TM Treatment Research Articles

Benefi cial role of spironolactone, telmisartan and their combination on isoproterenol-induced cardiac hypertrophy

Introduction Although spironolactone and telmisartan are reported to reduce the risk of morbidity and death, direct studies on their eff ects on isoproterenol-induced cardiac hypertrophy are scanty. Hence the present investigation was carried out to study the eff ect of spironolactone, telmisartan and their combination on isoproterenol-induced cardiac hypertrophy.Methods Isoproterenol was administered intra-peritoneally in a dose of 5 mg/kg once daily for 10 days to Wistar rats. Spironolactone (20 mg/kg/day) (SL), telmisartan (5 mg/kg/day) (TM) or their combination (SLTM) was administered for 10 days after which various biochemical and cardiac parameters were measured.Results Isoproterenol produced dyslipidaemia, hypertension, elevated cardiac enzyme and C-reactive protein levels (CRP), worsened haemodynamic parameters and produced cardiac hypertrophy, left ventricular (LV) hypertrophy and oxidative stress. Chronic treatment with SL, TM or SLTM signifi cantly controlled dyslipidaemia and produced a signifi cant reduction in the elevated creatine-kinase (CK) and CRP levels. TM or SLTM produced a decrease in elevated lactate de-hydrogenase levels; however, SL failed to produce this eff ect. Hypotension, tachycardia, and decreased rate of pressure development and decay were prevented by SL, TM and SLTM treatment. Chronic treatment with SL, TM or SLTM also produced signifi cant reduction in LV collagen levels, cardiac and LV hypertrophy index and prevented oxidative stress.Conclusions Our data suggests that SL, TM and SLTM produced a benefi cial eff ect on cardiac hypertrophy.

Endoplasmic reticulum stress induces sarco/endoplasmic reticulum calcium ATPase and alters calcium homeostasis in the vasculature

Calcium homeostasis is tightly regulated in the vasculature. The sarco/endoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) is responsible for maintaining a relatively low cytosolic Ca2+ concentration. The ER is a multifunctional organelle and is easily perturbed resulting in ER stress. When ER stress occurs, the ER initiates a cellular signaling network, the unfolded protein response (UPR) to alleviate this stress. We tested the hypothesis that during ER stress increased SERCA function would lead to increased Ca2+‐induced contraction and elevated ER Ca2+ stores in the aorta of male mice. Male CD‐1 mice were treated with tunicamycin (TM, 100mg/kg, 8hrs, i.p.), an inducer of ER stress or vehicle (saline, 125ul, 8hrs, i.p.). TM treatment significantly upregulated aortic SERCA2b expression. Using a wire myograph, aortic rings of TM treated mice had an increased Ca2+‐induced contraction when compared to control (99.7%±2.6% vs. 69.4%±2.7%) in the presence of thapsigargin (TG, 1μM), an inhibitor of SERCA. Caffeine (10mM) induced depletion of intracellular Ca2+ stores measured as a transient contraction, was greater in aortic rings from TM treated mice (62.1%±5.1% vs. 44.1%±3.9%) compared to controls which was completely abolished in the presence of TG (6.8%±1.7% vs 7.4%±1.1%). Therefore augmented SERCA activity following ER stress may be a mechanisms altering Ca2+ homeostasis in the vasculature.

Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreERTm and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1PB-CreERTm;R26RlacZ mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.

Cloning and characterization of the stress-induced bZIP gene ZmbZIP60 from maize

ZmbZIP60 is a member of the bZIP transcription factor family in maize. Expression of ZmbZIP60 is strongly induced by a wide spectrum of stresses, including dehydration, high salinity, abscisic acid and tunicamycin treatments. A truncated form of ZmbZIP60, without a transmembrane domain (ZmbZIP60ΔC) and fused with GFP, is localized in the nucleus, suggesting the translocation of the native protein to the nucleus by release from the membrane. Yeast one-hybrid analysis showed that both ZmbZIP60 and ZmbZIP60ΔC had transcriptional activity. The expression of ZmbZIP60 in Arabidopsis bzip60 mutant partially restored the induction of BiP3 transcription under TM treatment, which indicated that ZmbZIP60 may play a role in the signal transduction of endoplasmic reticulum stress. Overexpression of ZmbZIP60 in wild-type Arabidopsis displayed enhanced bolting trends when subjected to dithiothreitol stress. Real-time PCR analysis revealed that some key genes in floral transition, including CO, FT, and AP1, were up- or down-regulated in ZmbZIP60-overexpressing plants, which may reveal the functional difference of ZmbZIP60 from AtbZIP60.

Abstract C161: Tumor cells selected for resistance to an antibody-drug conjugate modulate protein trafficking and signaling pathways.

Abstract Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several new ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. An anti-Her2 trastuzumab - maytansinoid conjugate (TM) was used. Human breast cancer cell lines sensitive (BT474) or insensitive (JIMT1 & MDA-MB-361) to unconjugated trastuzumab were used as models; all cells were highly responsive to TM conjugate. Cells were exposed to multiple cycles of TM at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, minimal resistance was observed in BT474 cells, however significant resistance developed in JIMT and 361. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20× & >400× in JIMT & 361, respectively). In JIMT-TM cells, cross-resistance was observed to other trastuzumab-ADCs, including T-mcMMAD and T-vcMMAD (non-cleavable & cleavable linkers with dolastatin, respectively). In contrast, 361-TM cells retained partial sensitivity to ADCs containing vc-linked dolastatin and auristatins, including ADCs to other antigens. JIMT-TM & 361-TM cells showed low level resistance to free maytansine payload (∼ 4 − 6×). JIMT-TM was partially cross-resistant to free auristatins (∼4 − 5×), while 361-TM retained sensitivity to auristatins. Neither cell line showed marked resistance (1 − 3×) to other standard-of-care therapeutics such as paclitaxel, docetaxel, vinblastine, camptothecin, oxaliplatin, etoposide, doxorubicin, or 5FU. Interestingly, increased sensitivity was observed to Her2 kinase inhibitor neratinib in 361-TM cells. Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively, however partial resistance to free payloads suggests a complex resistance profile independent of Her2 levels. Proteomic profiling was conducted on surface proteins and organelle-enriched fractions. In JIMT-TM cells, significant increases were observed in proteins involved in post-translational modification, including ubiquitinating enzymes, kinases, and phosphatases. Interestingly, elevated levels of endosomal and vesicle proteins were noted, including RAB family members. Proteins mediating microtubule and actin dynamics were also increased in JIMT-TM resistant cells. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. Alterations in pathways involved in vesicle transport, cytoskeletal function, and phospho-signaling suggest unique ways by which cells may evade chronic ADC-based therapy. The observed sensitivity of 361-TM cells to other vc-linked-auristatin ADCs offers the potential for alternative conjugate-based therapy for cancers which fail TM treatment. Hence, these new ADC-refractory cell models will be valuable tools to interrogate molecular mechanisms underlying resistance to immunoconjugate therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C161.

Regulation of Cathelicidin Antimicrobial Peptide Expression by an Endoplasmic Reticulum (ER) Stress Signaling, Vitamin D Receptor-independent Pathway

Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.

SOIL AND PLANT NITROGEN DYNAMICS OF A TOMATO CROP UNDER DIFFERENT FERTILIZATION STRATEGIES

A field experiment was conducted in 2007 to investigate the effects of the N fertilizer source on the soil and plant N dynamics of a tomato crop grown in a sandy loam soil. The fertilization treatments were: mineral N-fertilization applied by fertigation (TM); organic N-fertilization (TO) with compost obtained from the organic fraction of urban waste (OFUW); and a combined treatment (TC) with half organic and half mineral N. Compost was incorporated in November 2006. Plants were drip irrigated with well water. The nitrate concentrations in the irrigation solutions were determined on a weekly basis. Soil samples were taken before planting and at harvest from depths of up to 90 cm in order to determine moisture and N-NO 3 levels. The estimated amounts of total N-NO 3 - available in the different treatments, including the initial content in the 0-90 cm soil layer, were 560 (TC), 570 (TO) and 610 (TM) kg N ha -1 . The N contents of plants sampled on three occasions during the growing period and those of marketable fruits were also analyzed. Total marketable yield was determined at the end of the harvest period. The EU-Rotate_N model was used to predict the effects of the applied treatments. The model was calibrated using data from a previous experiment. No differences between treatments were observed with respect to yield or N content in marketable fruits. The amount of N left in the field at the end of the cropping period was significantly lower in TO than in TC and TM. Simulated plant growth, marketable yield and N uptake were similar to observed data. N leaching predicted by the model for the TM treatment was 50% higher than in the TC and TO treatments. However, an underestimation of residual N contents was found in all treatments. It was concluded that tomato fertilization with compost obtained from the OFUW could offer a suitable strategy for reducing N leaching without affecting yield when there is sufficient mineral N available throughout the growing period. The EU-Rotate_N model may be a valuable tool for evaluating N fertilizer practices for tomato crops.

Tunicamycin-induced cell death in the trigeminal ganglion is suppressed by nerve growth factor in the mouse embryo.

The effect of nerve growth factor (NGF) on tunicamycin (Tm)-treated neurons in the trigeminal ganglion was investigated by use of caspase-3 immunohistochemistry. In intact embryos at embryonic day 16.5, only a few caspase-3-immunoreactivity were detected in the ganglion neurons. Mean +/- SE of the density of the immunoreactivity was 0.22 +/- 0.03%. In contrast, the number of the immunoreactive neurons was increased at 24 h after injection of 0.5 microg Tm in 1 microl of 0.05 N NaOH solution into mouse embryos at embryonic day 15.5. The density of immunoreactivity was also increased (mean +/- SE = 1.44 +/- 0.11%) compared to intact and 0.05 N NaOH-treated embryos (mean +/- SE = 0.35 +/- 0.03%). The Tm treatment caused increase of the number of trigeminal neurons representing apoptotic profiles (intact, mean +/- SE = 79.3 +/- 8.5; 0.05 N NaOH, mean +/- SE = 132 +/- 11.5; 0.5 microg Tm, mean +/- SE = 370.2 +/- 64.8). In addition, NGF significantly prevented the increase of density of the immunoreactivity (mean +/- SE = 0.54 +/- 0.16%) and the number of apoptotic cells (mean +/- SE = 146.2 +/- 11.3). Saline application (without NGF) had no effect on Tm-induced increase of the immunoreactivity (mean +/- SE = 1.78 +/- 0.23%) or the apoptotic profiles (mean +/- SE = 431.9 +/- 80.5). These results indicate that Tm-induced cell death in the trigeminal ganglion is suppressed by NGF in the mouse embryo.

A1 A NOVEL SMALL THIENOINDAZOLE-DERIVATIVE COMPOUND PROMOTES CHONDROGENIC DIFFERENTIATION WITHOUT INDUCING HYPERTROPHY THROUGH PRODUCTION OF RUNX1

Purpose: Aiming at regeneration of permanent cartilage like joint cartilage, this study screened natural and synthetic compound libraries to discover a novel compound inducing chondrogenic differentiation without hypertrophy. We further investigated the underlying molecular mechanism. Methods: As an efficient monitoring system for chondrogenic differentiation, we established stable lines of mouse chondrogenic ATDC5 cells expressing green fluorescent protein under the control of type II collagen promoter fused with four repeats of a Sox9 enhancer (COL2GFP). Chondrogenic differentiation was assessed by real-time RT-PCR for COL2, aggrecan, chondromodulin-1 and COL10, toluidine blue and Alcian blue stainings, and quantitative GAG assay in cultures of mouse embryonic stem cells or immature mesenchymal C3H10T1/2 cells. The downstream molecules were screened by a microarray analysis using C3H10T1/2 cells. The COL2 promoter activity was determined using HuH-7 cells transfected with a luciferase-reporter gene construct containing the COL2 promoter above, and the specific binding to the identified region was verified by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). For functional analyses, we performed adenoviral overexpression of the gene or the small interfering RNA in C3H10T1/2 cells, and compared the chondrogenic differentiation as described above with respective empty-vector controls. Molecular interactions were examined by immunoprecipitation, mammalian two-hybrid system, and immunohistochemistry in the mouse growth plate. Finally, cartilage formation in full-thickness defects of mouse knee cartilage was histologically evaluated after transplantation of cell-sheets of mouse rib chondrocytes with or without TM treatment. Results: The COL2-GFP system showed that a small thienoindazolederivative compound T-198946 (TM) most strongly induced the GFP fluorescence as early as after 48 h of treatment. TM was confirmed to enhance chondrogenic differentiation but inhibit the further hypertrophic differentiation in the cultures of precursor cells. The microarray screening revealed that Runx1 was most strongly induced by TM among 581 up-regulated genes including Sox5 and Sox6. Deletion, mutagenesis, and tandem-repeat analyses of the luciferase assay identified the core responsive element of Runx1 in the COL2 promoter to be between the −293 and −288 bp region containing a putative Runx-binding motif. EMSA and ChIP assays confirmed the specific binding of Runx1 to this region. Although chondrogenic differentiation of C3H10T1/2 cells was little enhanced by the Runx1 overexpression alone, it was much enhanced by co-transfection with Sox5, 6, and 9 (the Sox trio), without inducing the hypertrophy, as true of the effect of TM treatment. Gene-silencing of Runx1, Sox5/6, or Sox9 suppressed the TM effect on chondrogenic differentiation. In fact, Runx1 and the Sox trio were co-localized in the proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, and their physical interaction was confirmed by immunoprecipitation and two-hybrid analysis. Finally, cell-sheets of TM-treated chondrocytes filled the defects with cartilaginous tissue, while the control cell-sheets did not. Conclusions: A novel small compound TM promotes chondrogenic differentiation without inducing hypertrophy, through production of Runx1 that cooperatively functions with the Sox trio. TM will herald a new era of regenerative medicine of permanent cartilage, thus realizing an epochal treatment of osteoarthritis. A2 IMPACT OF GENDER ON QUALITY-ADJUSTED LIFE EXPECTANCY LOSSES DUE TO KNEE OSTEOARTHRITIS IN THE US ELDERLY POPULATION

AIP1 Is Critical in Transducing IRE1-mediated Endoplasmic Reticulum Stress Response

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

A Subgenomic Segment of Theiler's Murine Encephalomyelitis Virus RNA Causes Demyelination

The DA strain of Theiler's murine encephalomyelitis virus (TMEV) causes a persistent central nervous system (CNS) infection of mice with a restricted virus gene expression and induces an inflammatory demyelinating disease that is thought to be immune mediated and a model of multiple sclerosis (MS). The relative contribution of virus vis-à-vis the immune system in the pathogenesis of DA-induced white matter disease remains unclear, as is also true in MS. To clarify the pathogenesis of DA-induced demyelination, we used Cre/loxP technology to generate a transgenic mouse that has tamoxifen (Tm)-inducible expression of a subgenomic segment of DA RNA in oligodendrocytes and Schwann cells. Tm-treated young transgenic mice developed progressive weakness leading to death, with abnormalities of oligodendrocytes and Schwann cells and demyelination, but without inflammation, demonstrating that DA virus can play a direct pathogenic role in demyelination. Tm treatment of mice at a later age resulted in milder disease, with evidence of peripheral nerve remyelination and focal fur depigmentation; surviving weak mice had persistent expression of the recombined transgene in the CNS, suggesting that the DA subgenomic segment can cause cellular dysfunction but not death, possibly similar to the situation seen during DA virus persistence. These studies demonstrate that DA RNA or a DA protein(s) is toxic to myelin-synthesizing cells. This Cre/loxP transgenic system allows for spatially and temporally controlled expression of the viral transgene and is valuable for clarifying nonimmune (and immune) mechanisms of demyelination induced by TMEV as well as other viruses.

Comparison of a Targeted and General Massage Protocol on Strength, Function, and Symptoms Associated with Carpal Tunnel Syndrome: A Randomized Pilot Study

Carpal tunnel syndrome (CTS) is a major, costly public health issue that could be dramatically affected by the identification of additional conservative care treatment options. Our study aimed to evaluate the effectiveness of two distinct massage therapy protocols on strength, function, and symptoms associated with CTS. This was a randomized pilot study design with double pre-tests and subjects blinded to treatment group assignment. The setting for this study was a wellness clinic at a teaching institution in the United States. Twenty-seven (27) subjects with a clinical diagnosis of CTS were included in the study. Subjects were randomly assigned to receive 6 weeks of twice-weekly massage consisting of either a general (GM) or CTS-targeted (TM) massage treatment program. Dependent variables included hand grip and key pinch dynamometers, Levine Symptom and Function evaluations, and the Grooved Pegboard test. Evaluations were conducted twice during baseline, 2 days after the 7th and 11th massages, and at a follow-up visit 4 weeks after the 12th massage treatment. A main effect of time was noted on all outcome measures across the study time frame (p < 0.001); improvements persist at least 4 weeks post-treatment. Comparatively, TM resulted in greater gains in grip strength than GM (p = 0.04), with a 17.3% increase over baseline (p < 0.001), but only a 4.8% gain for the GM group (p = 0.21). Significant improvement in grip strength was observed following the 7th massage. No other comparisons between treatment groups attained statistical significance. Both GM and TM treatments resulted in an improvement of subjective measures associated with CTS, but improvement in grip strength was only detected with the TM protocol. Massage therapy may be a practical conservative intervention for compression neuropathies, such as CTS, although additional research is needed.

Decreased Expression of Phospholipase C-.BETA.1 Protein in Endoplasmic Reticulum Stress-Loaded Neurons

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

Molecule of the Month

Molecule of the Month

Role of p53 in neurotoxicity induced by the endoplasmic reticulum stress agent tunicamycin in organotypic slice cultures of rat spinal cord

The endoplasmic reticulum (ER) is important for maintaining the quality of cellular proteins. Various stimuli can disrupt ER homeostasis and cause the accumulation of unfolded or misfolded proteins, i.e., a state of ER stress. Recently, ER stress has been reported to play an important role in the pathogenesis of neurological disorders such as cerebral ischemia and neurodegenerative diseases, but its involvement in the spinal cord diseases has not been fully discussed. We conducted this study using tunicamycin (Tm) as an ER stress inducer for rat spinal cord in organotypic slice culture, a system that we have recently established. Tm was shown to induce ER stress by increased expression of GRP78. The viability rate of spinal cord neurons decreased in a dose-dependent manner with Tm treatment, and dorsal horn interneurons were more vulnerable to Tm-induced neurotoxicity. A p53 inhibitor significantly increased the viability of dorsal horn interneurons, and immunofluorescence studies showed nuclear accumulation of p53 in the dorsal horns of Tm-treated spinal cord slices. These findings suggest that p53 plays an important role in the killing of dorsal horn interneurons by Tm. In contrast, motor neurons were not protected by the p53 inhibitor, suggesting that the role of p53 may vary between different cell types. This difference might be a clue to the mechanism of the stress-response pathway and might also contribute to the potential application of p53 inhibitors for the treatment of spinal cord diseases, including amyotrophic lateral sclerosis.

Evaluation of the effectiveness of abate™ used for the treatment of community drinking ponds in the Nigeria guineaworm eradication program

A study was carried out to ascertain the effectiveness of the Abate™ (0-0-0 1-0 1-tetramethyl –0-0 1-thiodi-P-phenylene phosphorothiote) an organophosphorous compound used for the treatment of community drinking ponds in the Nigeria Guinea Worm Eradication Programme in South-West Zone of Nigeria. The Abate TM was used in treating 3 large rectangular concrete tanks lined with pond mud, and filled with pond water harbouring cyclops, the crustacean vector of guinea worm disease. A fourth tank containing untreated water served as control. The initial and final cyclops populations of each tank was determined daily for seven days and then weekly up to the 28 th day after Abate TM treatment. The standard concentration used in guinea worm eradication program (0.1ml of Abate TM to 50 litres of water) was added. At this concentration, cyclops is known to become paralysed within 72 hours. It was deduced that the population of cyclops, Thermocyclops decipiens, was not effectively controlled since it took more than 5, 6 and 14 days to eliminate them from tanks A, B and C respectively. The population of cyclops however, increased in the control tank over the study period. In the control of guinea worm disease vectors, it is necessary to take into cognisance the potency of the chemical used in order to achieve the desired result.Keywords: Abate™, community drinking ponds, cyclops, Thermocyclops decipiens, guinea worm disease International Journal of Natural and Applied Sciences Vol. 1(1) 2005: 53-56

English

The Nevada Stewart Mine is an abandoned lead-zinc mine located within the Coeur d'Alene Mining District, Idaho. In 2002, a passive Apatite II TM Treatment System (ATS) was installed by MSE Technology Applications, Inc. (MSE) to treat metal-laden adit discharge (pH values are circum-neutral). The treatment medium consisted of a mixture of fish bone (Apatite II TM ) and gravel. Monthly performance monitoring of the ATS over a 21-month period showed attenuation of cadmium, lead, zinc, iron and manganese. In comparison to the ATS inflow, redox conditions in the outflow were more reduced, with higher calcium, sulfide, nitrogen and phosphorus concentrations. Geochemical modeling using the United States Geological Survey code PHREEQC was conducted to identify the reaction mechanisms responsible for the observed changes in water quality. Modeling results were reviewed in the context of available solid-phase data (i.e., chemical composition and detailed mineralogy) and experience at other sites to identify the most likely attenuation mechanisms. Zinc attenuation was attributed to precipitation as zinc sulfide, subsequently confirmed by mineralogical analysis. Collagen was identified as the most likely source of nitrogen release from the ATS, and was likely responsible for reducing conditions in the effluent. The increase in phosphorus concentrations was attributed to dissolution of the Apatite II TM and subsequent formation of insoluble metal phosphates. Manganese phosphate (MnHPO4) and chloropyromorphite (Pb10(PO4)6Cl2) were identified as possible controls on manganese and lead concentrations, respectively.

Thrombomodulin plays an important role in arterial remodeling and neointima formation in mouse carotid ligation model

Thrombomodulin (TM) is a cell membrane-bound glycoprotein that functions as a thrombin cofactor in the activation of protein C. Its protein structure includes a N-terminal lectin-like domain (D1), 6 epidermal growth factor repeats (D2), a serine-threonine-rich region (D3), a transmembrane domain (D4) and a short cytoplasmic tail (D5). Recent studies have demonstrated the direct effect of TM on cellular proliferation, adhesion and inflammation. In the study, we investigated the role of TM in vascular remodeling and neointima formation in a mouse carotid ligation model. TM expressions on the endothelium, neointima and media were examined in the ligated carotid artery by immunohistochemistry and quantitative real-time reverse transcription PCR. Endothelial TM expression decreased after ligation and appeared later in the media and neointima, which is quite similar to the appearance of TM in the human atherosclerotic process. Recombinant TMD123 was prepared. It was effective for thrombin-dependent protein C activation and the inhibition of leukocyte adhesion to the vessel wall after carotid ligation. Recombinant TMD123 and saline was administered immediately before and after carotid ligation. The TM-treated arteries demonstrated significantly less arterial dilatation (30279 +/- 12605 vs 73789 +/- 15073 microm(2), p < 0.05) in response to less neointima formation (14179 +/- 6538 vs 42227 +/- 8754 microm(2), p < 0.05) at 4 weeks after ligation. Our data indicated that there was a compensatory increase in TM expression in the media and neointima in relation to the reduced endothelial TM after carotid ligation. Early recombinant TM treatment in mice undergoing carotid ligation altered vascular remodeling and decreased the severity of neointima formation.

REGULATION OF FRUIT FIRMNESS, MATURITY AND QUALITY OF LATE MATURING CULTIVARS OF PEACH WITH PREHARVEST APPLICATION OF RETAINTM

ISHS XXVI International Horticultural Congress: Issues and Advances in Postharvest Horticulture REGULATION OF FRUIT FIRMNESS, MATURITY AND QUALITY OF LATE MATURING CULTIVARS OF PEACH WITH PREHARVEST APPLICATION OF RETAINTM

Oxycom TM treatment suppresses Pseudomonas syringae infection and activates a mitogen-activated protein kinase pathway in tobacco

Commercial field use of Oxycom TM is correlated with improved plant performance in diverse crops. Oxycom TM conferred disease resistance to Pseudomonas syringae pv. tabaci ( Pst) in tobacco. Symptoms were reduced and bacterial multiplication was inhibited in leaves pretreated with Oxycom TM in comparison to the levels in leaves treated with water. Oxycom TM treatment enhanced the accumulation of PR-1 protein occurring after challenge by Pst. Oxycom TM-treated plants showed greater accumulations of transcripts from the defence genes PR-1a, PR-1g and PR-3a than plants treated with salicylic acid (SA). Oxycom TM treatment caused rapid phosphorylation of a 48 kDa protein with MAP kinase activity. Phosphorylation of the same protein was caused by SA treatment of leaves and suspension cultured tobacco cells. Thisprotein was recognized by a SIPK-specific antibody. These results suggest to us that Oxycom TM stimulates expression of a broad range of defence genes in plants, including those connected to the SA-activated MAPK pathway.