Determination of chiral bioactive molecules in Justicia adhatoda leaves by GC-MS.

Chiral compounds find importance as drugs and therapeutic targets. Enantiomers of chiral drugs have been found to show different biological properties like pharmacokinetics, toxicology, pharmacology, metabolism, and so forth. In this study, we have identified the chiral compounds present in the medicinal plant Adhatoda vasica Nees (Justicia adhatoda Linn). Phytochemical investigation on the leaves of Justicia adhatoda resulted in the identification of 27 chiral compounds. We report diverse compounds identified in the crude methanolic extract of Justicia adhatoda leaves by GC-MS analysis exhibiting diverse biological activities. Quantitative analysis of anticancer compound dihydroxycolchicine from the methanolic extract of J. adhatoda leaves was done by external standard method, and the amount of anticancer compound dihydroxycolchicine was found to be 87.823 mg/l indicative of moderate production in the leaves. Therefore, the extract of leaves of Justicia adhatoda can be used as a potential source of chiral bioactive molecules of pharmacological importance for drug synthesis.

Sialylation and sialyltransferase in insects.

Sialic acids are negatively charged nine carbon monosaccharides located terminally on glycoproteins and glycolipids that control cellular physiological processes. Sialylation is a post translational modification (ptm) regulated by enzymes and has been studied in prokaryotes including bacteria, dueterostomes including vertebrates, Cephalochordates, Ascidians, Echinoderms and protostomes including Molluscs and Arthropods and Plant. Although diverse structures of sialylated molecules have been reported in different organisms, unravelling sialylation in insect biology is a completely new domain. Within protostomes, the study of sialylation in members of Phylum Arthropoda and Class Insecta finds importance. Reports on sialylation in some insects exist. Genetically engineered components of sialylation pathway in Spodoptera frugiperda (Sf9) cell lines have enabled our understanding of sialylation and expression of mammalian proteins in insects. In this study we have summarised the finding on (i) sialylated molecules (ii) processes and enzymes involved (iii) function of sialylation (iv) genetic engineering approaches and generation of mammalian protein expression systems (v) a comparison of sialylation machinery in insects with that of mammals (vi) genes and transcriptional regulation in insects. At present no information on structural studies of insect sialyltransferase (STs) exist. We report minor differences in ST structure in insects on complete protein sequences recorded in Genbank through in silico approaches. An indepth study of all the components of the sialylation pathway in different insect species across different families and their evolutionary significance finds importance as the future scope of this review.

Sialic acids: biomarkers in endocrinal cancers.

Sialylations are post translational modification of proteins and lipids that play important role in recognition, signaling, immunological response and cell-cell interaction. Improper sialylations due to altered sialyl transferases, sialidases, gene structure and expression, sialic acid metabolism however lead to diseases and thus sialic acids form an important biomarker in disease. In the endocrinal biology such improper sialylations including altered expression of sialylated moieties have been shown to be associated with disorders. Cancer still remains to be the major cause of global death and the cancer of the endocrine organs suffer from the dearth of appropriate markers for disease prediction at the early stage and monitoring. This review is aimed at evaluating the role of sialic acids as markers in endocrinal disorders with special reference to cancer of the endocrine organs. The current study is summarized under the following headings of altered sialylations in endocrinal cancer of the (i) ovary (ii) pancreas (iii) thyroid (iv) adrenal and (v) pituitary gland. Studies in expression of sialic acid in testis cancer are limited. The future scope of this review remains in the targeting of endocrinal cancer by targeting altered sialylation which is a common expression associated with endocrinal cancer.

Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells.

Iron-based compounds possess the capability of inducing cell death due to their reactivity with oxidant molecules, but their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in mouse and human leukemia cell lines. Fe(salen)Cl induces cell death specifically in tumor cells and not in primary cells. Mouse and human T-cell leukemia cell lines, EL4 and Jurkat cells are found to be susceptible to Fe(salen)Cl and undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug.

Prevalence of mouthpart sensilla and protease producing symbiotic gut bacteria in the forensic fly Chrysomya megacephala (Fabricius, 1794): Insight from foraging to digestion.

The blow fly, Chrysomya megacephala (Fabricius, 1794) is a globally prevalent forensically important species that helps to estimate accurate postmortem interval since the death. This fly occasionally causes cutaneous myiasis and transmits several pathogenic bacteria. To understand their ability of corpse detection and digestion of protein-rich meal, the present study describes the mouthpart sensilla and assessment of protease producing symbiotic gut bacteria. Scanning electron microscopy (SEM) showed the prevalence of trichoid sensilla (Tr), basiconic sensilla (Ba) and microtrichia (Mr) on labellar lobes, haustellum and maxillary palps of mouthparts. Bacterial particles of both rod (small and large) and spherical shaped were detected in the gut of C. megacephala using SEM. The bacterial density was higher on the foregut and midgut in comparison to the hindgut. From 72 bacterial isolates, 10 isolates from the foregut region showed considerable protease-producing efficacy ranging between 3.98 - 6.83 GHR and 9.73 - 34.68 U/ml protease. Among these, the most promising protease-producing bacterial isolate showed 16S rDNA sequence similarity (99.85%) with Chryseobacterium artocarpi DNA. This bacterium was the first report from flies. The findings of the study might help in better understanding of the role of sensilla in host perception and foregut symbiotic bacterial association in protein digestion in C. megacephala.

Environmental pollutants, pathogens and immune system in earthworms.

Earthworms also known as farmer's friends are natural tillers of soil. They belong to Phylum Annelida and class Oligochaeta. Acid soils with organic matter and surface humus maintain the largest fauna of worms and earthworms. Due to their habitat in soil, they are constantly exposed to microbes and pollution generated by anthropogenic sources. Studies have revealed that damage of the immune system of earthworms can lead to alterations of both morphological and cellular characteristics of worms, activation of signalling pathways and can strongly influence their survival. Therefore, the understanding of the robust immune system in earthworms has become very important from the point of view of understanding its role in combating pathogens and pollutants and its role in indicating the soil pollution. In this article, we have outlined the (i) components of the immune system and (ii) their function of immunological responses on exposure to pollutants and pathogens. This study finds importance from the point of view of ecotoxicology and monitoring of earthworm health and exploring the scope of earthworm immune system components as biomarkers of pollutants and environmental toxicity. The future scope of this review remains in understanding the earthworm immunobiology and indicating strong biomarkers for pollution.

Application of Computational Methods in Planaria Research: A Current Update.

Planaria is a member of the Phylum Platyhelminthes including flatworms. Planarians possess the unique ability of regeneration from adult stem cells or neoblasts and finds importance as a model organism for regeneration and developmental studies. Although research is being actively carried out globally through conventional methods to understand the process of regeneration from neoblasts, biology of development, neurobiology and immunology of Planaria, there are many thought provoking questions related to stem cell plasticity, and uniqueness of regenerative potential in Planarians amongst other members of Phylum Platyhelminthes. The complexity of receptors and signalling mechanisms, immune system network, biology of repair, responses to injury are yet to be understood in Planaria. Genomic and transcriptomic studies have generated a vast repository of data, but their availability and analysis is a challenging task. Data mining, computational approaches of gene curation, bioinformatics tools for analysis of transcriptomic data, designing of databases, application of algorithms in deciphering changes of morphology by RNA interference (RNAi) approaches, understanding regeneration experiments is a new venture in Planaria research that is helping researchers across the globe in understanding the biology. We highlight the applications of Hidden Markov models (HMMs) in designing of computational tools and their applications in Planaria decoding their complex biology.

Scanning electron microscopic studies on antenna of Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae)-A blow fly species of forensic importance.

Blow flies (Diptera: Calliphoridae) are one of the foremost organisms amongst forensic insects to colonize corpses shortly after death, thus are of immense importance in the domain of forensic entomology. The blow fly Hemipyrellia ligurriens (Wiedemann, 1830) (Diptera: Calliphoridae) is considered as a forensically important fly species globally and is also known for its medical and veterinary importance. In the present study, we report for the first time scanning electron microscopic studies on the morphology of sensilla of antenna of adult male and female of H. ligurriens is with profound importance in better understanding of the insect morphology from forensic entomological perspective, and also could aid in proper identification of the species from other calliphorid flies. The structural peculiarities observed in the (i) antenna of H. ligurriens with three segments- scape, pedicel and flagellum with dorso-laterally placed arista (ii) densely covered microtrichia and most abundant trichoid sensilla identified on the antenna (iii) observation of only one type of sensilla, chaetic sensilla (ChI) on the scape (iv) two types of chaetic sensilla (ChI and ChII) and styloconic sensilla on the pedicel (v) the flagellum with three types of sensilla- trichoid, basiconic and coeloconic sensilla (vi) Basiconic sensilla with multiporous surfaces with characteristic olfactory function. Moderate sexual dimorphism in the width of the flagellum, the females with wider flagella than the males, bear significance to the fact that they bear more multi-porous sensilla than the males, thus suffice their need to detect oviposition sites. Significant difference was observed in the length and width of coeloconic sensilla between the two sexes, the females showed bigger coeloconic sensilla, suggesting their function in oviposition site detection and successful colonization in corpses.

Indoor air pollution: impact on health and stem cells.

Nearly 2 million people annually die prematurely from various illness contributed by indoor air pollutants (IAP). Such pollutants affect the lungs leading to diseases ranging from bronchial diseases to malignant lung cancer. Stem cells (SC) with the property of self-renewal, pluripotency, and capability of homing into tumors and metastases, have been reported to be promising in treatment of lung cancer. In this review, we have tried to understand the role of components of IAP affect the SC. Although very few studies have been conducted in these lines, existing reports suggest that IAP causes damage to stem cells and their niches thereby reducing successful chances of autologous stem cell transplantation and therapy. The mechanism by which components of IAP affects the functioning of stem cells thus conferring toxicity remains unexplored. The future scope of this review lies in revealing answer to underlying questions of repair and modulation of stem cells in therapeutic treatment of lung diseases.

C-reactive protein and the biology of disease.

The C-reactive protein (CRP) is a plasma protein of hepatic origin, belonging to pentraxin family and forms a major component of any inflammatory reaction. A key component of the innate immunity pathway, the concentration of CRP may rapidly increase to levels more than 1,000-folds above normal values as a consequence to tissue injury or infection. Although functioning as a classical mediator of innate immunity, it functions via interaction of components of both humoral and cellular effector systems of inflammation. Initially considered as an acute-phase marker in tissue injury, infection and inflammation, it now has a distinct status of a disease marker in cardiovascular diseases and is well known of its clinical and pathological significance. The present torrent of studies in a large number of diseases and associated conditions has highly elucidated the role of CRP as a therapeutic and research reagent. In this review, we focus our attention to role of CRP in health and disease. The future prospect of this review lies in the applicability of CRP as a molecule in understanding and monitoring of the biology of disease.

Effect of heavy metals on germination of seeds.

With the expansion of the world population, the environmental pollution and toxicity by chemicals raises concern. Rapid industrialization and urbanization processes has led to the incorporation of pollutants such as pesticides, petroleum products, acids and heavy metals in the natural resources like soil, water and air thus degrading not only the quality of the environment, but also affecting both plants and animals. Heavy metals including lead, nickel, cadmium, copper, cobalt, chromium and mercury are important environmental pollutants that cause toxic effects to plants; thus, lessening productivity and posing dangerous threats to the agro-ecosystems. They act as stress to plants and affect the plant physiology. In this review, we have summarized the effects of heavy metals on seeds of different plants affecting the germination process. Although reports exist on mechanisms by which the heavy metals act as stress and how plants have learnt to overcome, the future scope of this review remains in excavating the signaling mechanisms in germinating seeds in response to heavy metal stress.

Tinospora cordifolia: One plant, many roles.

Natural products with medicinal value are gradually gaining importance in clinical research due to their well-known property of no side effects as compared to drugs. Tinospora cordifolia commonly named as "Guduchi" is known for its immense application in the treatment of various diseases in the traditional ayurvedic literature. Recently the discovery of active components from the plant and their biological function in disease control has led to active interest in the plant across the globe. Our present study in this review encompasses (i) the genetic diversity of the plant and (ii) active components isolated from the plant and their biological role in disease targeting. The future scope of the review remains in exploiting the biochemical and signaling pathways affected by the compounds isolated from Tinospora so as to enable new and effective formulation in disease eradication.

O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL).

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(alpha2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-gamma due to specific stimulation of Neu5,9Ac(2)-GPs on PBMC(ALL) with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA(-)) revealed decreased nitric oxide production as compared to 9-O-AcSA(+) cells on exposure to IFN-gamma. Under this condition, a decrease in viability of 9-O-AcSA(-) cells as compared to 9-O-AcSA(+) cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac(2)-GPs are also capable of inducing disease-specific anti-Neu5,9Ac(2)-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac(2)-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac(2)-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.

Altered erythrocyte membrane characteristics during anemia in childhood acute lymphoblastic leukemia.

Anemia is a prominent feature in children with acute lymphoblastic leukemia (ALL). To investigate the erythrocyte features during anemia in these patients, we studied the altered characters of these cells and oxidative stress imposed in their serum. This investigation reveals that erythrocytes from ALL patients show (1) increased membrane fluidity detected by fluorescence anisotropy studies, increased osmotic fragility detected by hemolysis of erythrocytes in different saline concentrations, and increased hydrophobicity as measured by binding with 8-anilino-1-naphthalenesulfonic acid, (2) enhanced (approximately threefold) glycosylation and sialylation, monitored by digoxigenin enzyme assay, and (3) expression of disease-specific 210, 105, 83, 54, and 28 kDa 9-O-acetyl sialoglycoconjugates (9-O-AcSGs) demonstrated by Western blot analysis and fluorescence-activated cell sorter (FACS) analysis studies using Achatinin-H with specificity towards 9-O-AcSAalpha2-6GalNAc as the analytical probe. (4) In addition, induced oxidative stress was observed in the sera of these children as indicated by increased nitric oxide (approximately fourfold) and thiobarbituric acid (TBA) reactive species (twofold) as detected by Griess reaction and TBA assay, respectively. For all the experiments, erythrocytes from normal individuals served as controls. Thus, the altered membrane characteristics together with their exposure to induced oxidative stress in serum are found to be a few features restricted to diseased erythrocytes. Taken together, our results are suggestive of their interplay in the contribution to the observed anemia in these patients, which may be exploited for better management of the disease.

Increased interferon gamma production by peripheral blood mononuclear cells in response to stimulation of overexpressed disease-specific 9-O-acetylated sialoglycoconjugates in children suffering from acute lymphoblastic leukaemia.

Disease-specific over-expression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia (ALL, PBMC(ALL)) has been demonstrated using a lectin, Achatinin-H, with specificity towards 9-O-AcSAalpha2-6GalNAc. This study investigated the contributory role of 9-O-AcSGs induced on PBMC(ALL). Stimulation of PBMC(ALL) with Achatinin-H through 9-O-AcSGs led to a lymphoproliferative response with a significantly increased interferon-gamma (IFN-gamma) production when compared with unstimulated cells as demonstrated by enzyme-linked immunosorbent assay and mRNA expression. Under identical conditions, PBMC(ALL) ablated of O-acetylations did not respond to such stimulation. In summary, it may be concluded that stimulation of over-expressed 9-O-AcSGs regulate signalling for proliferation, leading to the release of IFN-gamma. Controlled expression of these molecules may be exploited as potential targets for therapy, promising beneficial effects to children with ALL.

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL).

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.

Differential expression of 9-O-acetylated sialoglycoconjugates on leukemic blasts: a potential tool for long-term monitoring of children with acute lymphoblastic leukemia.

Earlier studies have demonstrated overexpression of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) on lymphoblasts, concomitant with high titers of anti-9-O-AcSG antibodies in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the correlation between expression of different 9-O-AcSGs during chemotherapeutic treatment. Accordingly, expression of 9-O-AcSGs on lymphoblasts of ALL patients (n = 70) were longitudinally monitored for 6 years (1997-2002), using Achatinin-H, a 9-O-acetylated sialic acid (9-O-AcSA) binding lectin with preferential affinity for 9-O-AcSGs with terminal 9-O-AcSA alpha 2-->6GalNAc. Western blot analysis of patients (n = 30) showed that 3 ALL-specific 9-O-AcSGs (90, 120 and 135 kDa) were induced at presentation; all these bands disappeared after treatment in patients (n = 22) who had disease-free survival. The 90 kDa band persisted in 8 patients who subsequently relapsed with reexpression of the 120 kDa band. FACS analysis revealed that at presentation (n = 70) 90.1 +/- 5.0% cells expressed 9-O-AcSGs, which decreased progressively with chemotherapy, remained <5% during clinical remission and reappeared in relapse (80 +/- 10%, n = 18). Early clearance of 9-O-AcSG(+) cells, during 4-8 weeks of treatment showed a good correlation with low risk of relapse. Sensitivity of detection of 9-O-AcSG(+) cells was 0.1%. Numbers of both high- and low-affinity binding sites were maximum at presentation, decreased with treatment and increased again in clinical relapse. We propose that close monitoring of 90 and 120 kDa 9-O-AcSGs may serve as a reliable index for long-term management of childhood ALL and merits therapeutic consideration.

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia.

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-O-acetylneuraminic acid-alpha2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac(2)-GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac(2)-GPs(ALL) and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac(2)-GPs(ALL) were affinity-purified, and three distinct leukemia-specific molecular determinants (135, 120, and 90 kDa) were demonstrated by SDS-PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac(2)-GPs(ALL) was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac(2)-GP(ALL) antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac(2)-GPs(ALL) as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac(2)-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n = 50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma as well as normal healthy individuals (n = 21). This study demonstrated the potential of purified Neu5,9Ac(2)-GPs(ALL) as an alternate tool for detection of anti-Neu5,9Ac(2)-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients.