Molecular characterization of tumor associated antigen in mice exposed to a hepatocarcinogen

The present investigation is aimed to identify and characterize tumor-associated antigen (TAA) in animals exposed to hepatocarcinogen. Swiss albino mice showed an enhanced expression of an approximately 58 kDa glycoprotein in liver cells upon exposure to a potential hepatocarcinogen N-nitrosodibutylamine (DBN). Carcinogenesis induction in mice was monitored by assays of -glutamyl transpeptidase (GGT), acetylcholine esterase (AChE), glutathione-S-transferase (GST) activities and the level of glutathione (GSH) in liver. DBN treated animals showed cell distortion and extensive necrosis as observed by histological examination. The over-expressed TAA was purified by ion-exchange chromatography and further characterized by SDS-PAGE. The carbohydrate contents and glycan linkage to the polypeptide backbone was analyzed by using the DIG glycan differentiation and de-glycosylation kits. The glycoprotein has glycan chains that are N-linked via asparagines to the polypeptide backbone. It was also observed that the molecule is rich in sialic acid residues with a significantly high carbohydrate to protein ratio (2:1). The over-expressed high molecular weight glycoprotein TAA was found to be highly immunogenic and could eventually be used to induce immune response in order to counter tumor regression. (Mol Cell Biochem 271: 177–188, 2005)     

Effect of a total solar eclipse on the surface crowding of zooplankton in a freshwater lake ecosystem

Zooplankton surface crowding in a freshwater lake ecosystem during a total solar eclipse (maximum eclipse at 06:28:43 Indian Standard Time, 22 July 2009) was studied in relation to ambient physicochemical conditions and compared with the crowding that occurred during pre- and post-eclipse days. Rapid light attenuation on the eclipse day led to changes in zooplankton surface crowding, which manifested as alterations to community structure and statistical parameters. Zooplankton diversity and density varied depending on the day (either the pre-eclipse day, the eclipse day, or the post-eclipse day) and the sampling time considered. A total of 20 zooplankton species were recorded during the study. On the day of the eclipse, the highest zooplankton density in the surface water was recorded just after the end of totality at 06:30 IST. The populations of two adult cladoceran species (Alona rectangula rectangula and Chydorus sphaericus) were particularly prominent in the zooplankton, whereas rotifers were almost absent from the surface water during the eclipse. Rather than decreasing, the primary production of the phytoplankton increased on the day of the TSE compared to that seen on the control days. Comparatively high Lindeman’s efficiency values were observed during the eclipse, indicating particularly efficient utilization of photons in photosynthesis.     

Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.     

Reversal of T-cell unresponsiveness through serine-esterase inhibitors mediated enhanced lymphokine induced microbicidal activities in kala-azar.

After presenting processed glycoprotein of Leishmania donovani to T-cell, macrophage seeks the help of a panel of T-cells lymphokines to transform from a state that sustains intra cellular replication of parasite to an effector state for destructing parasites. But esterase and trypsin of macrophage membrane prevent T-cells to release MIF. Role of soya-bean trypsin inhibitor (STI) has been exposed in the present study with a view to alter esterase functional behaviour of macrophage for control of T-cell activation and also, if T-cells once made responsive to antigen by STI do alter macrophage response to T-cells or not. Results establish STI as potent effector molecule, which can serve as an adjuvant to candidate T-cell epitope and synthetic peptide for development of anti-Kala-azar vaccine protocol in future.     

Stress induced MAPK genes show distinct pattern of codon usage in Arabidopsis thaliana,Glycine max and Oryza sativa

Mitogen activated protein kinase (MAPK) genes provide resistance to various biotic and abiotic stresses. Codon usage profiling of the genes reveals the characteristic features of the genes like nucleotide composition, gene expressivity, optimal codons etc. The present study is a comparative analysis of codon usage patterns for different MAPK genes in three organisms, viz. Arabidopsis thaliana, Glycine max (soybean) and Oryza sativa (rice). The study has revealed a high AT content in MAPK genes of Arabidopsis and soybean whereas in rice a balanced AT-GC content at the third synonymous position of codon. The genes show a low bias in codon usage profile as reflected in the higher values (50.83 to 56.55) of effective number of codons (N_c). The prediction of gene expression profile in the MAPK genes revealed that these genes might be under the selective pressure of translational optimization as reflected in the low codon adaptation index (CAI) values ranging from 0.147 to 0.208.     

Downregulation of Mitochondrial Porin Inhibits Cell Growth and Alters Respiratory Phenotype in Trypanosoma brucei

Porin is the most abundant outer membrane (OM) protein of mitochondria. It forms the aqueous channel on the mitochondrial OM and mediates major metabolite flux between mitochondria and cytosol. Mitochondrial porin in Trypanosoma brucei, a unicellular parasitic protozoan and the causative agent of African trypanosomiasis, possesses a β-barrel structure similar to the bacterial OM porin OmpA. T. brucei porin (TbPorin) is present as a monomer as well as an oligomer on the mitochondrial OM, and its expression is developmentally regulated. In spite of its distinct structure, the TbPorin function is similar to those of other eukaryotic porins. TbPorin RNA interference (RNAi) reduced cell growth in both procyclic and bloodstream forms. The depletion of TbPorin decreased ATP production by inhibiting metabolite flux through the OM. Additionally, the level of trypanosome alternative oxidase (TAO) decreased, whereas the levels of cytochrome-dependent respiratory complexes III and IV increased in TbPorin-depleted mitochondria. Furthermore, the depletion of TbPorin reduced cellular respiration via TAO, which is not coupled with oxidative phosphorylation, but increased the capacity for cyanide-sensitive respiration. Together, these data reveal that TbPorin knockdown reduced the mitochondrial ATP level, which in turn increased the capacity of the cytochrome-dependent respiratory pathway (CP), in an attempt to compensate for the mitochondrial energy crisis. However, a simultaneous decrease in the substrate-level phosphorylation due to TbPorin RNAi caused growth inhibition in the procyclic form. We also found that the expressions of TAO and CP proteins are coordinately regulated in T. brucei according to mitochondrial energy demand.Trypanosoma brucei belongs to a group of parasitic protozoa that possess a single tubular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast (30). T. brucei is the infectious agent of the disease African trypanosomiasis, which is spread from one mammal to another by the bite of the tsetse fly (53). During transmission from the insect vector to the mammalian host and vice versa, the parasite undergoes various developmental stages accompanied by dramatic changes in mitochondrial activities (15). The bloodstream form that grows in mammalian blood uses glucose as its energy source and suppresses many mitochondrial activities. The bloodstream-form mitochondria lack cytochromes; thus, respiration in this form is solely dependent on the cytochrome-independent trypanosome alternative oxidase (TAO) (15). In contrast, the procyclic form that lives in the insect''s midgut possesses a developed mitochondrion with a full complement of the cytochrome-dependent respiratory system and a reduced level of TAO. The procyclic-form mitochondria produce ATP by both oxidative and substrate-level phosphorylations (7). On the other hand, the bloodstream-form mitochondria do not produce ATP but hydrolyze ATP to maintain the inner membrane (IM) potential (10, 33, 39, 48). Many of the mitochondrial IM- and matrix-localized proteins in T. brucei are well characterized (11, 29, 34, 43, 45). However, the mitochondrial outer membrane (OM) proteins in this group of parasitic protozoa have been poorly explored.Mitochondrial porin, which is also known as the voltage-dependent anion-selective channel (VDAC), is the most abundant protein in the OM (17, 28). The sizes and the secondary structures of this protein are very similar among different organisms. The VDAC possesses a N-terminal α-helical domain, and the rest of the protein consists of a number of amphiphilic β-strands, which form a barrel-like structure that integrates into the lipid bilayer (16, 17, 28). Recently, the three-dimensional structure of the human VDAC has been elucidated by nuclear magnetic resonance spectroscopy and X-ray crystallography, which showed a β-barrel architecture composed of 19 β-strands and the N-terminal α-helix located horizontally midway in the pore (5). Saccharomyces cerevisiae and Neurospora crassa VDACs also possess 16 to 19 β-strands, similar to the mammalian VDAC (17).The VDAC exists as different isomeric forms in different species (16, 19). In yeasts, there are two forms: VDAC1 and VDAC2. Only VDAC1 has the channel activity and is abundantly expressed (22, 23). Animals have three isoforms: VDAC1 to VDAC3. These isoforms showed more than 80% sequence homology among themselves. However, their expression levels and tissue specificities are different (16). Plants also have multiple isoforms of the VDAC with various expression levels under different pathological conditions (19). The VDAC plays a crucial role in regulated transport of ADP, ATP, Ca²⁺, and other metabolites in and out of mitochondria (17, 28, 41). Two ATP-binding sites found at the N- and C-terminal regions in the VDAC are critical for its function (54). Downregulation of VDAC expression disrupts mitochondrial energy production (22, 25). In contrast, overexpression of the VDAC in metazoa induces apoptosis, which can be blocked by compounds that inhibit its channel activity (1, 47).The OM of gram-negative bacteria also consists of various types of porins (24, 32, 40). Based on their structures and functions, they are divided into five groups. OmpA belongs to the small β-barrel integral membrane protein family, which is composed of eight β-strands. It is highly abundant and ubiquitous among most gram-negative bacteria (21). Other types of porins include general porin OmpF, which consists of 16 β-strands; substrate-specific porins, such as LamB or maltoporin, which contains 18 β-strands; receptor-type porin FhuA, the largest β-barrel, with 22 β-strands; and phospholipase A or OMPLA, an integral membrane enzyme containing 12 β-strands (21, 24, 32, 40). The OmpA plays important roles in bacterial conjugation, adhesion, invasion, and immune evasion and also acts as the receptor for several bacteriophages through its surface-exposed loops (44).Here, we show that the T. brucei mitochondrial porin (TbPorin) possesses a predicted β-barrel structure that has fewer β-strands than other mitochondrial porins but is similar to bacterial OmpA. TbPorin is crucial for mitochondrial energy production via both oxidative and substrate-level phosphorylations. The depletion of TbPorin reduced cell growth of the procyclic form as well as the bloodstream form. Furthermore, it reveals that depletion of mitochondrial ATP level by downregulation of porin alters the electron flow via TAO and the cytochrome-dependent pathway (CP) as well as the levels of proteins in these pathways.     

Comparison of metal-amino acid interaction in Phe-Ag and Tyr-Ag complexes by spectroscopic measurements

In this article, we have compared the metal–amino acid interactions in Tyr–Ag and Phe–Ag complexes through pH dependent SERS measurements. By analyzing the variation in relative intensities of SERS bands with the pH of the amino acid solution, we have obtained the orientation and conformation of the amino acid molecules on the Ag surface. The results obtained from our experimental studies are supported by the energy minimized structures and the observed charge distributions in different terminals of the molecules. This, in a way, shows that SERS measurements not only exhibit the interaction of the amino acid molecules with Ag clusters but also demonstrate their orientation around it. We have addressed a long standing query on whether the amine group is directly attached to the Ag surface along with the carboxylate group and π-electrons in these systems. In addition, pH dependent optical absorption and transmission electron microscopy measurements have been performed to understand the required conditions for the appearance of the SERS spectra in the light of the aggregation of metal particles and the number of hot sites in the sol. Our results confirm that the formation of hot sites in the sol plays a direct role in forming a stable Ag–ligand complex. Furthermore, the interaction kinetics of metal–amino acid complexes have been analyzed via both Raman and absorption measurements.     

Ficus cunia agglutinin for recognition of bacteria

Interaction of bacteria with lectin using anti-lectin antibody by ELISA is an established method. In the present study, we have devised a simple ELISA using a biotinylated lectin and antibiotin-HRP. Ficus cunia agglutinin (FCA), which has shown the specificity towards alpha/beta anomers of GlcNAc and other-NAc containing sugars like LacNAc and GlcNAcbeta(1-4/6)GlcNAc, was used as a model lectin for the study of interaction with immobilized microorganisms on ELISA plate. The bacterial cells of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus showed binding with FCA and the degree of binding was dependent on the bacterial surface antigen. This method is considered a simple technique to study the lectin-bacteria interaction.