Fluorescent gold clusters as nanosensors for copper ions in live cells.

This paper reports the use of fluorescent gold nanoclusters synthesized using bovine serum albumin (Au-BSA) for the sensing of copper ions in live cells. The fluorescence of the clusters was found to be quenched by Cu(2+) enabling its detection in cells. The selectivity of the nanosensor was demonstrated in the presence of several cations excluding Hg(2+). We did not study the effect of Hg(2+) since it was reported earlier. The present study suggests that Cu(2+) induced fluorescence quenching is due to its binding to BSA rather than the fluorescence quenching by metal-metal interaction as in the case of Hg(2+). The Au-BSA showed excellent selectivity to Cu(2+) at various pH conditions. The 'turn off' of fluorescence can be retrieved by a Cu(2+) chelator glycine. Our results showed that gold clusters can be used as a 'turn off' sensor for copper and a 'turn on' sensor for glycine. Under the experimental conditions, the probe showed a response for Cu(2+) over a range of 100 µM to 5 mM with a detection limit of 50 µM. The role of Cu(2+) in the misfolding and disassembly of Prion Protein (PrP) leading to various maladies is well ascertained. The methodology we reported here seems to be useful in supplementing other techniques in predicting disease conditions involving Cu(2+).

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF.

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.

Pollution level in distillery effluent and its phytotoxic effect on seed germination and early growth of maize and rice.

The effluent from a Lucknow- based distillery (Mohan Meakin Distillery) was analyzed for physico-chemical and biological parameters of pollution and concentration of potentially toxic heavy metals (Cd, Cr, Ni and Zn) and the effect of the distillery effluent, as such and on 50% dilution with tap water was studied on seed germination and seedling growth of maize (Zea mays L.) and rice (Oryza sativa L.). The effluent was wine red in colour and highly acidic (pH approximately 55) and possessed decaying alcoholic smell. The effluent contained high values of different pollution parameters, particularly total solids, 3450 mgl(-1) (soluble plus suspended solids), alkalinity 1500 mgl(-1), biological oxygen demand (BOD, 1649 mgl(-1)) and chemical oxygen demand (COD, 2036 mgl(-1)). It had very low values of dissolved oxygen (DO, 0.34 mgl(-1)). The heavy metals (Cd, Cr, Ni and Zn) content, particularly the nickel concentration (0.029 mg l(-1)) was high. Use of the distillery effluent, even on 1:1 dilution with tap water inhibited germination and early seedling growth of maize and rice. In both maize and rice, more so in the former germination % of seeds, length of radicle and plumule and the fresh and dry weight of the seedlings were significantly reduced. The emerging leaves of the seedlings also developed visible effects of toxicity some of which resembled the symptoms of nickel toxicity. Our observations suggest that the effluent, as discharged from the distillery carry a heavy load of pollutants. Its discharge into the river Gomti poses a potential threat to the aquatic life, perticularly during the summer months when the water flow in the river is drastically reduced. The distillery effluentis also harmful for irrigating crops grown along the drain carrying it.

Nano-anisotropic surface coating based on drug immobilized pendant polymer to suppress macrophage adhesion response.

Exploring drug molecules for material design, to harness concepts of nano-anisotropy and ligand-receptor interactions, are rather elusive. The aim of this study is to demonstrate the bottom-up design of a single-step and bio-interactive polymeric surface coating, based on drug based pendant polymer. This can be applied on to polystyrene (PS) substrates, to suppress macrophage adhesion and spreading. The drug molecule is used in this coating for two purposes. The first one is drug as a "pendant" group, to produce nano-anisotropic properties that can enable adhesion of the coatings to the substrate. The second purpose is to use the drug as a "ligand", to produce ligand-receptor interaction, between the bound ligand and receptors of albumin, to develop a self-albumin coat over the surface, by the preferential binding of albumin in biological environment, to reduce macrophage adhesion. Our in silico studies show that, diclofenac (DIC) is an ideal drug based "ligand" for albumin. This can also act as a "pendant" group with planar aryl groups. The combination of these two factors can help to harness, both nano-anisotropic properties and biological functions to the polymeric coating. Further, the drug, diclofenac (DIC) is immobilized to the polyvinyl alcohol (PVA), to develop the pendant polymer (PVA-DIC). The interaction of bound DIC with the albumin is a ligand-receptor based interaction, as per the studies by circular dichroism, differential scanning calorimetry, and SDS-PAGE. The non-polar π-π* interactions are regulating; the interactions between PVA bound DIC-DIC interactions, leading to "nano-anisotropic condensation" to form distinct "nano-anisotropic segments" inside the polymeric coating. This is evident from, the thermo-responsiveness and uniform size of nanoparticles, as well as regular roughness in the surface coating, with similar properties as that of nanoparticles. In addition, the hydrophobic DIC-polystyrene (PS) interactions, between the PVA-DIC coating and PS-substrate produce improved coating stability. Subsequently, the PVA-DIC coated substrate has the maximum capacity to suppress the macrophage (RAW 264.7 cell line) adhesion and spreading, which is partly due to wavy-surface topography of hydrophilic PVA and preferential albumin binding capacity of PVA bound DIC. Our result shows that, such surfaces suppress the macrophages, even under stimulation with lipopolysaccharide (LPS). The modified tissue culture plates can be used as an in vitro tool, to study the macrophage response under low spatial cues.

Chitosan matrix for oral sustained delivery of ampicillin.

Ampicillin was embedded in a chitosan matrix to develop an oral release dosage form. The in vitro release profile of ampicillin from chitosan beads and microgranules of chitosan was monitored, as a function of time, using a UV Spectrophotometer. The releasing studies were performed in a rotating shaker at 100 r.p.m., containing 0.1 M HCI buffer, pH 2.0, or 0.1 M phosphate buffer, pH 7.4, solutions, and a comparison was made between the drug loaded microbeads and microgranules. It seems that the amount and percentage of drug release was much higher in HCI solution compared with the phosphate solution, probably due to the gelation properties of the matrix at acid pH. The release rate of ampicillin from the chitosan matrix was slower for the beads as compared with the granules. From scanning electron microscopic studies, it appears that the drug forms a crystal structure within the chitosan beads, which dissolves out slowly to the dissolution medium through the micropores of the chitosan matrix. The results propose the possibility of modifying the formulation in order to obtain the desired controlled release of the drug for a convenient oral sustained delivery system.

Effect of plasma glow, glutaraldehyde and carbodiimide treatments on the enzymic degradation of poly (L-lactic acid) and poly (gamma-benzyl-L-glutamate) films.

The hydrolytic and enzymic degradation of poly(L-lactic acid) (PLA) and poly(gamma-benzyl L-glutamate) (PBGA) films, together with a series of surface treatments, were studied, as a function of exposure time. The degradation of these polymers was monitored by weight loss, contact angle, pH changes and tensile strength studies. Glutaraldehyde treatment retained the maximum strength of PLA in buffer, followed by carbodiimide, compared with control films. On the other hand, plasma glow reversed the effect. The ability of alpha-chymotrypsin, carboxypeptidase, ficin, esterase, bromelain and leucine aminopeptidase to modulate the degradation of PLA and PBGA was also investigated. Addition of these enzymes to the polymer-buffer system reduced the tensile strength of these polymers variably. Among the six enzymes studied, leucine aminopeptidase showed the highest enzymic effect on the degradation of the glutaraldehyde-treated and bare PLA or bare PBGA films. However, glutaraldehyde-cross-linked PLA demonstrated maximum stability in buffers or in all other enzyme systems studied compared with bare PLA. It is conceivable that surface treatments on these polymers might have altered their physical and chemical configuration and the subsequent degradation properties. Surface modifications may provide new ways of controlling the biodegradation of polymers for a variety of biomedical applications.

Chitosan beads and granules for oral sustained delivery of nifedipine: in vitro studies.

Nifedipine was embedded in a chitosan matrix to develop a prolonged-release form. The in vitro release profiles of nifedipine from chitosan beads and microgranules were monitored by UV spectrophotometer. The studies were performed in a rotating shaker (100 rev min-1) in 0.1 M HCl buffer (pH 2.0) or 0.1 M phosphate buffer (pH 7.4). Comparison was made between drug-loaded microbeads and microgranules. The amount and percentage of drug release were much higher in HCl than in phosphate buffer, probably due to the salt formation of the matrix (chitosan hydrochloride) at acid pH. The release rate of nifedipine from chitosan matrix was slower for beads than granules. These findings suggest the possibility of modifying the formulations to obtain the desired controlled release of the drug in an oral sustained-delivery system.

Effect of liposome-albumin coatings on ferric ion retention and release from chitosan beads.

Ferric chloride was embedded in a chitosan matrix to develop a prolonged-release form. The in vitro release profiles of ferric ions from chitosan beads were monitored in 0.1 M Tris-HCl buffer, pH 7.4, using a UV spectrophotometer. The amount of drug release was much higher initially, followed by a constant slow release profile for a prolonged period. The initial burst release was substantially modified with liposome and albumin coatings. From scanning electron microscope studies, it appears that the ferric ions diffuse out slowly to the dissolution medium through the micropores of the chitosan matrix. Further, the liposome forms a phospholipid membrane layer in the pores of chitosan beads and encapsulates the ferric ions within their vesicles and controls the release profile. The chitosan beads loaded with ferric ions substantially inhibited the polyurethane-associated calcification, in an in vitro model system. The released ferric ions, appeared to alter the protein-surface binding and improved the biocompatibility of the matrix. The results propose the possibility of modifying the polymer matrix to obtain a desired controlled release of the drug for a prolonged period.

Albumin adsorption on to aluminium oxide and polyurethane surfaces.

The changes in protein adsorption onto aluminium surfaces coated with different thicknesses of oxide layers were examined. The oxide layers on aluminium substrates were derived by the anodizing technique. Protein adsorption studies were conducted using 125I-labelled albumin and the amount of albumin adsorbed was estimated with the help of a gamma counter. An increase in albumin adsorption was observed on oxide layer coated aluminium surfaces. The effect of anti-Hageman factor on albumin and fibrinogen adsorption on to bare aluminium, oxide layer coated aluminium and bare polyether urethane urea surfaces was also investigated. It was observed that the presence of anti-Hageman factor increased the adsorption of albumin and fibrinogen on to all these substrates.

Influence of steroid hormones on protein-platelet interaction at the blood-polymer interface.

To develop artificial materials for prolonged use in the vascular system, the complicated process of surface-induced thrombosis needs to be better understood. Steroidal hormones have a profound role in thrombosis and haemostasis, although adequate studies are not available to demonstrate their part in the thromboembolic phenomena that occur at the blood-foreign material interface. We studied the interfacial phenomena of five steroid hormonal drugs, Sustanon, Menstrogen, Mixogen, Durabolin and Ovral and their interaction with proteins and platelets toward an artificial surface using contact angle, polyacrylamide gel electrophoresis, trace labelling methods, etc. This study demonstrates the effect of these hormones to modulate platelet-surface attachment in the presence of platelet inducers. The addition of steroid hormones to the polymer-protein system can inhibit the level of surface-bound albumin where the fibrinogen binding to an artificial surface has been enhanced or unaltered. Steroids also increase platelet-surface attachment to variable degrees. Prolonged use of steroids or the oestrogen-containing oral contraceptive agents may not be advisable for patients having an artificial implant in contact with blood.

Changes in protein adsorption on polycarbonate due to L-ascorbic acid.

When a synthetic material is introduced into blood, plasma proteins are rapidly adsorbed on to its surface, followed by the attachment of formed elements of blood, leading to thrombus formation. Previous research reported that vitamin C (L-ascorbic acid) prolongs the clotting time due to the formation of vitamin C-calcium complexes, which reduce the availability of Ca2+ ions for the clotting mechanism in in vitro conditions. Contact angle and platelet adhesion studies have indicated that vitamin C modifies the surface-protein interaction and surface-platelet binding at the interface. In this paper an increased thickness of protein layer deposited on the polycarbonate substrate has been observed in the presence of vitamin C (approximately 50 A) using ellipsometric measurements. Further polyacrylamide gel electrophoresis (PAG) and infrared attenuated total reflection spectroscopy (IR-ATR) techniques have provided a better understanding of the interfacial phenomena. It seems, that the adsorption of albumin is increased, relatively, in the presence of vitamin C, when compared with that of fibrinogen and gamma-globulin from an equal amount of protein mixture.

Development of chitosan/polyethylene vinyl acetate co-matrix: controlled release of aspirin-heparin for preventing cardiovascular thrombosis.

Aspirin and heparin were embedded in chitosan/polyethylene vinyl acetate co-matrix to develop a prolonged release form. The in vitro release profiles of these drugs from the co-matrix system were monitored in Tris HCl buffer pH 7.4, using a UV spectrophotometer. The amount of drug release was initially much higher. followed by a constant slow release profile for a prolonged period. The initial burst release was substantially modified with styrenebutadiene coatings. From scanning electron microscopy studies it appears that the drugs diffuse out slowly to the dissolution medium through the micropores of the co-matrix. The released aspirin-heparin from the co-matrix system had shown their antiplatelet and anticoagulant functions. The results propose the possibility of delivering drug combinations, having synergestic effects for therapeutic applications.

Structural studies on bovine bioprosthetic tissues and their in vivo calcification: prevention via drug delivery.

Cardiovascular calcification, the formation of calcium phosphate deposits in cardiovascular tissue, is a common end-stage phenomenon affecting a wide variety of bioprostheses. To study the process of calcification in tissue prosthetics, glutaraldehyde-treated bovine pericardium, dura mater and fascialata were implanted subcutaneously in rats and retrieved 21 days later and thereby morphological findings were correlated with biochemically determined levels of calcium. Transmission electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the interfibrillar spaces. The deposition of calcium was higher with dura and fascia prostheses compared to pericardium. However, the release of Fe3+ ions from chitosan matrix had substantially inhibited the deposits of calcium in all implanted tissues. It seems that the structural and anatomical features of the tissue is one of the important factors for tissue-associated calcification. It is also confirmed that glutaraldehyde-preserved pericardium is the most suitable material for the development of cardiac prosthesis, with an appropriate drug combination therapy for prevention of pathological calcification.

Protein/platelet interaction with an artificial surface: effect of vitamins and platelet inhibitors.

Protein adsorption and platelet adhesion are two important biological processes arising at the blood-prosthetic interface. The effect of Vitamins and antiplatelet drugs to modulate the surface induced platelet adhesion to polycarbonate was investigated using washed calf platelets in presence and absence of fibrinogen. This study also demonstrated the effects of Vitamins and antiplatelet drugs towards protein adsorption to an artificial surface. It seems Vitamin B6, Vitamin E, combinations of Aspirin-Persantine, Aspirin-Vitamin C, a synthetic Polyelectrolyte and Galactosamine reduced the fibrinogen (fg) surface concentration from a mixture of proteins. These antiplatelet agents also enhanced the albumin surface concentration. This itself may be one of the parameters to reduce the platelet adhesion towards an artificial surface. A combination of Aspirin-Vitamin C-Vitamin B6-Vitamin E inhibited the fibrinogen surface binding, which might be beneficial to improve the blood compatibility of an artificial surface.

Structural relevance of adsorbed aminosugars on polycarbonate - antithrombogenicity.

The effects of glucosamine, galactosamine, and mannosamine on platelet adhesion and coagulation induced by an artificial surface were investigated. Glucosamine and galactosamine showed both antiplatelet and anticoagulant effects, but in case of mannosamine none of the above effects was found significant. The three analogs studied were having the same molecular weight and common molecular structure, however from our preliminary observations it could be suggested, that the spatial arrangement of functional groups in the glucopyranose ring at C2, C4 positions were important for it's activity.

Effect of household exposure to environmental tobacco smoke on airflow mechanics in asymptomatic healthy women.

BACKGROUND & OBJECTIVES: Exposure to environmental tobacco smoke (ETS) can lead to airflow limitation, similar to that seen in smokers. However, the effects have not been conclusively proven. In the present study an attempt was made to characterize the effect of ETS exposure at home on airflow mechanics in asymptomatic healthy women. METHODS: Fifty women volunteers with no apparent health related problem, exposed to household ETS (group I), and 50 age-matched women not exposed (group II) were studied. Vital capacity (VC), forced expiratory flow in first second (FEV1), FEV1/VC ratio, peak expiratory flow (PEF), maximal midexpiratory flow (FEF(25-75%)), airway resistance (R(aw)) and specific airway conductance (sG(aw)) were measured, and compared between the two groups. Conditional logistic and linear regression analysis were done to assess contribution of household ETS exposure to decreased lung function. RESULTS: FEV1 and PEF values were marginally lower among women in group I (mean difference 0.13 l and 0.20 l/sec respectively). FEF(25-75%), R(aw) and sG(aw) were significantly impaired in this group. Ten (20.0%) women in group I and five (10.0%) in group II had abnormal R(aw) (adjusted odds ratio 6.72, 95% confidence limits 1.15-39.42), while eight (16.0%) women in group I and one (2.0%) in group II had abnormal sG(aw) (adjusted odds ratio 21.08, 95% confidence limits 1.30-341.05). Cumulative life time ETS exposure was, not significantly related to a reduction in FEV1, VC, PEF, FEF(25-75%), R(aw) or sG(aw) after adjustments for potential confounders. INTERPRETATION & CONCLUSION: Exposure to household ETS resulted in subtle impairment of airflow mechanics in asymptomatic women, possibly attributed to small airway narrowing. Further investigations are required to study the progression of this impairment with time.

On the regulation of beta 2 integrins.

The complex functions played by beta 2 integrins in mediating a large variety of adhesive interactions of leukocytes are highly regulated. This regulation results in transient adaptations/associations, permitting physical and functional recycling of these receptors during chemotaxis, phagocytosis and target-cell killing. The structural definition of these adaptations will lead not only to a better understanding of how these receptors are regulated in leukocytes but also shed valuable light on how these integrins integrate diverse extracellular signals into spatially and temporaly coordinated cellular responses.

Phytotoxic lesions of chromium in maize.

Chromium (Cr) is fairly abundant in the earth's crust and ranks fourth among the 29 elements of biological importance. Besides natural sources, Cr enters biotic components of the ecosystem in various ways. Of other major industrial sources, tanning and chrome-plating industries are prominent sources. Cr(VI) form of chromium is highly reactive and influences both plants and animals. Due to Mn present in soil, Cr(III) is oxidized to Cr(VI) which remains in soil for a long time and can affect plant growth and development. Since maize is an important food and fodder plant for human beings and cattle, a study was conducted to investigate the effects of Cr on some metabolic activities of maize (Zea mays L. cv. Ganga 5). Chromium caused visible lesions of interveinal chlorosis. Young leaves showed vein clearing. Also, a papery appearance was observed in leaves. Margins of leaves were curled and the leaves appeared pale at greater Cr exposure. Concentrations of both chlorophyll a and b were reduced by exposure to Cr, the activities of ribonuclease and phenyl phosphatase were greater while the activity of iron-porphyrin enzyme catalase was less and the activity of amylase was also much less in plants exposed to Cr. Chromium also caused retardation of soluble protein. Accumulation of Cr in roots was much at all the levels of chromium supply. Exposure to Cr resulted in reduction in grain production and quality.

Blood-compatible materials: a perspective.

The interrelation of physicochemical properties with biological interactions at the interface to understand blood compatibility of materials in contact with blood has been discussed. The bioethics for human experimentation and future research outlook has been projected.

Adhesion and stability of blood cells onto polymer substrates: effect of glow discharge.

The adhesion of platelets, red blood cells, and lymphocytes onto various polymer substrates, hydrophobic to hydrophilic in nature, has been studied. Cell adhesion is found to be higher on hydrophilic substrates. The stability of these adhered cells has also been studied under a flow rate of 20 ml/min. Further the effect of glow discharge treatment onto various substrates is investigated. It seems the stability is more on glow discharge treated substrates due to increased surface free energy.