Inverted binding due to a minor structural change in berberine enhances its phospholipase A2 inhibitory effect

Biotransformation of berberine by Rhizopus oryzae leads to its demethylation, producing hydroxyl derivatives, as revealed by Fourier Transform Infra Red spectroscopy, Nuclear Magnetic Resonance and Electro Spray Ionization-Mass Spectrometric analyses. Surface Plasmon Resonance and enzyme kinetic studies showed that biotransformed derivatives of berberine had a higher inhibitory potential than berberine towards phospholipase A(2). X-ray crystal structures demonstrated that biotransformed berberine binds to PLA(2) in an entirely different, inverted orientation with respect to the binding of berberine. This study brings out the significance of biotransformation in generation of better drug-lead compounds.     

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.     

Ascorbic acid supplementation down-regulates the alcohol induced oxidative stress, hepatic stellate cell activation, cytotoxicity and mRNA levels of selected fibrotic genes in guinea pigs

Both oxidative stress and endotoxins mediated immunological reactions play a major role in the progression of alcoholic hepatic fibrosis. Ascorbic acid has been reported to reduce alcohol-induced toxicity and ascorbic acid levels are reduced in alcoholics. Hence, we investigated the hepatoprotective action of ascorbic acid in the reversal of alcohol-induced hepatic fibrosis in male guinea pigs (n = 36), and it was compared with the animals abstenting from alcohol treatment. In comparison with the alcohol abstention group, there was a reduction in the activities of toxicity markers and levels of lipid and protein peroxidation products, expression of α-SMA, caspase-3 activity and mRNA levels of CYP2E1, TGF-β(1), TNF-α and α(1)(I) collagen in liver of the ascorbic acid-supplemented group. The ascorbic acid content in liver was significantly reduced in the alcohol-treated guinea pigs. But it was reversed to normal level in the ascorbic acid-supplemented group. The anti-fibrotic action of ascorbic acid in the rapid regression of alcoholic liver fibrosis may be attributed to decrease in the oxidative stress, hepatic stellate cells activation, cytotoxicity and mRNA expression of fibrotic genes CYP2E1, TGF-β(1), TNF-α and α(1) (I) collagen in hepatic tissues.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.     

Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics

Phytoremediation — the use of plants to clean up polluted soil and water resources — has received much attention in the last few years. Although plants have the inherent ability to detoxify xenobiotics, they generally lack the catabolic pathway for the complete degradation of these compounds compared to microorganisms. There are also concerns over the potential for the introduction of contaminants into the food chain. The question of how to dispose of plants that accumulate xenobiotics is also a serious concern. Hence the feasibility of phytoremediation as an approach to remediate environmental contamination is still somewhat in question. For these reasons, researchers have endeavored to engineer plants with genes that can bestow superior degradation abilities. A direct method for enhancing the efficacy of phytoremediation is to overexpress in plants the genes involved in metabolism, uptake, or transport of specific pollutants. Furthermore, the expression of suitable genes in root system enhances the rhizodegradation of highly recalcitrant compounds like PAHs, PCBs etc. Hence, the idea to amplify plant biodegradation of xenobiotics by genetic manipulation was developed, following a strategy similar to that used to develop transgenic crops. Genes from human, microbes, plants, and animals are being used successfully for this venture. The introduction of these genes can be readily achieved for many plant species using Agrobacterium tumefaciens-mediated plant transformation or direct DNA methods of gene transfer. One of the promising developments in transgenic technology is the insertion of multiple genes (for phase 1 metabolism (cytochrome P450s) and phase 2 metabolism (GSH, GT etc.) for the complete degradation of the xenobiotics within the plant system. In addition to the use of transgenic plants overexpressed with P450 and GST genes, various transgenic plants expressing bacterial genes can be used for the enhanced degradation and remediation of herbicides, explosives, PCBs etc. Another approach to enhancing phytoremediation ability is the construction of plants that secrete chemical degrading enzymes into the rhizosphere. Recent studies revealed that accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as major limitation in improving phytoremediation efficiency. However, this can be overcome by the selective expression of bacterial ACC deaminase (which regulates ethylene levels in plants) in plants together with multiple genes for the different phases of xenobiotic degradation. This review examines the recent developments in use of transgenic-plants for the enhanced metabolism, degradation and phytoremediation of organic xenobiotics and its future directions.     

Influence of the application of sugarcane bagasse on lindane (gamma-HCH) mobility through soil column: implication for biotreatment

In the present study we employed sugarcane bagasse for biotreatment of soil containing 50mgkg(-1) of lindane. Garden soil were treated with lindane and amended with varying concentrations of sugarcane bagasse (10%, 20%, 30%, 40% and 50%; w/w). Data on dissipation and degradation of lindane in soil columns (0-15, 15-30cm) were studied at six consecutive samplings (0, 3, 7, 45 and 60 days). Treatment with 50% sugarcane bagasse resulted in >53% degradation of lindane in upper soil column with minimal leaching to lower soil column (0.002%) while highest leaching of lindane from upper soil column to lower soil column was occurred in garden soil (35.8%). Similarly, a substantial microbial biomass input has detected in amended soil than garden soil. Our results provide evidence that sugarcane bagasse can accelerate lindane degradation by enhanced microbial activity and prevent pesticide mobility through soil column by adsorption. Sugarcane bagasse could be useful as cheaper, easy available alternative for the biotreatment of lindane impacted soil.     

Reaction mechanism,evolutionary analysis,and role of zinc in Drosophila methionine-R-sulfoxide reductase

Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.     

QSAR studies on psychotomimetic phenylalkylamines

Quantitative Structure-Activity Relationship (QSAR) studies on a series of psychotomimetic phenylalkylamines have been made using a combination of Minimum Topological Difference (MTD) method and topological methodology. The topological indices used being a pool of distance-based topological indices. The regression analyses have shown that excellent results are obtained in multiparametric model containing MTD parameters, topological indices in that quantum chemical parameters has to be introduced. The predictive power of the proposed model is discussed on the basis of cross-validation parameters.     

T cell-activated macrophages are capable of both recognition and rejection of pancreatic islet xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.