Modulation of anxiety behavior by intranasally administered vaccinia virus complement control protein and curcumin in a mouse model of Alzheimer's disease.

Widespread neuroinflammation in the central nervous system (CNS) of Alzheimer's disease (AD) patients, involving pro-inflammatory mediators such as complement components, might be responsible for AD associated behavioral symptoms such as anxiety. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are the bioactive compounds of natural origin shown to inhibit the in-vitro complement activation. In order to develop complement regulatory compounds which could be delivered to the CNS by a non-invasive route, VCP, its truncated version (tVCP), and Cur were administered to Wistar rats intranasally. The distribution of these compounds in cerebrospinal fluid (CSF) was studied using an enzyme linked immunosorbent assay (ELISA), using VCP and tVCP as antigens and a modified fluorimetric method (Cur). VCP and tVCP were also detected in the olfactory lobes of the rat brain using immunohistochemical analysis. These compounds were then compared for their ability to attenuate the anxiety levels in APPswePS1δE9 mice using an elevated plus maze (EPM) apparatus. VCP treatment significantly improved the exploratory behavior and reduced the anxiety behavior in APPswePS1δE9 mice. tVCP however showed an opposite effect to VCP, whereas Cur showed no effect on the anxiety behavior of these mice. When these mice were subsequently tested for their cognitive performance in the Morris water maze (MWM), they showed tendencies to collide with the periphery of the walls of MWM. This unusual activity was termed "kissperi" behavior. This newly defined index of anxiety was comparable to the anxiety profile of the VCP and tVCP treated groups on EPM. VCP can thus be delivered to the CNS effectively via intranasal route of administration to attenuate anxiety associated with AD.

Hsp90 mediates activation of the heme regulated eIF-2 alpha kinase during oxidative stress.

The heme-regulated inhibitor (HRI), a member of the eIF-2 alpha kinase family is crucial for regulating protein synthesis during stress. In addition to heme, stress proteins Hsp90 and Hsp70 are known to regulate HRI. The present study aims to determine the physical association of these Hsps in the regulation of HRI activation during oxidative stress using human K562 cells as a model. Extracts from the stress-induced cells were used for determining HRI kinase activity by measuring eIF-2 alpha phosphorylation, and Hsp-HRI interaction by immunoprecipitation and immunoblot analyses. The results indicate a significant increase in both Hsp70 and Hsp90 expression during AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride)-induced oxidative stress. Further, their interaction with HRI, which correlates well with its increased HRI kinase activity leads to inhibition of protein synthesis. Thus, we demonstrate that Hsps play an important role in the regulation of initiation of protein synthesis during oxidative stress.

Birth weight and anthropometry of newborns.

OBJECTIVE: To determine the relationship between birth weight and the anthropometric measurements of newborn to screen for low birth weight. METHODS: 1,252 newborns have been enrolled in the study. The birth weight, mid-arm, head and chest circumferences were measured by standard techniques. RESULT: The highest correlation of birth weight was found with chest circumference (r = 0.70). The maximum sensitivity of detecting low birth weight was seen with chest circumference (94.26%). CONCLUSION: Measurement of chest circumference being simple, easy, cheap and reliable method for identification of low birth weight in the community.

Iodine induced thyrotoxicosis following povidine-iodine dressings: a case report.

A 40-year-old woman was admitted to the intensive care unit after a radical resection of a recurrent abdominal malignancy. Her postoperative course was complicated by sepsis requiring numerous abdominal operations with the abdomen being left open for drainage and dressings. Use of povidone-iodine soaked abdominal packs to reduce secondary infection led to development of thyrotoxicosis, which resolved following the cessation of the iodine dressings. A high index of suspicion is needed for the diagnosis of this condition in intensive care patients as the usual haemodynamic signs of thyrotoxicosis may be misinterpreted as being caused by sepsis.

Role of biotransformation in conceptal toxicity of drugs and other chemicals.

Many developmental toxicants and teratogens require prior metabolism to reactive species or free radicals to exert toxicity. Thus the knowledge of conceptal biotransformation is absolutely critical in understanding toxicity of these chemicals. Due to extremely low content of cytochrome P450 in the embryo and other conceptal tissues, the research focus in recent years has steadily shifted toward enzymes capable of peroxidative oxidation of xenobiotics. At least three enzymes viz. lipoxygenase, peroxidase and prostaglandin synthase, each capable of peroxidative xenobiotic metabolism, occur in conceptal tissues of man and laboratory animals in biologically significant amounts. This review mainly summarizes the available information on the enzymatic bioactivation of teratogens and developmental toxicants belonging to diverse classes such as drugs, pesticides, environmental contaminants, industrial and other chemicals. Additionally, some discussion is devoted toward issues such as drug-drug interactions. The emerging new information on the peroxidative glutathione conjugate formation from xenobiotics is also presented. A critical need for gathering more information on this subject using different enzyme preparations from human conceptal tissues is stressed to avoid tragedies in the future.

Lipoxygenase--a versatile biocatalyst for biotransformation of endobiotics and xenobiotics.

Lipoxygenase, a member of the arachidonate cascade enzymes, dioxygenates polyenoic fatty acids to finally yield products with profound and distinct biological activity. This review summarizes the available evidence for another role played by lipoxygenases in the metabolism of endobiotics and xenobiotics. Although other mechanisms exist, a direct hydrogen abstraction by the enzyme and the peroxyl radical-dependent chemical oxidation appear to be central to the co-oxidase activity of lipoxygenases. Besides polyunsaturated fatty acids, H2O2, fatty acid hydroperoxides, and synthetic organic hydroperoxides support the lipoxygenase-catalyzed xenobiotic oxidation. The major reactions documented thus far include oxidation, epoxidation, hydroxylation, sulfoxidation, desulfuration, dearylation, and N-dealkylation. It is noteworthy that lipoxygenases are also capable of glutathione conjugation of certain xenobiotics. The enzyme system appears to be inducible following exposure to chemicals. Lipoxygenases are inhibited by a large number of chemicals, some of which also serve as co-substrates. Available data suggest that lipoxygenases contribute to in vivo metabolism of xenobiotics in mammals.

Human term placental lipoxygenase-mediated N-demethylation of phenothiazines and insecticides in the presence of linoleic acid.

This study investigated the hypothesis that human term placental lipoxygenase (HTPLO) and soybean lipoxygenase (SLO) are capable of mediating N-demethylation of selected phenothiazines and insecticides in the presence of linoleic acid (LA). In addition to being LA dependent, the N-demethylation reaction mediated by HTPLO and SLO was limited by incubation time, pH of the medium, concentration of the enzyme and the substrate. Using Nash reagent to monitor formaldehyde production, the specific activity for LA-dependent N-demethylation of chlorpromazine, a model phenothiazine, was determined to be 1.7+/-0.3 nmoles/min/mg HTPLO. Besides chlorpromazine, N-demethylation of promazine, promethazine and trimeprazine was also observed. The insecticide, aminocarb, displayed a specific activity of 2.2+/-0.3 nmoles/min/mg HTPLO for N-demethylation. Other insecticides, namely chlordimeform, dicrotophos and zectran, were oxidized in a similar manner. As compared with HTPLO, the rates of N-demethylation of phenothiazines and insecticides mediated by SLO were higher. Classical inhibitors of lipoxygenase, as well as antioxidants and free radical scavengers, caused a dose-dependent reduction in the production of formaldehyde from chlorpromazine and aminocarb by HTPLO. These results clearly demonstrate the ability of polyunsaturated free fatty acids to support N-demethylation of xenobiotics via the lipoxygenase pathway.

Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines.

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117+/-12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9+/-0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106+/-11 nmol HCHO/min/mg SLO, and 3.2+/-0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.

Metabolic fate of chemical mixtures. I. "Shuttle Oxidant" effect of lipoxygenase-generated radical of chlorpromazine and related phenothiazines on the oxidation of benzidine and other xenobiotics.

Many carcinogens, mutagens, teratogens, and other toxicants are known to be oxidized by lipoxygenases to potentially deleterious free radical intermediates. In this study, we tested for the first time the possibility that certain efficient substrates for lipoxygenase produce shuttle oxidants that stimulate the generation of reactive species from other chemicals. To evaluate the hypothesis, we investigated the metabolic interaction of two well-known substrates, chlorpromazine and benzidine, which have been shown to be oxidized by soybean lipoxygenase in the presence of hydrogen peroxide. The evidence presented here clearly indicates that the chlorpromazine cation radical generated by the lipoxygenase triggers a rapid oxidation of benzidine to benzidine diimine. Under the experimental conditions employed, the metabolic interaction resulted in a 42-fold stimulation in the rate of benzidine oxidation. The magnitude of stimulation of benzidine oxidation exhibited a dependence on the pH of the reaction medium, amount of the enzyme, and concentration of chlorpromazine, benzidine, and hydrogen peroxide. A number of other phenothiazines were also found to stimulate benzidine oxidation, albeit to a lesser degree. The chlorpromazine cation radical stimulated the oxidation of all six other xenobiotics tested. The highest stimulation (94-fold) was noted with tetramethyl phenylenediamine oxidation to the Wursters blue radical, while the lowest stimulatory response (2-fold) was observed with guaiacol. Preliminary data suggest that purified human term placental lipoxygenase also displays a similar stimulatory response in the benzidine oxidation in the presence of chlorpromazine. Although the toxicological significance of these in vitro findings remains to be established, it is worth pondering whether such a synergistic interaction occurs in humans in vivo. Teratogenesis Carcinog. Mutagen. 20:195-208, 2000.

A simple and efficient method for hemoglobin removal from mammalian tissue cytosol by zinc sulfate and its application to the study of lipoxygenase.

A simple and efficient method is described to remove hemoglobin (Hb) from human term placental cytosol to study dioxygenase and co-oxidase activities of lipoxygenase. In the untreated samples, 70%-80% of the linoleic acid-dependent dioxygenase and co-oxidase activities were found to be associated with the pseudo-lipoxygenase activity of Hb. Zinc sulfate (0.5 mM) precipitated >97% of the Hb present in the cytosol. The dioxygenase activity of the ZnSO4 treated cytosol exhibited a Vmax value of 313 nmoles linoleic acid hydroperoxide formed/min/mg protein and a K(M) of 1.4 mM for linoleic acid. The ZnSO4 treated cytosol displayed co-oxidase activity toward benzidine, dimethoxybenzidine, guaiacol, pyrogallol, tetramethylbenzidine and tetramethyl-p-phenylenediamine. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, butylated hydroxyanisole, butylated hydroxytoluene and gossypol caused concentration dependent inhibition of dioxygenase and co-oxidase activities. These results suggest ZnSO4 precipitation of Hb from cytosol does not alter the functional characteristics of the human term placental lipoxygenase.

Oxidation of eugenol by purified human term placental peroxidase.

The oxidation of eugenol by purified human term placental peroxidase (HTPP) was examined. Spectral analyses indicated that, similar to horseradish peroxidase, HTPP is capable of catalyzing the oxidation of eugenol. The accumulated stable product in the reaction medium due to eugenol oxidation by HTPP was tentatively identified as quinone methide of eugenol (EQM). The EQM formation exhibited a pH optimum of 8.0 and was dependent on incubation time, amount of HTPP and the concentration of both eugenol and hydrogen peroxide. The specific activity of approx 2.8 micromoles of EQM/min/mg protein was observed with different preparations of HTPP. The EQM formation was significantly suppressed by glutathione and ascorbic acid. The classical peroxidase inhibitors viz. potassium cyanide and sodium azide blocked the reaction in a concentration manner. Collectively, the results suggest that eugenol may undergo peroxidative metabolism in human placenta.

Lipoxygenase-mediated hydrogen peroxide-dependent N-demethylation of N,N-dimethylaniline and related compounds.

To date, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases. In this study we investigated the ability of non-heme iron proteins, namely soybean lipoxygenase (SLO) and human term placental lipoxygenase (HTPLO) to mediate N-demethylation of N,N-dimethylaniline (DMA) and related compounds in the presence of hydrogen peroxide. In addition to being hydrogen peroxide dependent, the reaction was also dependent on incubation time, concentration of enzyme and DMA and the pH of the medium. Using Nash reagent to estimate formaldehyde production, we determined the specific activity for SLO mediated N-demethylation of DMA to be 200 + 18 nmol HCHO/min per mg protein or 23 +/- 2 nmol/min per nmol of enzyme, while that of HTPLO was 33 +/- 4 nmol HCHO/min per mg protein. Nordihydroguaiaretic acid (NDGA), a classical inhibitor of lipoxygenase (LO), as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of production of formaldehyde from DMA by SLO. Besides N,N-dimethylaniline, N-methylaniline, N,N,N',N'-tetramethylbenzidine, N,N-dimethyl-p-phenylenediamine, N,N-dimethyl-3-nitroaniline and N,N-dimethyl-p-toluidine were also demethylated by SLO. The formation of a DMA N-oxide was not detected. Preliminary experiments suggested SLO-mediated hydrogen peroxide-dependent S-dealkylation of methiocarb or O-dealkylation of 4-nitroanisole does not occur.

Lipoxygenase-mediated biotransformation of p-aminophenol in the presence of glutathione: possible conjugate formation.

This study tested a hypothesis that soybean lipoxygenase (SLO), a model enzyme, may be capable of generating a glutathione (GSH) conjugate(s) from p-aminophenol (PAP). Horseradish peroxidase was employed as a positive control. GSH depletion or an increase in the absorption at 327 nm with time due to GS-PAP formation was used to quantitate the reaction. The rate of GS-PAP formation was dependent on the incubation time and the amount of SLO and exhibited Km values of 0.44 and 0.71 mM for PAP and H2O2, respectively. Classical inhibitors of lipoxygenase and free radical scavengers markedly decreased the rate of GS-PAP formation in a concentration-dependent manner. PAP-dependent GSH depletion from the reaction medium occurred at a rate of 2.37 +/- 0.18 micromol/min/mg protein. Collectively, the results suggest that lipoxygenase pathway may be involved in the enzymatic formation of GSH conjugate(s) from PAP.

Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.

Failed intubation in the intensive care unit managed with laryngeal mask airway and percutaneous tracheostomy.

We report the management of failed intubation in a critically ill, hypoxic and catabolic patient with sepsis and acute lung injury. Insertion of a laryngeal mask airway restored ventilation and corrected hypoxia. As the laryngeal mask provides only a temporary airway, it was essential to secure the airway by percutaneous tracheostomy to initiate mechanical ventilation.

N-demethylation of phenothiazines by lipoxygenase from soybean and human term placenta in the presence of hydrogen peroxide.

Several phenothiazine derivatives have been shown to cause reproductive toxicity. The biochemical mechanisms responsible for these effects are not fully understood at present. In this study, we investigated hydrogen peroxide-dependent oxidation of six phenothiazines by purified lipoxygenase from soybean (SLO) and human term placenta (HTPLO). Chlorpromazine was employed as the prototype phenothiazine drug. Chlorpromazine was easily demethylated releasing formaldehyde when incubated at pH 7.0 and 6.5 with SLO or HTPLO, respectively, in the presence of hydrogen peroxide. The reaction was linear with respect to time, exhibited dependence on the amount of enzyme, and the concentration of chlorpromazine and hydrogen peroxide. Under the optimal assay conditions, the estimated Vmax values for chlorpromazine N-demethylation were 139 and 7.2 nmoles/min/mg of SLO and HTPLO, respectively. Collectively, the results suggest an enzymatic nature of the reaction. In the presence of gossypol and NDGA, the classical inhibitors of different lipoxygenases, the formaldehyde production was significantly decreased, as expected. Similar to SLO, the generation of chlorpromazine cation radical, an initial oxidation product with an absorption maximum at 525 nm, was also observed with HTPLO. The radical generation was detectable only under acidic conditions (pH 3.5-4.5). The formaldehyde production was also decreased by BHT and BHA, suggesting a radical nature of the SLO-mediated chlorpromazine N-demethylation. Reduced glutathione, ascorbate, and dithiothreitol suppressed the rate of SLO-dependent formaldehyde generation, presumably due to the reduction of the cation radical back to chlorpromazine in a concentration-dependent manner. Besides chlorpromazine, SLO also oxidized promazine, triflupromazine, trifluperazine, trimeprazine, and perphenazine, albeit at different rates, in the presence of hydrogen peroxide. The evidence gathered in this in vitro study suggests that phenothiazines can undergo peroxidative N-demethylation via lipoxygenase pathway. The role of this biochemical mechanism in the in vivo developmental toxicity of phenothiazines remains to be established.

Co-oxidation of acrylonitrile by soybean lipoxygenase and partially purified human lung lipoxygenase.

1. Human lung lipoxygenase (HLLO) was partially purified by concanavalin-A (Con-A) affinity chromatography that provided an easy and rapid one-step procedure for the removal (> or = 96%) of haemoglobin from cytosol. 2. HLLO exhibited dioxygenase activity towards arachidonic acid (AA) and linoleic acid (LA). The dioxygenase activity towards LA varied approximately 12-fold (48-591 nmol/min/mg protein) among different human lung samples examined. 3. Reverse-phase HPLC analysis of AA metabolites indicated the predominance of 15-lipoxygenase in human lung cytosol. 4. HLLO exhibited co-oxidase activity towards benzidine (BZD) and several other model compounds. The co-oxidase activity towards BZD was significantly inhibited by several lipoxygenase inhibitors. 5. HLLO and soybean lipoxygenase (SLO), used as a model enzyme, metabolized acrylonitrile (ACN) to 2-cyanoethylene oxide (CEO) and ultimately to cyanide. 6. HLLO was a approximately 6-fold better catalyst than SLO in converting ACN to cyanide. The generation of cyanide by HLLO was dependent on the concentration of enzyme and the reaction was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-oxidant butylated hydroxytoluene (BHT). 7. Under optimal assay conditions, the covalent binding of HLLO-generated reactive intermediate(s) from [14C]ACN to protein and DNA (nmol equivalent bound/15 min/mg HLLO/mg bovine serum albumin or calf thymus DNA) was observed at approximately 1.20+/-0.13 and 2.20+/-0.50 respectively. Both protein and DNA binding were inhibited by NDGA, butylated hydroxyanisole (BHA) and BHT.

Awareness of urban slum mothers regarding home management of diarrhoea and symptoms of pneumonia.

A total of 635 mothers of under five children from urban slum area of Nanded city were assessed to know their awareness about home management of diarrhoea and symptoms of pneumonia. 48.5% of the mothers were unaware of any method of rehydrating the child with diarrhoea at home level followed by 36.2% mothers who were knowing home available fluids for rehydrating the child. 50.4% of the mothers were not knowing a single symptom of pneumonia followed by 35.1% mothers who were aware rapid abdominal movements (Pet Udna) as a symptom of pneumonia.