrophages, thereby preventing infection.

Fatty acylation of a 55 kDa membrane protein of human erythrocytes

The major palmitoylated human erythrocyte membrane protein has an M(r) of 55,000. It is distinct from the glucose transporter and is not derived from band 3 or ankyrin. It resists salt extraction suggesting a high affinity for the membrane. Pulse chase experiments demonstrate that palmitoylation is a dynamic process, and it may therefore have regulatory significance in membrane protein-protein or protein-lipid interaction. Slower dynamics of palmitoylation in erythrocytes from patients suffering from chronic myelogenous leukemia, which are less stable than normal erythrocytes, strengthen this view.

Abnormal erythrocyte membrane cytoskeleton structure in chronic myelogenous leukaemia

Chronic myelogenous leukaemia (CML) is a haematologic malignancy characterised by excessive growth of myeloid cells and their progenitors. Our studies show that there are several abnormalities in CML red blood cells. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Decreased spectrin tetramer formation observed in several hereditary anaemias has been associated with decreased red cell deformability leading to splenic sequestration. This could also be one of the causes of the severe anaemia observed in CML. Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3.

The structure and stereochemistry of lantanilic acid, the β,β-dimethylacryloyl ester of lantaninilic acid, isolated from Lantana camara

Abstract The structure of lantanilic acid, a new triterpene isolated from the leaves of Lantana camara , has been determined as the β,β-dimethylacryloyl ester of lantaninilic acid.