Kinetics of metribuzin release from bentonite-polymer composites in water.

A series of bentonite polymer-composites (BPCs) loaded with metribuzin were studied for their controlled release in aqueous medium. The release of active ingredient from BPCs was significantly lower as compared to commercial metribuzin formulation. The results revealed that the cumulative metribuzin release was highest (81%) from the BPCs containing 8% clay (commercial bentonite) and 2% metribuzin which correspond to the lowest (14 days) half-life values i.e., time required for 50% release of active ingredient (t1/2). The metribuzin release from the BPCs decreased with increased concentration of clays in polymer matrix and the release was further decreased with BPCs prepared with pure nano-bentonite. BPCs containing 12% clay and 2% metribuzin showed maximum t1/2 values i.e., 25 and 51 days for commercial bentonite and pure nano-bentonite as clay sources, respectively. The differential behaviour in the metribuzin release rates from BPCs was ascribed due to variations in crosslinking of metribuzin in the composites. As metribuzin release was found to be slower in BPCs compared to commercial formulation, it could be used for control of weeds tailored to different crops.

Comparison of properties and aquatic arsenic removal potentials of organically modified smectite adsorbents.

Arsenic (As) poses a tremendous threat to human health due to exposure through arsenic-contaminated drinking water and/or food. We aimed to develop organically modified clay adsorbents for the removal of As from aqueous solution. We modified a smectite sample using three organic agents, namely hexadecyl trimethylammonium (HDTMA), chitosan and citric acid, and characterized the products using X-ray diffraction, infrared spectroscopy, and scanning electron microscopy techniques. The characterization techniques suggested successful organic modifications of the smectite sample. The surfactant-modified smectite was the most efficient (66.9%) As removing adsorbent with a maximum adsorption capacity of 473.2 µg g-1. Kinetic study showed that the adsorbents reached As adsorption equilibrium within 3 h, and the data fitted reasonably well to power function and simple Elovich equations (R2 > 0.89). The adsorption data were explained well by the Freundlich and Sips isothermal models. The surfactant-modified and chitosan-grafted organoclays adsorbed As by electrostatic attraction and anion exchange, whereas the citric acid activated smectite followed ligand exchange and simple anion exchange mechanisms. This study thus demonstrated the potential of surfactant-modified clays in removing As from contaminated waters.

Long-term fertilization and manuring with different organics alter stability of carbon in colloidal organo-mineral fraction in soils of varying clay mineralogy.

Majority of organic matter is bound to clay minerals to form stable colloidal organo-mineral fraction (COMF) in soil. Stability of carbon (C) in COMF is crucial for long-term C sequestration in soil. However, information on the effect of long-term fertilization and manuring with various organic sources on C stability in such fraction in soils with varying clay mineralogy is scarce. The present study was, therefore, carried out to assess the effect of thirty-one years of continuous fertilization and manuring with different organics on C-stability in COMF extracted from an Inceptisol, a Vertisol, a Mollisol, and an Alfisol. The treatments comprised of control (no fertilization), 100% NPK (100% of recommended N, P and K through fertilizer), 50% NPK+ 50% of recommended N supplied through either farm yard manure (FYM) or cereal residue (CR) or green manure (GM). The stability of C (1/k) in COMF was determined from desorption rate constant (k) of humus-C by sequential extraction and correlated with extractable amorphous Fe-Al-Si-oxides, and crystallite size of illite minerals. Long-term fertilization and manuring with the above sources of organic altered the contents of amorphous Fe-Al-Si-oxides, and decreased the crystallite size of illite in all the soil orders. Fifty percent substitution of fertilizer N by various organics significantly increased C-stability in COMF by 27-221% (mean 111%) over full dose of NPK (100% NPK). Smectite dominating Vertisol exhibited highest stability of C followed by the Mollisol, the Inceptisol and the Alfisol. Stability of such C in soil was correlated positively with the amount of amorphous Fe and Al oxides but negatively with crystallite size of illite (r = -0.46, P < 0.01). Application of NPK + GM or NPK + FYM in Inceptisol, Vertisol and Mollisol and NPK + GM or NPK + CR in Alfisol emerged as the best management practices for higher stabilization of C in COMF for long-term C sequestration.

Active carbon-pools in rhizosphere of wheat (Triticum aestivum L.) grown under elevated atmospheric carbon dioxide concentration in a Typic Haplustept in sub-tropical India.

Study on active and labile carbon-pools can serve as a clue for soil organic carbon dynamics on exposure to elevated level of CO2. Therefore, an experimental study was conducted in a Typic Haplustept in sub-tropical semi-arid India with wheat grown in open top chambers at ambient (370 micromol mol-1) and elevated (600 micromol mol-1) concentrations of atmospheric CO2. Elevated atmospheric CO2 caused increase in yield and carbon uptake by all plant parts, and their preferential partitioning to root. Increases in fresh root weight, volume and length have also been observed. Relative contribution of medium-sized root to total root length increased at the expense of very fine roots at elevated CO2 level. All active carbon-fractions gained due to elevated atmospheric CO2 concentration, and the order followed their relative labilities. All the C-pools have recorded a significant increase over initial status, and are expected to impart short-to-medium-term effect on soil carbon sequestration.

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine.

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.

Retinoic acid receptor-gamma in human epidermis preferentially traps all-trans retinoic acid as its ligand rather than 9-cis retinoic acid.

The biologic activity of retinoids is mediated through nuclear retinoic acid receptors (RAR), which are ligand-activated transcription factors. RAR directly bind and are activated by two naturally occurring isomers of retinoic acid (RA), all-trans retinoic acid (t-RA) and 9-cis retinoic acid (9c-RA). Human skin predominantly expresses RAR-gamma (approximately 87%) and RAR-alpha makes up the remainder. Recombinant RAR-gamma preferentially binds t-RA over 9c-RA in cell-free assays containing mixtures of the two retinoic acid isomers. We have investigated the ligand-binding properties of RAR in human epidermis. [3H]All-trans retinol (t-ROL) added to suspensions of intact epidermal cells was metabolically converted to [3H]t-RA, which bound to RAR. No binding of [3H]9c-RA to RAR was detected. Binding of [3H]t-RA, formed from [3H]t-ROL, was abolished by adding unlabeled t-RA, but was unaffected by adding unlabeled 9c-RA. Intact epidermal cells were incubated with mixtures of [3H]9c-RA and [3H]t-RA in varying ratios, and the amount of each labeled retinoid bound to RAR was measured. At ratios of 9c-RA to t-RA of 3:1 or lower, only [3H]t-RA was bound by RAR. Incubation of cells with [3H]9c-RA alone resulted in substantial (38%) binding of [3H]t-RA to RAR, in addition to binding of [3H]9c-RA, due to isomerization of [3H]9c-RA to [3H]t-RA. RAR in nuclear extracts from epidermal cells also displayed strong preferential binding of t-RA over 9c-RA. Competition studies revealed that 9c-RA was 6-fold less effective than t-RA at displacing [3H]t-RA bound to RAR in nuclear extracts. At ratios of 9c-RA to t-RA of 4:1 or lower, RAR in nuclear extracts bound t-RA exclusively. At higher ratios, [3H]9c-RA binding increased steeply. RAR-alpha in nuclear extracts bound both 9c-RA and t-RA without preference, whereas RAR-gamma displayed strong preferential binding of t-RA over 9c-RA. The level of endogenous t-RA exceeds that of 9c-RA in human skin in vivo, and significant isomerization of topically applied 9c-RA and 13c-RA to t-RA occurs. The relative abundance of t-RA in human skin, and preferential binding of t-RA by RAR-gamma, indicate that t-RA is the primary ligand mediating RAR-dependent responses in human skin under physiologic conditions, and under pharmacologic conditions when t-RA, 9c-RA, or 13c-RA are applied to skin.

All-trans retinoic acid induces cellular retinol-binding protein in human skin in vivo.

We examined the regulation of cellular retinol-binding protein (CRBP) mRNA and protein expression in human skin in vivo by all-trans retinoic acid and all-trans retinol. Treatment of human skin for 24 h with all-trans retinoic acid (0.1%) or all-trans retinol (1.6%) induced CRBP mRNA 5.5-fold (p < 0.01, n = 10) and 5.7-fold (p < 0.01, n = 5), respectively, compared with skin treated with vehicle or sodium lauryl sulfate (used as an irritant control). In vitro translation of poly A+ RNA from all-trans retinoic acid, all-trans retinol, sodium lauryl sulfate, and vehicle-treated human skin demonstrated that the observed increased CRBP mRNA in all-trans retinoic acid- and all-trans retinol-treated skin was able to direct increased (2.3-2.9-fold) CRBP protein synthesis. Riboprobe in situ hybridization revealed that CRBP mRNA was uniformly elevated throughout the epidermis and in dermal cells after all-trans retinoic acid treatment of human skin. Western analysis revealed that CRBP protein was elevated 3.2-fold (p < 0.01, n = 6) and 3.0-fold (p < 0.01, n = 6) after all-trans retinoic acid treatment of human skin in vivo for 24 and 96 h, respectively, compared with vehicle- and sodium lauryl sulfate-treated skin. In addition, functional CRBP levels measured by [3H]all-trans retinol binding were elevated 1.9-fold (p < 0.01, n = 6) and 3.5-fold (p < 0.01, n = 6) at 24 and 94 h, respectively, after all-trans retinoic acid treatment, compared with vehicle- or sodium lauryl sulfate-treated skin. Gel mobility shift analysis revealed that retinoid receptors in nuclear extracts from human skin formed a specific complex with a DNA probe containing the retinoic acid response element in the mouse CRBP gene. Monoclonal antibodies to nuclear retinoid receptors demonstrated that predominantly retinoic acid receptor-alpha/retinoid X receptor-alpha heterodimers bound to the CRBP retinoic acid response element. These data demonstrate that CRBP expression in human skin in vivo is regulated by exogenous all-trans retinoic acid and all-trans retinol.

Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation.

We investigated the clinical, histologic, and molecular responses of normal human skin to all-trans-retinol (ROL) application, compared to those induced by topical all-trans-retinoic acid (RA), and measured ROL-derived metabolites. Up to 1.6% ROL, 0.025% RA in vehicle (70% ethanol/30% propylene glycol), or vehicle alone were applied in a double-blind fashion to normal buttock skin and occluded for 4 d. ROL produced from none to only trace erythema, which was clinically and statistically insignificant, whereas RA induced a significant 3.7-fold increase in erythema score compared to vehicle (n = 10, p < 0.01). However, ROL induced significant epidermal thickening (1.5-fold at 1.6% ROL, p < 0.01), similar to RA (1.6-fold at 0.025% RA, p < 0.01), relative to the vehicle. ROL, compared with vehicle, also increased mRNA levels of cellular retinoic acid binding protein (CRABP-II) and cellular retinol binding protein (CRBP) genes as determined by Northern analysis (5-6-fold and 6-7-fold, respectively) and riboprobe in situ hybridization. CRABP-II and CRBP protein levels were also higher following ROL than vehicle treatment, as measured by ligand binding (3.2-fold, p < 0.001; n = 7) and Western analysis (3.6-fold, p < 0.003; n = 6), respectively. Epidermal retinyl ester (RE) content, measured after removal of stratum corneum, rose 240-fold (p < 0.005, n = 5) by 24 h of ROL occlusion. RA content, however, was undetectable or detectable only at trace amounts in all samples obtained at 0, 6, 24, and 96 h after ROL occlusion. Detectability of RA was not correlated with ROL treatment (compared to untreated normal skin, p = 0.86) or baseline skin ROL levels (average r = -0.1, p > 0.3). These data demonstrate that ROL application 1) produces trace erythema not significantly different from vehicle, whereas RA causes erythema; 2) induces epidermal thickening and enhances expression of CRABP-II and CRBP mRNAs and proteins as does RA; 3) causes marked accumulation of retinyl ester; and 4) does not significantly increase RA levels. Taken together, the data are compatible with the idea that ROL may be a prohormone of RA, because it produces changes in skin similar to those produced by RA but without measurable RA or irritation.

Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.

Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18-29 y, 30-59 y, 60-79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60-79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin.

Effects of emetine on the metabolism of ascorbic acid.

Treatment with emetine lowered the ascorbic acid concentrations of serum, liver and kidney, while the ascorbic acid concentration of adrenal tissue remained unaffected. The ability of the liver to synthesize (-)-ascorbic acid from (+)-glucuronolactone was also reduced after emetine treatment. The reduced concentration of ascorbic acid in liver and serum after emetine treatment may result from the diminished synthesis of ascorbic acid by the liver.

Human squamous carcinoma cell invasion in organ-cultured skin.

In a previous study we showed that normal human epidermal keratinocytes were capable of invading the dermis of organ cultured skin when the tissue was treated with an exogenous source of epithelial growth factors (Fligiel and Varani (1993) Invasion Metastasis, 13, 225-233). Here we examined human squamous carcinoma cells from three different tumors for ability to invade the dermis in the same model. Invasion occurred in 40-80% of the tissues, depending on the tumor line, and was observed with equal frequency under both growth factor-free conditions and in the presence of exogenous growth factors. These data demonstrate that malignant human epithelial cells have the capacity to invade the dermis of organ-cultured skin. Unlike normal keratinocytes, there appears to be no exogenous growth factor requirement for invasion by the malignant cells.

Transforming growth factor beta stimulates phosphoinositol metabolism and translocation of protein kinase C in cultured astrocytes.

Transforming growth factor beta (TGF-beta) is a regulatory peptide found in many normal and neoplastic tissues, including brain, with a diverse range of cellular effects. The transmembrane biochemical signals by which TGF-beta exerts these effects and the second messenger systems that may amplify them are unknown. We investigated the effects of TGF-beta upon membrane phosphoinositol metabolism and protein kinase C activity in cultured astrocytes. We found that exposure of astrocyte enriched cultures to TGF-beta resulted in the stimulation of phosphoinositol lipid turnover to inositol phosphates and in the apparent redistribution of protein kinase C from cytosol to membrane.

A convenient synthesis of dinorbile acids: oxidative hydrolysis of norbile acid nitriles.

We report a convenient method for the synthesis of dinorbile acids (23,24-dinor-5beta-cholan-22-oic acids, pregnane-20-carboxylic acids) in fair to good yields from norbile acid nitriles in one step by oxidative hydrolysis with oxygen in the presence of potassium-t-butoxide. The method results in stepwise overall removal of two carbon atoms in bile acid side chains in two steps. Dinorbile acids corresponding to several common bile acids have been prepared and their structures confirmed by spectroscopic methods. This simple method for synthesis of dinorbile acids may facilitate their study metabolically.

Platelet and erythrocyte-membrane adenosine triphosphatase activity in depressive and manic-depressive illness.

An investigation of Mg2+ and (Na+ + K+)-dependent adenosine triphosphatase (ATPase) activities in the platelet and erythrocyte membrane of 44 depressive and 44 manic-depressive patients was carried out before and during (for 3 weeks to less than 1 year) drug treatment. When compared with normal controls (n = 43), the patients showed significantly elevated enzyme activities during both the drug-free and drug treatment periods. No remarkable changes in enzyme activities were observed between the drug-free and drug treatment (even long-term drug treatment) periods. Results suggest an alteration of cell-membrane activity, which may reflect changes in (1) ATP level, (2) cationic balance, (3) membrane phospholipid, or some combination thereof.

Separation of creatine from arginine in biological fluids by thin-layer chromatography.

Creatine in biological fluids was estimated using a modified Sakaguchi procedure. Interference by arginine was eliminated by thin-layer chromatography.

Mechanism of inhibition of smooth muscle of guinea-pig taenia coli by chloramphenicol.

The effects of antibiotic chloramphenicol (CAP) on Ca(2+)-ATPase activity and muscle tension were examined in guinea-pig taenia coli. In general, when CAP was added to the resting tissue no inhibition was observed except when a tonus was present, caused by either ouabain, high K+ or acetylcholine. Ouabain and high K(+)-induced sustained contractions were concentration-dependently inhibited by CAP. The sustained contraction induced by high K+ was more strongly inhibited by CAP than ouabain (IC50 value: high K+ 0.29 mumol/ml; ouabain 0.34 mumol/ml). In Ca(2+)-free solution, inhibition of ouabain-induced sustained contracture by CAP was more pronounced. CAP increased the activity of Ca(2+)-ATPase in taenia coli in all experiments. In presence of cystine, CAP-induced inhibition and increase in Ca(2+)-ATPase activity could not be observed. CAP analogue thiamphenicol (TAP), devoid of p-NO2 group, showed insignificant response on smooth muscle inhibition and Ca(2+)-ATPase activity. These findings suggest that CAP inhibits smooth muscle contractility by decreasing cytosolic Ca2+ ([Ca2+]i) level through a cGMP mediated increase in Ca(2+)-ATPase activity and this action is possibly related with the p-NO2 group present in its molecule like other nitro-compounds.

The effect of chloramphenicol on mucosal transference of glucose in mice; the role of intestinal alkaline phosphatase in this process.

The effect of chloramphenicol (CAP) on the mucosal transference of glucose in mice and its relation with the activities of different small intestinal enzymes were studied. CAP produced an increase in the mucosal transference of glucose in jejunum and a decrease in ileum. However, CAP reduced the activity of Na(+)-K(+)-ATPase in both of these segments. Treatment with ouabain could not alter the effects of CAP. Under similar experimental conditions, the activity of alkaline phosphatase (AP) was reduced in jejunum but increased in ileum. In presence of theophylline, the CAP-induced increase in the transference in jejunum was further enhanced, whereas in acute experiments with ileum theophylline counteracted the reduced transference to the control level. In presence of Zn2+, CAP-induced changes in jejunum were reversed whereas in ileum the decrease was more pronounced. Like AP, CAP altered the activity of Ca(2+)-ATPase in both segments. It is proposed that in presence of CAP an inverse relationship exists between the activity of AP and the glucose transference in these segments. It is further revealed that such differential changes in the transference of glucose may be due to site specific alterations in the activity of AP.

Stimulation of liver growth and DNA synthesis by glucosylceramide.

The nature of the growth-stimulating effect of glucosylceramide was studied. Mice were injected intraperitoneally with emulsified glucosylceramide and conduritol B epoxide, an inhibitor of cerebroside glucosidase. Within one or two days, the liver grew 18-24%, as reported. Two enzymes involved in DNA synthesis also increased more than the weight. The total liver activity of thymidine kinase increased 46-73%, and the total activity of ornithine decarboxylase increased as much as 101%. It is suggested that elevated liver levels of glucocerebroside stimulate cell proliferation through a relatively direct mechanism.

Glucosylceramide and the level of the glucosidase-stimulating proteins.

The concentration of beta-glucosidase-stimulating proteins (called cohydrolase here) was measured in mouse liver and brain by immunoassay. Factors that might influence the levels of cohydrolase were examined. Injecting mice with an inactivator of glucosidase (conduritol B epoxide) rapidly produced elevations in liver glucosylceramide (the enzyme's substrate) and in liver and brain cohydrolase. Injection of glucosylceramide emulsified with Myrj 52 produced the same two effects in liver but not in brain. The increases in cohydrolase level induced by the enzyme inhibitor persisted in both organs for at least seven days, reaching 61-70% above the normal level. Injection of emulsified galactocerebroside, sphingomyelin and mixed glucosphingolipids but not of ceramide also produced rises in cohydrolase level. An increase in cohydrolase level resulted from injection of phenylhydrazine, which produces hemolysis and consequently an increased workload for the glucosidase of liver. When the enzyme inhibitor and/or larger amounts of glucosylceramide emulsion were injected (750 mg/kg body weight), increases in liver weight of 13 to 37% appeared within one day. The increased weight was characterized by increases in the weights of protein, total lipid and DNA and a very high increase in glucosylceramide level. These procedures have produced a rapidly developing model version of Gaucher disease in mice. Injected glucocerebroside also induced an elevated level of glucosidase activity.

Cyclic AMP mediates change in superoxide dismutase activity to monitor host-parasite interaction in Leishmania donovani.

The present study is designed to understand the role of cyclic AMP (cAMP) in host-parasite interaction involving Leishmania donovani, the causative agent for Kala-azar. When Leishmania promastigotes or macrophages were pretreated with dibutyryl cAMP or theophylline and epinephrine, which are well defined initiators for cAMP release, a key enzyme of the oxygen defense system, superoxide dismutase (SOD), was inhibited. At the same time, parasite interaction was considerably reduced to the level of 54.5% and 46.2%, respectively, for pretreated promastigotes. Internalization of the organisms in phagolysosomes was similarly affected. Dibutyryl cAMP-treated promastigotes in the presence of SOD, on the other hand, restored in vitro infection to the normal level. At least 50% less cAMP entered into Leishmania promastigotes when SOD was added to the incubation system containing dibutyryl cAMP. Data reveal that cAMP perturbs the Leishmania-macrophage interaction through inhibition of SOD, pointing to the importance of a promastigote enzyme for the survival of this pathogen within phagolysosomes.