Anantanarayanan Rajaram

Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes.

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6h exposure to 10μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC(50) value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.

Curcumin conjugated gold nanoparticle synthesis and its biocompatibility

Gold nanoparticles have gained much attention due to their widespread biological and technological applications, and consequently their simpler synthesis via green chemistry has also become of foremost importance. We report the room temperature synthesis of spherical gold nanoparticles using curcumin alone as the reducing and stabilizing agent. The pH is found to have an important role in curcumin solubilisation and subsequent formation of curcumin conjugated gold nanoparticles (cAuNPs). UV-visible studies show that the cAuNPs formed are of uniform size and HRTEM studies confirm spheres of average size 18 nm. The DLS measurements show a particle size of 58 nm. The crystallinity has been determined by HRTEM and XRD. The conjugation of stable curcumin on the cAuNPs is indicated by FTIR spectra which also suggest that the phenolic and enolic groups of curcumin bring about the reduction. The zeta potential value of cAuNPs is −23 mV which is stable for up to 6 months at room temperature. The mechanism of cAuNP formation is inferred to be through temporal evolution. This is the first demonstration where curcumin is solubilized at alkaline pH without using any external agent and is used for reducing HAuCl4 to form cAuNPs. The non toxic nature of the cAuNPs is evidenced through biocompatibility studies using human blood cells.

Rapid synthesis of gold nanoparticles with Cissus quadrangularis extract using microwave irradiation

The present study focuses on the rapid synthesis of gold nanoparticles (AuNP) using the aqueous extract of Cissus quadrangularis (CQE) by microwave irradiation. The UV-Visible spectroscopy of the solution obtained from reduction of hydrogen tetrachloroaurate (HAuCl4) by CQE revealed a sharp surface plasmon resonance (SPR) peak at 530 nm confirming the presence of AuNP. The formation of AuNP was optimal at a pH of 9. The AuNP was characterised by FT-IR, SEM, HR-TEM, SAED, XRD, TGA, DLS and Zeta potential measurements. The results indicated that microwave assisted synthesis produced well dispersed, small sized, uniform nanoparticles when compared to conventional room temperature synthesis. The spherical nanoparticle had an average size of 12.0±3.2 nm as revealed through TEM. The crystalline nature of AuNP was confirmed through HR-TEM, SAED and XRD. The FT-IR and TGA data revealed the presence of the CQE components on the surface of the AuNP particles which serve as the capping agent. Upon incubation, the particles did not lyse the red blood corpuscles (RBCs) indicating that they are biocompatible. A possible mechanism for the formation of AuNP in the presence of CQE is proposed.

Chromium picolinate induced apoptosis of lymphocytes and the signaling mechanisms thereof.

Cr(III)(picolinate)(3) [Cr(III)(pic)(3)] is currently used as a nutritional supplement and for treating Type-2 diabetes. The effect of Cr(III)(pic)(3) uptake in peripheral blood lymphocytes is investigated in this study. From the cytotoxicity data, DNA fragmentation pattern, Annexin V staining, TUNEL positivity and the ultrastructural characteristics such as chromatin condensation and formation of apoptotic bodies, it is clear that Cr(III)(pic)(3) induces a concentration dependent apoptosis. It is shown that reactive oxygen species (ROS) produced by treatment with Cr(III)(pic)(3) leads to apoptosis, since we find that pretreatment with N-acetyl cysteine inhibits the process. Using Western blotting technique and fluorescence measurements, the downstream signaling molecules have also been identified. Cr(III)(pic)(3) treatment leads to collapse of the mitochondrial membrane potential, Bax expression, increase in cytosolic cytochrome c content and active caspase-3 and DNA fragmentation and all these manifestations are reduced by pretreating the lymphocytes with N-acetyl cysteine. Thus, it is shown that Cr(III)(pic)(3) is cytotoxic to lymphocytes with ROS and mitochondrial events playing a role in bringing about apoptosis.

Zinc protects human peripheral blood lymphocytes from Cr(III)(phenanthroline)3-induced apoptosis.

We have studied the effect of Cr(III)(phen)3 [(tris(1,10-phenanthroline) chromium(III) chloride)] on lymphocytes in order to find out if metallothioneins (MTs) are produced in the process. We also investigated whether zinc pretreatment is able to protect cells from apoptosis reported to occur for this compound. Our results indicate that MT synthesis is induced by Cr(III)(phen)3, and it has been identified as the MT-3 isoform through RT-PCR which has not been reported earlier. By zinc pretreatment, this apoptosis is reversed as inferred from cytotoxicity studies, Annexin-V/PI staining, ethidium bromide/acridine orange staining and DNA fragmentation pattern and ultrastructural investigations using TEM and SEM. The zinc pretreatment reduces the amount of ROS produced by Cr(III)(phen)3. The MT-1a and 1b synthesized by zinc (also evidenced through RT-PCR experiments) is possibly able to scavenge ROS which is one of the early signaling molecules that lead to apoptosis. Zinc pretreatment also reverses the changes in downstream signaling events such as mitochondrial membrane potential, ATP levels and the activation of caspase-3. This is the first report on the induction of MT-3 in lymphocytes due to a metal stress or any other stimuli. Even though MT-3 is synthesized here, apoptosis still occurs due to ROS production on Cr(III)(phen)3 exposure when the cells have not been primed with zinc.

Autoschizis of T-cells is induced by the nutritional supplement, Cr(III)picolinate

In recent times, Cr(III)(picolinate)(3) [Cr(III)(pic)(3)] a nutritional supplement, is gaining attention because of its clastogenic and mutagenic properties. Earlier studies of ours indicated that Cr(III)(pic)(3) is cytotoxic to lymphocytes with ROS and mitochondrial events playing a role in bringing about apoptosis. Now, we report that, autoschizis is induced in lymphocytes in a concentration and time dependent manner which is confirmed through TEM and SEM. Lymphocytes treated with concentrations of 100microM of Cr(III)(pic)(3) exhibit features such as cytoplasmic bleb, self excision of cytoplasm, cytoplasmic leakage and membrane bound bodies formed from the excised pieces apart from apoptosis and necrosis. Though autoschizis has been described in tumor cell lines treated with menadione and ascorbate, occurrence of this cell death in normal T-lymphocytes is reported here. The cellular events that accompany autoschizis are found to be increase in intracellular Reactive Oxygen Species (ROS) and cytoplasmic lactate dehydrogenase, loss of mitochondrial membrane potential (MMP) and depletion of ATP. Further, autoschizis is effected through increases in DNase I and DNase II activity with a concomitant decrease in caspase-3 activity which leads to a random cleavage of the DNA as demonstrated by a smear like pattern after electrophoresis on agarose gel.

Silver ion impregnated composite biomaterial optimally prepared using zeta potential measurements.

Biodegradable, antimicrobial composite of various silver ion concentrations was synthesized using zeta potential and isoelectric point measurements, for a controlled release of silver ions, and in addition to assess the effect of protein adsorption with the increase of the silver ion concentration. The interaction between hydroxyapatite (HAp) and silver incorporated hydroxyapatite (AgHAp) with gelatin was increased by optimally adjusting the zeta potential and isoelectric point of the ceramic (HAp and AgHAp), and bio-polymer individually. The electrostatic interactions between the ceramic and biopolymer were confirmed, through shifts in N-H stretching, decrease in the swelling ratio, and increase in the degradation temperature observed by the derivative thermo-gravimetric analysis (DTG). These results substantiate that, the zeta potential is a novel tool to increase the ceramic-biopolymer interaction. Increasing electrostatic interaction between the biopolymer and ceramic, decreases the release of silver ions in the simulated body fluid, due to the controlled degradation of the biopolymer. The isoelectric point decreases with the increase of the silver ion concentration, which evidenced the change in the net surface charge. With the increase of the silver ion concentration, the protein adsorption decreases due to an increase in hydrophilic character of the composite. This study examines the minimum concentration of silver ion essential for maximum protein adsorption, antimicrobial and hemocompatibility. This study provides a novel route to control the release of silver ions by enhancing the ceramic-polymer interaction and estimate the silver ion concentration suitable for protein adsorption. The prepared composite is nontoxic, degradable, and antimicrobial, with the controlled release of silver ions in the simulated body fluid.

Effect of Cr(VI) and Ni(II) metal ions on human adipose derived stem cells

Environmental exposure of Cr(VI) and Ni(II) due to rapid industrialization causes adverse effects in living tissues. Small quantities of these ions also find their way into tissues when metal alloys are used as implants. Even though considerable research has been done on the effects due to their exposure in animal cells, there are only very few reports on how they can affect stem cells which have been shown to be found in adult tissues as well, albeit in small quantities. Hence this study was aimed at understanding how Cr(VI) and Ni(II) affect human adipose derived stem cells (hADSCs) in a cell culture environment. Our results indicate that both ions induce apoptosis in a concentration and time dependent manner with loss of mitochondrial membrane potential (MMP) and corresponding increase in caspase-3 activity. With regard to Ni(II), apoptosis seems to occur only in a small percentage of cells while necrosis is predominant. It can be inferred that the long term exposure of these metals may cause adverse effects in stem cell proliferation and differentiation.

Properties of porous calcium apatite rods

Calcium apatite powder was formed and a definite quantity was mixed separately with 1% solutions of gelatin, chitosan and collagen to form a paste. The gelatin apatite mixture flowed easily while the collagen apatite one was too viscous. The paste formed by the different mixtures was packed individually in thin plastic tubes and calcined at 800/spl deg/C. The ashing away of the organic matter and the phase transformation of the phosphate resulted in rod shaped calcium phosphate rods. The chitosan apatite paste gave the most uniform rods of good porosity. The calcined samples were analysed for their chemical and physical properties. Characterisation was done by FT IR and X-ray diffraction. The material was stable in water and in a phosphate buffer and did not disintegrate. The water uptake and chitosan solution uptake were determined. The porous rods were brittle with comparatively poor bending strength. The apatite rods loaded with an antibiotic like gentamicin sulphate is intended to be used for drug delivery at the sites of orthopaedic implants for the prevention of osteomyelitis.