Differential response of antioxidant defense in HepG2 cells on exposure of Livotrit®, in a concentration dependent manner.

Livotrit®, a polyherbal formulation (Zandu, India) is commonly prescribed for liver health. The present study was undertaken to elucidate possible mechanism of antioxidant potential of Livotrit®. Livotrit® exhibited concentration dependent radical scavenging activity, inhibition of lipid peroxidation as well as activation and gene expression of antioxidant enzymes. Interestingly, lower concentration of Livotrit® (0.05%) significantly increased activities and gene expression of catalase, Glutathione reductase (GR) and Gluthathione peroxidase (GPx), while higher concentration of Livotrit® (0.5%) significantly increased antioxidant enzyme Heme-oxygenase 1(HO-1) and not catalase (CAT), GR and GPx. Transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2) required for expression of catalase, GR, GPx and HO-1 was efficiently translocated into the nucleus at both concentrations. Inspite of this, concentration dependent activation of these enzymes was found to be mediated through miRNAs involved in regulation of their gene expression.

DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India.

We have checked the utility of DNA barcoding for species identification of nymphalid butterflies from Western Ghats of India by using 650 bp sequence of mitochondrial gene cytochrome c oxidase subunit I. Distinct DNA barcoding gap (i.e. difference between intraspecies and interspecies nucleotide divergence), exists between species studied here. When our sequences were compared with the sequences of the conspecifics submitted from different geographic regions, nine cases of deep intraspecies nucleotide divergences were observed. In spite of this, NJ (Neighbour Joining) clustering analysis successfully discriminated all species. Observed cases of deep intraspecies nucleotide divergences certainly warrant further study.

Pre-treatment of Syndrex protects mice from becoming diabetic after streptozotocin injection.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia due to either insufficiency of insulin or inability of cells to respond to insulin. Many clinical and experimental evidence have suggested the strong association between hyperglycemia, oxidative stress and diabetic complications. Therefore, the antidiabetic drugs with antioxidant potential would have a higher therapeutic value. To check its antidiabetic and antioxidant properties in vivo, experiments were done wherein mice were fed with Syndrex in different schedules and/or made diabetic by intraperitoneal injection of streptozotocin. Animals fed with Syndrex prior to the induction of diabetes by streptozotocin injection showed resistance to an increase in blood glucose levels. This treatment increased the activities of antioxidant enzymes namely, catalase, glutathione reductase and superoxide dismutase and reduced serum triglyceride and cholesterol levels as compared to those found in uncontrolled diabetic mice. Among the three different schedules used for Syndrex treatment, the best effect was seen in the case of mice pretreated with Syndrex prior to STZ injection. In our opinion, Syndrex given along with insulin may reduce the amount of insulin dose required and because of its strong antioxidant activity would certainly help to reduce the development of diabetic complications.

Pterocarpus marsupium extract reveals strong in vitro antioxidant activity.

Diabetes mellitus is a complex chronic disease characterized by hyperglycemia, which via several mechanism leads to an increase in production of reactive oxygen species (ROS) leading to various secondary complications. Thus, a drug having both antidiabetic and antioxidant properties would have great therapeutic value for overcoming the oxidative load in diabetes. The present study was aimed at extensively evaluating the antioxidant properties of an anti-diabetic plant extract of stem bark of Pterocarpus marsupium using various in vitro radical scavenging assays as well as by using liver slice cultures as a model system. Our results demonstrate that the whole aqueous extract showed high antioxidant activity in all different assays used and also protected mitochondria against oxidative damage. Ethanol was used as an inducer of oxidative stress in liver slice culture and cytotoxicity was estimated by quantitating release of cytotoxicity marker enzymes such as lactate dehydrogenase (LDH). Additionally, levels of antioxidant enzymes (AOEs) namely superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were also estimated. The whole aqueous extract significantly reduced LDH release along with reduction of lipid peroxidation compared to ethanol treated slices. These results indicate that the P. marsupium extract may serve as a potential source of natural antioxidant for treatment of diabetes.

Enhancement of vertebrate cardiogenesis by a lectin from perivitelline fluid of horseshoe crab embryo.

Cardiac myocytes are the first cells to differentiate during the development of a vertebrate embryo. A wide variety of molecules take part in various steps in this process. While exploring biologically active molecules from marine sources, we found that a constituent of perivitelline fluid from embryos of the Indian horseshoe crab can enhance growth and differentiation of chick embryonic heart. We have purified the factor and identified the cardiac promoting molecule to be a novel lectin. We show that this molecule influences cardiac development by increasing the number of cells constituting the heart and by modulating the expression of several cardiac development regulatory genes in chick embryos. Using mouse embryonic stem cells we show that the cardiac myocyte-enhancing capacity of this molecule extends to mammals and its effects can be blocked using methylated sugars. This molecule may prove to be an important tool in the study of cardiomyocyte differentiation.

Activin disrupts somitogenesis in cultured chick embryos.

The anatomical and cell biological aspects of somite formation in the chick embryo have been rather well studied. Molecular regulation of somitogenesis in vertebrates is just beginning to be understood. We have studied the effects of human recombinant activin on somitogenesis in gastrulating chick embryos cultured in vitro with a view to assessing the possible role of activin-related molecules in this phenomenon. Activin disrupted somitogenesis in treated embryos, resulting in the formation of abnormal, split or ectopic somites. Light microscopic examination indicated that the ability of activin to interfere with somitogenesis might be partly due to initiation of somite formation at ectopic sites. We show that these cells are indeed somitogenic by their expression of one of the earliest somite-specific marker genes, Pax3. Scanning electron microscopic examination of control and treated embryos revealed direct effects of activin on cell-cell interactions. Cells from treated embryos exhibited disrupted intercellular adhesion leading to large intercellular spaces, altered cell shapes and modification of cell surface protrusions. The effects of activin on somitogenesis appear to be specific, since the neural structures, which are generally more susceptible to chemical insults during gastrulation, were relatively less affected. The results clearly point to a role of activin-related molecules in somitogenesis in the chick embryo.

Hydra constitutively expresses transcripts involved in vertebrate neural differentiation.

The diploblastic Hydra is among the most primitive multicellular organisms. Using cross-hybridization with Xenopus probes, noggin-like transcripts were detected in the hypostome and basal disc of adult Hydra (Pelmatohydra oligactis), regions with properties similar to that of the amphibian organizer. This points to the possibility of a close molecular similarity between the Xenopus and Hydra organizers. The constitutive expression of a noggin-like gene in Hydra may be responsible for its regenerative capacity.

FGF signaling is essential for the early events in the development of the chick nervous system and mesoderm.

Fibroblast growth factor (FGF) belongs to a family of polypeptides with diverse biological functions. In the present study we have assessed the role of FGF signaling in the development of nervous system and mesodermal tissues in chick embryo. Treatment of in vitro cultured embryos with exogenous, human recombinant FGF led to abnormalities in neural induction and development, notochord formation and somitogenesis as studied by gross morphology and histology. Overall growth and development was also adversely affected as seen from the measurement of body axis length. Further, treatment of embryos with FGF resulted in differential modulation of expression of two genes important in normal development as studied by whole mount in situ hybridization using DIG-labeled riboprobes. The expression of Brachyury, which is necessary for mesoderm formation, was down-regulated in FGF-treated embryos. The expression of noggin, the product which participates in the patterning of the chick neural tube was, on the other hand, up-regulated within 2 h. We also studied development of neural and mesodermal tissues in conditions where FGF signaling was defective. This was achieved by culturing the embryos in the presence of suramin. In the presence of low doses of suramin (100-150 nmole/culture), abnormalities were detected mainly in the mesodermal structures while at higher doses (200-400 nmole/culture), the nervous system too was found to be abnormal in a large proportion of embryos. Treatment of chick embryos with suramin (200 nmole/culture) also modulated the expression of Brachyuryand noggin within a 2 h period. The results showthat FGF signaling plays an important role in the molecular events leading to the development of nervous system and mesodermal tissues in the chick embryo.

Reproductive toxicity of piperine in Swiss albino mice.

Piperine (CAS 94-62-2) is a constituent of various spices which are used as common food additives all over the world. The reproductive toxicity of piperine was studied in Swiss albino mice. Relevant short-term tests were employed to assess the effect on estrous cycle, mating behaviour, toxicity to male germ cells, fertilization, implantation and growth of pups. Piperine (10 and 20 mg/kg b.w.) increased the period of the diestrous phase which seemed to result in decreased mating performance and fertility. Post-partum litter growth was not affected by the piperine treatment. Sperm shape abnormalities were not induced by piperine at doses up to 75 mg/kg b.w. Considerable anti-implantation activity was recorded after five days post-mating oral treatment with piperine. The sex ratio and post-implantation loss were unaffected after treatment with piperine. Intrauterine injection of piperine caused the total absence of implants in either of the uterine horns (16.66%) or one of the horns (33%) of treated females. No histopathological changes were detected in the ovary and the uterus at the cellular level. Prostaglandin E1-induced acute inflammation of rat paw was significantly reduced after piperine treatment. Our results show that piperine interferes with several crucial reproductive events in a mammalian model.

Assessment of genotoxic effect of piperine using Salmonella typhimurium and somatic and somatic and germ cells of Swiss albino mice.

Piperine (CAS 94-62-2) is a constituent of various spices and is used as a common food additive all over the world. The genotoxic potential of piperine was assessed using four different test systems, namely, Ames test using Salmonella typhimurium, micronucleus test, sperm shape abnormality test and dominant lethal test using Swiss albino mice. In the Ames test, six different doses of piperine, in the range of 0.005-10 mumol/plate, did not induce his+ revertants, with or without metabolic activation, indicating its nonmutagenic nature. In the bone narrow micronucleus test using two doses in the range of therapeutic usage (10 and 20 mg/kg body weight), piperine itself was non-mutagenic. Like in somatic cells, piperine (10 and 50 mg/kg body weight) failed to induce mutations in male germ cells of mouse as assessed by using the sperm shape abnormality and dominant lethal tests. Piperine thus appears to be a non-genotoxic chemical.

Cell surface alterations by taxol associated with abnormal morphogenesis in the chick embryo.

The anticancer drug taxol brings about its biological effects by altering the stability of microtubules. We have examined the effects of taxol on early morphogenesis in chick embryos cultured in vitro. Taxol induced various abnormalities in the developing nervous system, heart and somites as well as general retardation of development. SEM studies revealed that taxol treatment leads to dramatic alterations in the embryonic cell surfaces. Time-course experiments demonstrated that the action of taxol is very rapid and becomes evident within a few minutes at the ultrastructural level. Taxol thus throws embryonic cell adhesion and motility out of balance. This appears to be the major cause of abnormal morphogenesis in taxol-treated embryos.

Inhibition of cleavage by restriction endonucleases due to modifications induced in SV40 DNA by methyl methanesulfonate.

Methyl methanesulfonate (MMS), a direct mutagen, methylates DNA bases and causes distortions in DNA structure. Supercoiled SV40 DNA was treated in vitro with varying concentrations of MMS from 0.001 mM to 10 mM MMS either for 30 min or 3 h and analysed by electrophoresis in 1% neutral and alkaline agarose gels. The electrophoretic mobility (EPM) of native DNA did not change after treatment with the mutagen, while alkaline gels revealed low MW DNA fragments due to single strand breaks at alkali-sensitive sites generated by the action of MMS. By two-dimensional electrophoresis, we find that all three native DNA forms contain alkali-sensitive sites after treatment with MMS. To examine the effect of base modification by MMS on DNA-protein interactions, we have used as probes, restriction endonucleases. These cleave DNA in a sequence-specific manner, and their activity is dependent upon the methylation status of the substrate DNA. We find that cleavage by these restriction endonucleases is inhibited due to methylation by MMS.

Fast inducible repair of microinjected UV-irradiated SV40 DNA in monkey kidney cells.

In monkey kidney cells (TC-7), microinjected with UV-irradiated (103-362 J/m2) SV40 DNA, the expression of viral antigens decreases in a UV-dose-dependent manner and the viral genes are not repaired constitutively. When the viral DNA is microinjected 4 h after UV-irradiation (40 J/m2) of host cells, the expression of viral antigens is restored in all cells. The time course of restoration of viral gene expression function shows that in UV-irradiated cells the repair is induced rapidly and fully within 2 h and the induced state is maintained for 24 h. Intact viral DNA molecules, microinjected during the period of induction of cellular UV repair, are expressed less efficiently than UV-irradiated viral genomes.

Ultrastructural cell surface alterations in developing embryos of frog (Microhyla ornata) treated with cytochalasins.

Scanning electron microscopic examination of M. ornata embryos treated with cytochalasins A, B and H (CA, CB and CH) showed extensive cell disaggregation resulting in large intercellular spaces and apparent loss of intercellular communication. All the three cytochalasins significantly reduced surface features, such as, filopodia and membrane ruffling which are considered essential for normal morphogenetic movements. Appreciable qualitative differences could not be detected in effects exerted by CA, CB and CH although potency of the three drugs clearly differed. The results demonstrate that in spite of the differences in their primary mechanisms of action, treatment with all the three cytochalasins culminates in comparable effects on the cell surface architecture resulting into abnormal morphogenesis.

Mesoderm enhancing effect of human seminal plasma inhibin and its synthetic C-terminal nonapeptide fragment in the chick embryo.

Isomers of fibroblast growth factor and members of the transforming growth factor beta family have been identified as potent mesoderm inducing factors, particularly in amphibians. Activins belonging to the latter group are capable of inducing all types of mesoderm. Inhibins, also belonging to the same family of proteins have an exactly opposite biological action than activins in the adult organism. We have examined the effects of human seminal plasma inhibin on the early development of the chick embryo, where also activins appear to be important in mesoderm induction. Contrary to expectations, inhibin brought about stimulation of development of somites and heart, structures of mesodermal origin, and increase in the body length in more than 50% of the treated chick blastoderms. A synthetic fragment of human seminal plasma inhibin, a nonapeptide fragment of C-terminal end, also exhibited similar effects. In some cases the treatments resulted in completely abnormal development while in some increase in the number of somites was associated with abnormality in the anterior region. Our results demonstrate that human seminal plasma inhibin does not act as an inhibitor of mesoderm induction in the chick embryo but in amniotes inhibin-related molecules may have a role as mesoderm enhancers.

Genotoxicity assessment of the antifungal antibiotic aureofungin in Salmonella typhimurium and Swiss albino mice.

The widely used agricultural antifungal agent aureofungin (ARF) was subjected to genotoxicity assessment using the Ames Salmonella assay as well as the in vivo micronucleus test and dominant lethal test in Swiss mice. In the Ames Salmonella spot test, ARF slightly elevated the number of histidine revertants after metabolic activation over a wide dose range (1-1000 micrograms/plate) in TA102 but not in TA97a, TA98 or TA100. In the preincubation plate incorporation assay with TA102, ARF increased the number of revertants in a dose-dependent manner only after metabolic activation. ARF failed to significantly elevate the frequency of micronucleated polychromatic erythrocytes (PE) in the bone marrow of Swiss mice. It elevated the frequency of dominant lethal mutations in the 7th and 8th weeks at 30 mg/kg body weight, a concentration much higher than the actual concentration used in the field. We conclude that ARF is non-mutagenic in somatic cells in vivo at doses used in the present study, probably mutagenic in stem-cell spermatogonia and may be classified as an equivocal promutagen, possibly acting as a cross-linker.

Essential role of insulin during early prepancreatic development of the frog Microhyla ornata.

We undertook the present study to examine the role of insulin during early development of frog when functional pancreas does not exist. Two approaches were adopted to achieve this objective. In the first approach, influence of exogenous insulin on the early embryonic development of the frog Microhyla ornata was studied. In the second approach, the effects of antiserum to insulin on embryonic development were studied. Exogenous insulin stimulated the embryonic development while immunoneutralization of endogenous insulin not only resulted in retardation of development but also induced developmental abnormalities. These results demonstrate the essential role of insulin during early embryonic development of this frog. To our knowledge, such findings have not been reported so far in any amphibian.

Modulation of cyclophosphamide mutagenicity by vitamin C in the in vivo rodent micronucleus assay.

The modulatory effect of vitamin C (Vit C) on the mutagenic effect of the antineoplastic drug cyclophosphamide (CP) was assessed in the in vivo micronucleus test in Swiss mice. Simultaneous oral administration of Vit C with i.p. administration of CP was found to decrease the frequency of micronucleated polychromatic erythrocytes elevated by CP. Vit C exhibited a significant antimutagenic effect over a wide dose range (1.56-200 mg/kg). The dose-response relationship was highly significant. These results demonstrated the ability of the in vivo micronucleus test to detect in vivo modulation of CP mutagenicity by Vit C. Our earlier results and those from other laboratories also indicate that this model system is suitable for primary in vivo screening of modulation of mutagenesis.

Studies on modulation of the effects of colchicine by L-cysteine using bone marrow of Swiss mice.

Colchicine (COL) elevates the frequency of micronucleated polychromatic erythrocytes (PE), the ratio of normochromatic to polychromatic erythrocytes (N/PE) and the frequency of large PE due to spindle disruption. Simultaneous i.p. injection of L-cysteine (CYS) does not influence the effects of COL while if administered 1 h prior to COL, CYS suppresses the N/PE ratio and frequency of large PE but not the frequency of micronucleated PE elevated by COL. Preincubation of CYS with COL at 37 degrees C for 1 h results in a significant decrease in all the COL effects. The modulatory effect of exogenous CYS appears to be due to its competition with the endogenous tubulin cysteine residues for interacting with COL.

In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline.

We have previously shown that the common antiamebic drug diiodohydroxyquinoline (DIHQ) exhibits mutagenic activity in the in vivo micronucleus test in Swiss albino mice. Results of experiments undertaken to study the influence of ascorbic acid (vitamin C) on the mutagenicity of DIHQ in this model system showed that ascorbic acid acts as an antimutagen against DIHQ. The effective antimutagenic doses of ascorbic acid themselves do not show any genotoxic effects in this in vivo system. It will be necessary, however, to elucidate the mechanism of action of ascorbic acid as well as its effects on the therapeutic properties of DIHQ before a practical use of ascorbic acid is contemplated for this purpose.