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Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (>10 microM) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK(75)) isolated for its approximately 20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1-3 microM) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2-4 Gy) of 60Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches.

We have examined the role of stress-activated p38 MAP kinase in regulating human immunodeficiency virus (HIV) gene expression in response to ultraviolet light (UV). We found that UV activated p38 in HeLa cells harboring stably integrated copies of an HIVcat plasmid to levels similar to those obtained by hyperosmotic shock. However, hyperosmotic shock resulted in one order of magnitude smaller increase in CAT activity than treatment with UV. The specific p38 inhibitor SB203580 significantly decreased (>80%) UV activation of HIV gene expression whereas PD98059, a specific MEK-1 inhibitor did not, suggesting that p38 is specifically involved in the HIV UV response and little to no contribution is provided by MEK-1 and the p42/p44 MAP kinase pathway. Whereas increased binding of NF-kappaB to an oligonucleotide spanning the HIV enhancer was observed after UV, as expected, this binding was not affected by SB203580. Furthermore, UV activation of HIV gene expression in cells having the cat reporter gene under control of an HIV promoter deleted of the enhancer (-69/+80) produced results indistinguishable from those using HIVcat/HeLa cells with an intact HIV promoter (-485/+80), suggesting that SB203580 acts through the basal transcription machinery. Northern blot analysis of steady-state RNA from HIVcat/HeLa cells revealed an almost complete inhibition of UV activation with SB203580 at the RNA level. Similarly, the UV response was almost completely obliterated at the CAT and RNA levels in HIVcat/HeLa cells stably transfected with a plasmid expressing a kinase-inactive mutant of p38 (isoform alpha), without affecting NF-kappaB activation, providing strong genetic evidence that p38, at least the alpha isoform, is necessary for UV activation of HIV gene expression and that NF-kappaB activation alone is insufficient. These results firmly establish p38 MAP kinase as a key modulator of HIV gene expression in response to UV that acts independently of NF-kappaB.

The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

Thiol depleting agents phenylarsine oxide and N-ethylmaleimide significantly inhibited the phorbol ester induced protein kinase C activation in vascular smooth muscle cells. Phenylarsine oxide is a good protein tyrosine phosphatase inhibitor. Sodium orthovanadate, also a protein tyrosine phosphatase inhibitor, neither activated nor inhibited protein kinase C in vascular smooth muscle cells. Phenylarsine oxide, N-ethylmaleimide, orthovanadate, hydrogen peroxide and pervanadate [sodium orthovanadate + hydrogen peroxide] all significantly induced mitogen activated protein kinase in vascular smooth muscle cells. Phorbol ester and platelet derived growth factor induced mitogen activated protein kinase was inhibited by phenylarsine oxide pretreatment to vascular smooth muscle cells. However, hydrogen peroxide/pervanadate induced mitogen activated protein kinase was not prevented by phenylarsine oxide. These results suggest that oxidation of the cellular thiols inhibits the protein kinase C and activates mitogen activated protein kinase in vascular smooth muscle cells. In addition, peroxides induced, the activation of mitogen activated protein kinase in vascular smooth muscle cells which is independent of protein kinase C.

The study was conducted in three mungbean growing districts namely Barisal, Patuakhali and Noakhali of Bangladesh during the period of 2010-11 to assess the extent of technology adoption, profitability, farmers’ attitude, and constraints to mungbean. The study focuses the level of technology adoption for both input use and agronomic practices follow by most of the farmers were close to the recommendation which was very encouraging. All the farmers adopted improved mungben varieties and they were mostly influenced by DAE personnel and neighboring farmers. The yield of improved mungbean was found 824 kg/ha, which was more or less same with the national average of 820 kg/ha. The cultivation of improved mungbean was profitable since the net profit and BCR were Tk.38850 and 1.62 respectively. The variables such as experience, training, organizational membership, relation with different media, and mungbean suitable area had positive and significant influence in increasing the area under mungbean cultivation. Maximum number of farmers showed positive attitude towards improved mungbean cultivation of which 67% farmers wanted to increase its cultivation in the next year. The major constraints to improved mungbean production were; high price of insecticides, lack of labour and disease and insect infestation.

We have investigated oxidant-mediated stimulation of phospholipase D (PLD) activity in bovine pulmonary artery endothelial cells (BPAEC), prelabeled with [32P]orthophosphate or [32P]lysophospholipids. Treatment of cells incubated in Hanks' balanced salt solution (HBSS) containing 0.5% ethanol with hydrogen peroxide (H2O2) or linoleic acid hydroperoxide (18:2-OOH) enhanced the formation of 32P-labeled phosphatidylethanol (PEt) and phosphatidic acid (PA) in a dose- and time-dependent manner, indicating the activation of PLD. The H2O2- and 18:2-OOH-mediated PLD activation was not associated with cytotoxicity as determined by [3H]deoxyglucose release. The addition of ferrous chloride (50 microM) augmented H2O2-induced formation of [32P]PEt and [32P]PA about 2-fold, whereas the addition of the iron chelator desferoxamine blocked the potentiating effect of ferrous chloride. Replacement of the HBSS medium with Medium 199 containing 20% calf serum also potentiated the effect of H2O2-induced PLD activation. In addition to phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were readily hydrolyzed by PLD in response to H2O2 and 18:2-OOH treatment. The substrate specificity for oxidant-stimulated PLD activity differed from that observed in the presence of bradykinin or exhibited by agonist stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) where PC was the major phospholipid hydrolyzed by PLD. The formation of PEt in the presence of H2O2 and 18:2-OOH was not abolished by chelation of either extracellular Ca2+ with EGTA (5 mM) or intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (25 microM, 30 min). Furthermore, pretreatment of BPAEC with the protein kinase C (PKC) inhibitor staurosporine and down-regulation of PKC by chronic TPA treatment (100 nM, 18 hr) had no effect on H2O2-induced PLD activation, suggesting that PLD activation by H2O2 is independent of PKC activity. It is possible that H2O2- and 18:2-OOH-induced activation of PLD represents an important mechanism to produce PA and diacylglycerol in endothelial cells.

We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin-streptavidin interaction.

Hydrogen peroxide, individually, or in combination with potassium thiocyanate, inactivated milk folic acid (free and total) significantly. Addition of hydrogen peroxide to milk caused inactivation of folic acid added to milk and this inactivation was prevented by the addition of potassium thiocyanate. Potassium thiocyanate per se did not inactivate folic acid in milk. Lactoperoxidase, in the presence of hydroperoxides or GSH-Mn(II), was also found to degrade folic acid in vitro. Potassium thiocyanate and high concentrations of hydroperoxides inhibited this degradation. The role of lactoperoxidase, hydroperoxides and potassium thiocyanate (antimicrobial system) on the folic acid stability in milk is discussed.

Objective. To determine the impact of prognostic variables on local control in patients with aggressive fibromatosis treated with or without radiation. Materials and methods. Forty-two patients presenting to the combined sarcoma clinic at Johannesburg Hospital with aggressive fibromatosis from 1990 to 2003 were analysed retrospectively. There were 14 males and 28 females. The lesions involved the head and neck in 6 cases(14%), the thorax in 6 (14%), the extremities in 19 (45%) and the abdomen in 11 (26%). Thirty-seven patients (88%) presented to the clinic for the first time, whereas 5 (12%) had recurrent disease at presentation. Fifteen patients (36%) underwent excision only, 15 (36%) had excision followed by postoperative radiation, 8 (19%) had biopsy only, and 4 (9%) had radiation only. The median dose of radiation was 60 Gy (range 9 - 70 Gy). Results. One patient had local failure following excision and postoperative radiation therapy. The local control was 100% for surgery alone and 86% for surgery followed by postoperative radiation at ≥ 24 months. On univariate analysis, age, sex, positive margins, primary or recurrent presentation, site of involvement and initial treatment did not affect local control significantly. Eight of 19 patients (42%) receiving radiation developed severe moist desquamation following treatment, and all these patients had doses of 60 Gy or more. Conclusion. Surgery with or without radiation therapy gave excellent local control. The addition of radiation therapy to surgery as well as other known prognostic parameters did not impact on local control. The morbidity of radiation treatment is considerable, as noted in this series, and adjuvant radiation therapy should therefore be considered only in situations where the risk of recurrence and the morbidity of re-excision are high. South African Journal of Surgery Vol. 44 (1) 2006: pp. 6-11

The study was conducted in Satkania, Patiya and Hathazari upazilas of Chittagong district during 2008 to identify different pest problems and practices, input use and economic returns at farmers’ levels. About 95% of the farmers relied on the application of insecticides to control insect pests and they said that the insecticides use was profitable. Majority of the farmers of Patiya sprayed insecticides more than 40 times in brinjal cultivation. For other selected vegetables, farmer’s sprayed insecticide more than 15 times in a season. Especially for Satkania, majority of the farmers sprayed every alternative day while in the winter, the spraying frequency was reduced once a week. Pesticide dealers were the major source of information to farmers on the selection of chemicals and application methods. Very few farmers used protective measures or safety measures during pesticide application, only 39% of the respondents did not use any safety measures where 21% of the vegetable growers covered their body and faces. Eight percent covered their face and 32% covered their body at the time of spraying. On an average 61% believed that pesticide application are harmful to farm labour, 40% farmers expressed their views that pesticide application pollute water and air.

Subtraction hybridization applied to a 'differentiation therapy' model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10-family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/IL-24's unique properties, including cancer-specific apoptosis-induction, cell cycle regulation, an ability to inhibit angiogenesis, potent 'bystander antitumor activity' and a capacity to enhance the sensitivity of tumor cells to radiation, chemotherapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent 'bystander antitumor activity', ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers.

Familial hypercholesterolemia (FH) is an autosomal dominant disease predominantly caused by a mutation in the low-density lipoprotein receptor (LDLR) gene. Here, we describe two severely affected FH patients who were resistant to statin therapy and were managed on an apheresis program. We identified a novel duplication variant c.1332dup, p.(D445*) at exon 9 and a known silent variant c.1413A>G, p.(=), rs5930, NM_001195798.1 at exon 10 of the LDLR gene in both patients.

Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean ± SE, n = 6–8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 ± 3.1% vs. 27.6 ± 3.3% with saline control; P < 0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1α (HIF-1α) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1α and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-κB activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-κB-knockout (KO) mice (11.1 ± 3.0% vs. 25.1 ± 5.0% in saline-treated p50-KO mice; P < 0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 ± 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1α, AP-1, and iNOS but independent of NF-κB activation.

Recently, we reported that the herbal drug St. John's Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat/HeLa cells. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-kappaB are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism by which curcumin inhibits UV-activated HIV-LTR gene expression. We found that treatment of HIVcat/HeLa cells with micromolar concentrations of curcumin completely abolished UV activation of HIV gene expression. Curcumin treatment at similar doses as those used to inhibit HIV gene expression also effectively blocked UV activation of NF-kappaB, as demonstrated by electrophoretic mobility shift assay. In contrast, curcumin did not inhibit UV-induced phosphorylation of p38 MAP kinase. This observation was also supported by findings that curcumin did not inhibit UV-induced phosphorylation of CREB/ATF-1 and ATF-2. Although curcumin was ineffective in preventing UV-induced p44/42 MAP kinase phosphorylation, the JNK (1 and 2) and AP-1 activation were efficiently blocked by curcumin in HeLa cells. We conclude that the mechanism by which curcumin modulates UV activation of HIV-LTR gene expression mainly involves the inhibition of NF-kappaB activation.