Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.     

Calcitriol–copper interaction leads to non enzymatic,reactive oxygen species mediated DNA breakage and modulation of cellular redox scavengers in hepatocellular carcinoma

Calcitriol is the metabolically active form of Vitamin D and is known to kill cancer cells. Using the rat model of DEN induced hepatocellular carcinoma we show that there is a marked increase in cellular levels of copper in hepatocellular carcinoma and that calcitriol–copper interaction leads to reactive oxygen species mediated DNA breakage selectively in hepatocellular carcinoma cells. In vivo studies show that calcitriol selectively induces severe fluctuations in cellular enzymatic and non enzymatic scavengers of reactive oxygen species in the malignant tissue. Lipid peroxidation, a well established marker of oxidative stress, was found to increase, and substantial cellular DNA breakage was observed. We propose that calcitriol is a proxidant in the cellular milieu of hepatocellular carcinoma cells, and this copper mediated prooxidant action of calcitriol causes selective DNA breakage in malignant cells, while sparing normal (non malignant) cells.     

Assessment of Heavy Metals in Pharmacological Important Medicinal Plants Consumed in the Bannu District,Pakistan

This study assesses the contents of heavy metals in the four most commonly used indigenous medicinal plants in the Bannu District, Pakistan. The rapid appraisal approach was used along with semi-structured interviews with elderly people and herbalists for selection of indigenous plants. Heavy metals were determined via flame atomic absorption spectrometer by acid digestion of samples. Four medicinal plants were selected after interviews of 53 local residents and herbalists. Plant and soil samples were obtained for evaluations from a wastewater zone (WWZ) and a clean water zone (CWZ). Indigenous plants from the WWZ showed considerably higher metal contents compared to the CWZ. The trend of metals for indigenous medicinal plants grown in clean water was Mn > K > Na > Zn > Co > Fe > Cu while for wastewater the trend appeared as Co > K > Na > Zn > Fe > Mn > Cu. An alternative significant extent of research is needed to qualify the assessment of the human health insinuations of consumption of indigenous remedies. The heavy metal content of administering herbal medicine should be screened, as formulation and processing of medicine may affect heavy metal contents of the remedies.     

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.     

The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes

Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD₅₉₀ value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes.     

The Pathway of Leukemic Cell Death Caused by Glucocorticoid Receptor Fragment 465*

The truncated glucocorticoid receptor mutant gene 465* codes for a protein that is interrupted by a frame-shift mutation in the second zinc finger of the natural DNA binding domain. Thus, 465* represents the natural amino acid sequence 1-465 followed by 21 novel amino acids starting at position 466. The entire ligand binding domain is missing. Prior studies have shown that transient transfection of the glucocorticoid-resistant leukemic T-cell clone ICR-27 with a plasmid expressing 465* rapidly reduces the number of viable cells. This response does not require activation by a steroid, and a hybrid protein consisting of green fluorescent protein fused to 465* is found primarily in the cytoplasm. In the present study, we present evidence that the decrease in cell number is due to a form of cell death that bears many of the classic characteristics of apoptosis. Expression of the 465* protein can be detected a few hours after electroporation and is followed by activation of caspase-3 as well as reduction of the mitochondrial inner transmembrane potential. The caspase-3 inhibitor ZVAD-fmk blocks 465*-dependent cell death when added acutely after electroporation, but fails to do so later. We conclude that the novel 465* gene causes cell death by apoptosis.     

Zoonotic and non-zoonotic helminths in black rats of rain-fed and irrigated areas of Swat,Khyber Pakhtunkhwa,Pakistan

Present study was conducted to get information on helminth parasites of zoonotic importance among the black rats of district Swat, Pakistan. Two hundred and sixty nine rats were captured from agricultural ecosystem of the district using live captured traps from 2011 to 2013. Captured rats were anesthetized and surveyed for the presence of ectoparasites, then were carefully dissected for investigation of endoparsites. Helminth parasites of 8 species were identified. Presence of parasite was noticed in 23.7% of sampled rats. The infection rates of sampled rats was given in order of their infectivity as Syphacia obvelata 13(4.83%), Aspiculuris tetraptera 13(4.83%), Heterakis spumosa 12 (4.46%), Hymenolepis spp. 9(3.34%), H.diminuta 8(2.97%), Hymenolepis fusa 4(1.48%), Lutziella microacetabularae 4(1.48%) and Lutziella spp. 1 (0.37%). No significant difference (P < 0.4289) was found in prevalence of parasites among areas, crops, crop stages and sex of the host while adult rats were found more infected than sub-adults. S. obvelata and A. tetraptera were the most common species of helminths while Lutziella sp., 1 (0.37%) was found only in one host. Rattus rattus (the black rat) was regarded as the host of helminth parasites of zoonotic importance, therefore the hidden health hazards of this rodent species needed to be considered to prevent infectivity of humans. Current study was concluded that Rattus rattus harbored a wide variety of helminth parasites which shows a hidden risk to inhabitants of the region. Monitoring rats’ population in settle areas and educating the local community about the risk of rat borne parasitic diseases transmission through rats appears to be absolutely essential.     

The role of calcium ions for the expression of ricin toxicity in cultured macrophages

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited ³H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca²⁺-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg²⁺ did not affect protein synthesis or binding of ¹²⁵I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.