Antibacterial Compound Isolation and Characterization from the Plant Cynotis axillaris Schult

A novel flavone glycoside was isolated from the methanolic extract of Cynotis axillaris Schult. Various analysis and characterization techniques were used to determine its structure and properties. The compound exhibited a melting point range of 231–232 °C and had a molecular formula of C₂₇H₃₀O₁₄. Several spectral characterization techniques were employed to establish the isolated compound's structure. These included UV-visible spectroscopy, FT-IR, LC-ESI-MS, and NMR spectroscopy. Based on these analyses, the structure of the isolated compound was determined to be 5,7,4’-trihydroxyflavone-8-α-L-rhamnopyranoside-4’-O-β-D-galactopyranosyl. This structure indicates that it is a flavone glycoside consisting of a flavone (5,7,4’-trihydroxyflavone) moiety attached to a sugar molecule (galactopyranosyl) at position 4’, which further bears a rhamnose group at position 8 of the flavone. In addition, to the structural characterization, the compound also demonstrated significant antibacterial efficacy against various bacterial pathogens, including Gram-positive bacteria such as Bacillus subtilis MTCC441 and Gram-negative bacteria such as Escherichia coli MTCC1098, Proteus vulgarize MTCC426, and Salmonella Typhimurium MTCC3224. The antimicrobial activity was evaluated by measuring the zone of inhibition in millimetres, which provides an indication of the compound's ability to inhibit bacterial growth. The study successfully identified and characterized a novel flavone glycoside from Cynotis axillaris Schult. and its antimicrobial activity.     

Observations on Schizothrix mexicana Gomont (Cyanophyta) under culture conditions

Using agar gel électrophoresis, the number and relative mobility of seric protein fractions has been determined for twelve species of fishes belonging to the Elasmobranchii, Dipnoi and Actinopterygii.The study of relative mobilities has shown both similitudes and divergences between some of the proteinograms. Immunoelectrophoretic cross tests using the twelve sera and five antisera have shown that these similitudes did not result from protein homology and thus agar gel electrophoresis could not be used to determine phylogenetic relationships between the species considered.

Laboratoire d'Hydrobiologie et de Pisci culture, Université de Kinshasa (Zaïre)     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

Changes in litter decomposition and soil organic carbon in a reforested tropical deciduous cover (India)

Soil organic carbon (SOC) up to 1 m depth originates from contemporary vegetation cover dating from past millennia. Deforestation and reforestation with economically important species is influencing soil carbon sequestration. An attempt has been made in this study to evaluate the impact of vegetation cover change (due to replacement of natural heterogeneous cover by teak and bamboo) on SOC using carbon isotopes (δ¹³C, ¹⁴C) in a tropical system (India). A litter decomposition study was carried out to understand the impact of differences in vegetation characteristics (specifically of leaves) on decomposition. Both experiments were carried out to look at the impact of changes in vegetation characteristics (specifically of leaves) on litter decomposition, and how these influence near term litter decomposition rates (k values) and long-term SOC content of the soil system beneath. Leaves of teak, bamboo and eight other species were selected for this study. The proportion of structural carbohydrates (lignin and cellulose) in leaves significantly (at 5 % level) influenced k values. The SOC and carbon isotope data collected in this study indicate that C₃ vegetation cover in the study area could be contemporary and dominant for the past few centuries. This can be extended up to ~2,200 years from the recorded ¹⁴C values of teak cover. The study confirms that k values of leaf litter influence SOC present beneath the vegetation cover at the decadal/century time scale.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.Cancer cells favor glycolysis over oxidative phosphorylation (OXPHOS) to meet their energy demand,¹ suggesting that they have adapted to survive and proliferate in the absence of fully functional mitochondria. Research in the last two decades demonstrates that, in addition to generation of energy, mitochondria including cancer cell mitochondria regulate multiple cellular signaling pathways encompassing cell death, proliferation, cellular redox balance, and metabolism.^{2, 3} As cancer cells possess defects in these pathways that provide an opportunity to target this organelle for therapeutic purposes. Subsequently, several agents have been developed that target cancer cell mitochondria to induce apoptosis, a cell death pathway, and eradicate cancer cells.^{4, 5} Cancer cell mitochondria harbor several proapoptotic proteins including cytochrome c, which is released from mitochondria in response to anticancer agents and activates caspases to execute apoptosis.^{5, 6} Thus, anticancer agents that induce cytochrome c release from mitochondria will be beneficial for induction of apoptosis in cancer cells. Indeed, several such agents have been developed, which include inhibitors targeting prosurvival Bcl-2 family members including Bcl-2, Bcl-xL, and Mcl-1.^{7, 8, 9} Unfortunately, cancer cells have developed multiple mechanisms to resist or overcome cytochrome c release and evade apoptosis.Although underlying mechanisms of cancer cell resistance to apoptosis are still undefined, the OXPHOS defect is known to be one of the key reasons for the attenuation of apoptosis in cancer cells.^{10, 11} Multiple lines of evidence support the notion that cancer cell survival and proliferation commonly associate with an OXPHOS defect in cancer.^{1, 12} Active OXPHOS is an efficient form of respiration but also regulates apoptosis through the OXPHOS complexes. The OXPHOS system consists of five multimeric protein complexes (I, II, III, IV, and V). The components of these complexes (except complex-II) are encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA).^{12, 13} Thus mutations, deletions, and translocations in either mtDNA or nDNA can potentially result in OXPHOS deficiency. MtDNA mutations associate with inhibition of apoptosis, induction of angiogenesis, invasion and metastasis of various types of cancer.^{3, 12, 14} Thus, mtDNA could potentially be an important target to restore cell death in cancer and attenuate cancer growth. Therefore, there is an urgent need to investigate the role of OXPHOS in the molecular mechanisms underlying cancer cell death.We investigated the effects of several anticancer agents of different classes including DNA-damaging agents (etoposide and doxorubicin), protein kinase inhibitors (staurosporine and sorafenib), mitotic inhibitor (taxol), ER stressor/inhibitor of Ca²⁺-ATPases (thapsigargin), and histone deacetylase (HDAC) inhibitor (apicidin) on mtDNA. We also determined the impact of OXPHOS defects on apoptosis induction by these agents. Although most anticancer agents induced caspase activation and apoptosis, the mtDNA level was elevated maximally by etoposide and it was not modulated by a caspase inhibitor but reduced by an autophagy inhibitor. Induction of mtDNA is associated with increased reactive oxygen species (ROS) production and elevated mitochondrial mass. Pharmacologic inhibition of OXPHOS complexes reduced the etoposide-induced elevation in mtDNA, suggesting the involvement of these complexes in etoposide-induced apoptosis. Together, we define the impact of mtDNA and OXPHOS function on mitochondrial apoptosis, which has significance in restoring cancer cell apoptosis for therapeutic purposes.     

Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells

Coding sequences for a hammerhead ribozyme designed to cleave lexA mRNA in a targeted manner was cloned under phage T7 promoter and expressed in E. coli strain BL-21 (DE3) expressing T7 RNA polymerase under the control of IPTG-inducible lac UV-5 promoter. Ribozyme expression in vivo was demonstrated by RNase protection assay. Also, total RNA extracted from these transformed cells following induction by IPTG, displays site-specific cleavage of labeled lexA RNA in an In vitro reaction. The result demonstrates the active ribozyme in extracts of cell transformed with a recombinant cassette and goes beyond the earlier demonstration of the stability of In vitro synthesized ribozyme in cell extracts. The observed rise in lexA mRNA rules out any role for protease activity or resulting fragments of lexA protein in de-repression of RNA. (Mol Cell Biochem 271: 197–203, 2005)