Prolonged Pretreatment Research Articles

Prolonged insulin treatment inhibits GH signaling via STAT3 and STAT1

Growth hormone (GH) and insulin are important regulators of cellular and whole body metabolism as well as somatic growth and body composition. Studies have indicated complex feedback effects of GH on insulin action and of insulin on GH signaling pathways. Previous studies in our laboratory have shown that GH induction of signal transducers and activators of transcription (STAT)5B tyrosine phosphorylation is inhibited by prolonged insulin treatment, probably via downregulation of GHR. Here, we find that in rat H4IIE hepatoma cells GH-induced tyrosine phosphorylation of two other STATs (STAT3 and STAT1) was also greatly reduced following prolonged insulin pretreatment compared with that induced by GH alone. In the present work, total STAT5B and STAT1 protein levels were not altered by prolonged insulin treatment. However, prolonged insulin treatment (16 h; 10 or 100 nM) resulted in a 30-40% reduction of total STAT3 protein, with little change at 0.1 and 1.0 nM insulin. Thus, there is a selective reduction of total STAT3 protein levels by insulin, but only at high concentration of insulin. Basal tyrosine phosphorylated (PY)-STAT3 was also significantly reduced by prolonged insulin treatment, and to a greater extent than total STAT3 protein levels. The inhibitory effect of insulin on total STAT3 protein and basal PY-STAT3 levels was dependent on activation of the MEK-ERK pathway, rather than the PI3K pathway. In contrast, the MEK-ERK pathway did not play a major role in insulin's inhibition of GH-induced PY-STAT3 and PY-STAT1. The present studies indicate that prolonged hyperinsulinemia, such as that found in some obese patients or patients with Type 2 diabetes mellitus, may have profound effects on GH signaling via STAT3 and STAT1.

Protein Kinase C Modulates Agonist-sensitive Release of Ca2+ from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.

Plant defense activators potentiate the generation of elicitor-responsive photon emission in rice

Ultraweak photon emission from rice induced by an elicitor was measured to estimate the influence of pretreated plant defense activators on elicitor-responsive photon emission. Rice leaf segments treated with an elicitor transiently generated relatively high levels of elicitor-responsive photon emission. Pretreatment with plant defense activators increased elicitor-responsive photon emission. This increase was also observed in suspension-cultured rice cells. Prolonged pretreatment allowed the plant defense activators to accelerate photon generation and to act at lower doses. The activators themselves did not induce any marked photon emission in rice leaf segments or cells. The spectral compositions of the increased and nonincreased elicitor-responsive photon emissions from rice cells were almost the same. Therefore, plant defense activators probably potentiate the generation of elicitor-responsive photon emission itself. The elicitor-responsive expression of the PBZ1 gene was strongly enhanced by pretreatment of rice cells with plant defense activators, when elicitor-responsive photon emission was also enhanced. We could not identify a significant correlation between the degree of photon emission enhancement and the potentiation of PBZ1 expression with each treatment. However, the results indicate that the generation of elicitor-responsive photon emission is potentiated when rice cells are primed for disease resistance by plant defense activators.

Delivery of a Liposomal c-raf-1 Antisense Oligonucleotide by Weekly Bolus Dosing in Patients with Advanced Solid Tumors

Rapid cleavage in vivo and inefficient cellular uptake limit the clinical utility of antisense oligonucleotides (AON). Liposomal formulation may promote better intratumoral AON delivery and inhibit degradation in vivo. We conducted the first clinical evaluation of this concept using a liposomal AON complementary to the c-raf-1 proto-oncogene (LErafAON). A dose escalation study was done to determine the maximum tolerated dose and to characterize the toxicities of LErafAON given as weekly intravenous infusion for 8 weeks to adults with advanced solid tumors. Pharmacokinetic analysis and evaluation of c-raf-1 target suppression in peripheral blood mononuclear cells were included. Twenty-two patients received LErafAON (median 7 infusions; range 1-27) at doses of 1, 2, 4, and 6 mg/kg/week. Across all dose cohorts patients experienced infusion-related hypersensitivity reactions including flushing, dyspnea, hypoxia, rigors, back pain, and hypotension. Prolonged infusion duration and pretreatment with acetaminophen, H1- and H2-antagonists, and corticosteroids reduced the frequency and severity of these reactions. Progressive thrombocytopenia was dose-limiting at 6 mg/kg/week. No objective responses were observed. Two patients treated at the maximum tolerated dose of 4 mg/kg/week had evidence of stable disease, with dosing extended beyond 8 weeks. Pharmacokinetic analysis revealed persistence of detectable circulating rafAON at 24 hours in 7 of 10 patients in the highest 2 dose cohorts. Suppression of c-raf-1 mRNA was noted in two of five patients analyzed. Dose-independent hypersensitivity reactions and dose-dependent thrombocytopenia limited tolerance of LErafAON. Future clinical evaluation of this approach will depend on modification of the liposome composition.

Involvement of capsaicin-sensitive afferent nerves and cholecystokinin 2/gastrin receptors in gastroprotection and adaptation of gastric mucosa to Helicobacter pylori-lipopolysaccharide.

Lipopolysaccharide (LPS) is one of the virulence factors in the Helicobacter pylori (Hp)-infected stomach, but it remains unknown whether single and prolonged pretreatment with Hp-LPS can affect the course of gastric damage induced by aspirin (ASA). We compared the effects of Hp-LPS with those induced by LPSs isolated from intestinal Bacteroides fragilis, Yersinia enterocolitica, and Campylobacter jejuni applied for 4 days on acute ASA-induced gastric lesions in rats. The area of ASA-induced gastric lesions, gastric blood flow (GBF), expression of mRNA and protein of leptin and plasma leptin, gastrin, interleukin-1beta, and tumor necrosis factor-alpha levels were examined. Single (once) or repeated (five times) i.p. injections of Hp-LPS (1 mg/kg) or intestinal LPSs failed to produce macroscopic gastric damage and did not affect the GBF when compared with vehicle. Hp-LPS injected repeatedly suppressed the gastric acid secretion, up-regulated leptin mRNA and protein, and increased plasma leptin and gastrin levels. Hp-LPS significantly reduced the ASA-induced gastric damage and the accompanying decline in the GBF, and these effects were significantly attenuated by capsaicin denervation and selective antagonism of cholecystokinin-B (CCK2) receptors by RPR-102681 [N-(metoxy-3 phenyl) N-(N-methyl N-phenyl-carbamylmethyl) carbamoylmethyl]-3 ureido]-3 phenyl]-2 propronique] but not by loxiglumide, an antagonist of CCK1 receptors. We conclude that 1) daily application of Hp-LPS enhances gastric mucosal resistance against ASA damage due to the increase of GBF and the expression and release of leptin and gastrin exerting trophic and gastroprotective effects, and 2) this enhanced resistance to ASA damage in Hp-LPS-adapted stomach is mediated by the sensory afferents and specific CCK2/gastrin receptors.

Prolonged antithrombotic pretreatment increased risk for MI or death in unstable coronary syndromes

Source Citation Neumann FJ, Kastrati A, Pogatsa-Murray G, et al. Evaluation of prolonged antithrombotic pretreatment (“cooling-off” strategy) before intervention in patients with unstable coronary ...

Evaluation of prolonged antithrombotic pretreatment (“cooling-off” strategy) before intervention in patients with unstable coronary syndromes

Evaluation of prolonged antithrombotic pretreatment (“cooling-off” strategy) before intervention in patients with unstable coronary syndromes

Induction of cytokine tolerance requires internalization of Chylomicron-Bound LPS into hepatocytes

Background Chylomicron-bound LPS (CM-LPS) renders hepatocytes unresponsive to stimulation by proinflammatory cytokines, a process termed cytokine tolerance. We have shown that cytokine tolerance is a time- and dose-dependent process requiring functional low-density lipoprotein receptors (LDLR). Thus, we hypothesized that cytokine tolerance directly correlates with the internalization of CM-LPS complexes, and inhibition of lipoprotein binding and/or internalization inhibits the induction of cytokine tolerance in hepatocytes. Materials and methods We correlated the rate of internalization of radioiodinated CM-LPS complexes with hepatocellular NO production as a measure of cytokine responsiveness. In additional studies, we used four different strategies to inhibit binding/internalization of CM-LPS via LDLR and then determined the effect of each strategy on the induction of cytokine tolerance. Results There was a strong inverse correlation between the internalization of CM-LPS and the responsiveness of hepatocytes to proinflammatory cytokines (r 2 = −0.997). Furthermore, the greater the degree of LDLR inhibition, the less susceptible hepatocytes were to the induction of cytokine tolerance by CM-bound LPS. Accordingly, cytokine tolerance induction was inhibited in hepatocytes with decreased membrane expression of LDLR as compared to control cells (69 versus 12% control; P = 0.005). Competitive inhibition of CM-LPS binding prevented internalization of CM-LPS and resulted in loss of the cytokine-tolerant phenotype. Whereas CM-LPS successfully induced cytokine tolerance in ldlr −/− hepatocytes, it only occurred after a prolonged pretreatment period of 8 h. CM-LPS complexes containing apolipoprotein (apo) E 2 also required a prolonged pretreatment period to induce a level of cytokine tolerance comparable to that induced by CM-LPS complexes containing either apo E 3 or E 4. Conclusion Lipoprotein-bound LPS inhibits the responsiveness of hepatocytes to proinflammatory cytokines in a manner directly correlated with the internalization of LPS. Furthermore, inhibition of lipoprotein binding/internalization prevents this LPS-mediated induction of cytokine tolerance in rodent hepatocytes.

Cooperation between PKC-α and PKC-ε in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors Gö6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-α, -βII, and -ε isoforms, but not PKC-δ, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-ε was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-ε in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-α, suggesting that PKC-α and PKC-ε act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-α pathway is convergent with the PKC-ε/MEK1/2 pathway in terms of the activation of JNK by PMA.

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo.

RSR13, 2-[4-[2-[(3,5-dimethylphenyl)amino]-2-oxoethyl]phenoxy]-2-methylpropanoic acid monosodium salt, allosterically modifies hemoglobin to increase tumor pO(2), increases the effect of radiation in animal tumor models, and is in phase III clinical trials as an adjuvant to radiotherapy. Cisplatin and carboplatin, two commonly used anticancer drugs have been used in combination with radiotherapy. Some studies have suggested that the cytotoxic effects of these drugs are altered in hypoxia. This study tested whether RSR13 plus oxygen breathing increased the cytotoxicity of cisplatin and carboplatin in vivo. Solid EMT6 tumors in BALB/c Rw mice were treated with cisplatin (5-30 microg/g) or carboplatin (5-200 microg/g) along with 150 microg/g RSR13 in combination with oxygen breathing. Tumor cell survival was assayed using clonogenic assays. The effects of pre- and posttreatments with RSR13 and oxygen breathing on the cytotoxicity of cisplatin or carboplatin were also examined. To assess whether RSR13 had direct effects on the cytotoxicity of the drugs, exponentially growing monolayers of EMT6 mouse mammary carcinoma cells were treated with graded concentrations of cisplatin or carboplatin for 2 h along with simultaneous (2 h) RSR13 treatments or with prolonged (22 h) pre- or posttreatment incubations with 100 microM RSR13. Single or multiple treatments with 150 microg/g RSR13 plus oxygen breathing had no effect on the viability of cells in EMT6 tumors in mice. After treatment with cisplatin or carboplatin, the tumor cell survival tended to be lower in oxygen-breathing mice especially at higher doses of cisplatin. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents did not alter the cytotoxicity of cisplatin or carboplatin from that seen with oxygen breathing alone. Pretreatment with RSR13 plus oxygen at 22 and 14 h prior to administration of either cisplatin or carboplatin did not alter the effect of either alkylating agent. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents and lasting for 2 or 5 h did not alter the cytotoxicity of either drug from that seen with oxygen breathing alone. The cytotoxicity of cisplatin was not altered by treatment with oxygen alone or with RSR13 plus oxygen for 5 h after cisplatin injection. For carboplatin, treatment with oxygen alone and with RSR13 plus oxygen for 5 h after injection increased to similar extents the response of the tumor cells compared to that seen with assays at 2 h. Neither short simultaneous treatments, prolonged pretreatment incubations, nor prolonged posttreatment incubations with RSR13 altered the survival of EMT6 cells in cultures treated with cisplatin or carboplatin. These findings indicate that RSR13 in combination with oxygen breathing does not alter the cytotoxicity of cisplatin or carboplatin when used simultaneously, as a pretreatment or as a posttreatment in vitro or in vivo. Our in vivo findings indicate trends that support previous findings that cisplatin is more cytotoxic to well-oxygenated tumor cells than to hypoxic tumor cells, and that this effect can be improved by improving tumor oxygenation, but the differences seen in our studies did not achieve statistical significance.

Prolonged antithrombotic pretreatment not COOL in ACS

Prolonged antithrombotic pretreatment not COOL in ACS

Antibiotic treatment enhances C2 toxin production by Alexandrium tamarense in batch cultures

The effects of a mixture of penicillin G and streptomycin on the growth and C2 toxin production of a marine dinoflagellate, Alexandrium tamarense CI01, were investigated to determine if antibiotic treatment would increase the toxin yield of the cultured algae in batch cultures. Algal growth and toxin production were both enhanced markedly when the culture was supplemented with the antibiotics, each at an initial concentration of 100 unit ml −1 in medium, 2 Antibiotic activity: 1668unitmg −1 for penicillin G and 749unitmg −1 for streptomycin. 2 but were severely inhibited when the concentration was 500 unit ml −1 or higher. Short-term pretreatment of algal inocula with the antibiotics at 100, 500, and 1000 unit ml −1 all produced the enhancing effects on the algal cultures in an autoclaved medium. A prolonged antibiotic pretreatment of the algal culture followed by repeated sterile cultivation resulted in an algal culture free of cultivable bacteria. This “drug-treated” culture became more resistant to the toxicity and more responsive to the enhancing effects of the antibiotics. Our results indicated that the antibiotics can enhance growth and C2 toxin productivity not only through their inhibition of the growth of bacteria that compete for nutrients with the coexisting algae, but also through their direct effects on the physiology of the algae. Supplementation of the two antibiotics therefore is an efficient way to increase the yield of C2 toxin in the production cultures of A. tamarense CI01.

Ovarian suppression with oral contraceptive pills prior to ART: can you get too much of a “good” thing?

Objective: Women undergoing ART are frequently placed on oral contraceptive pills (OCP) for pre-cycle ovarian suppression and to assist in patient scheduling. In our military hospital-based ART program, patients are enrolled world-wide into quarterly cycles. This often necessitates a long duration of oral contraceptive ART pre-cycle suppression (> 42 days). The objective of this study is to compare the effect of prolonged oral contraceptive pre-treatment on ART cycle outcome. Design: Retrospective Cohort Analysis. Materials and Methods: Women undergoing their first ART cycle from January 2002 to September 2002 were included for analysis. All were pre-treated with 35 microgram monophasic oral contraceptive pills. Ovarian hyperstimulation was with 40 microgram GnRH-agonist flare gonadotropin protocols (Leondires et al., 1999). All infertility diagnoses, ART cycle types, and stimulation protocols were considered. Cycle cancellation and clinical pregnancy rates (CPR) per cycle start and embryo transfer were then compared between ART cycles in which OCP pre-ART cycle pretreatment was ≤ 42 and > 42 days. Statistical significance was determined by χ2, with a p < 0.05 considered significant. Results: 327 ART cycles met criteria for inclusion in the analysis. OCP pre-treatment of ≤ 42 days (mean 31.1) occurred in 166 cycles. OCP pre-treatment of > 42 days (mean 64.6) occurred in 161 cycles. Patient age and basal FSH were not significantly different in both groups. The cycle cancellation rate was 9.6% in women treated ≤ 42 days vs. 25.5% in women treated > 42 days (p < 0.05). The CPR per cycle start was 40.4% in women treated ≤ 42 days vs. 34.8% in women treated > 42 days. CPR per transfer was 44.7% in women treated ≤ 42 days vs. 46.7% in women treated > 42 days. Cancellations in both groups were restricted to poor ovarian response. The significant differences are noted in Table 1. Tabled 1 Conclusion: Prolonged oral contraceptive pill use for pre-cycle ovarian suppression increases the cancellation rate in ART, presumably by decreasing ovarian response. In patients who achieve embryo transfer, however, pregnancy rates are not affected.

A rapid protocol for the prevention of contrast-induced renal dysfunction: the RAPPID study

ObjectivesThis study was designed to test a rapid protocol of intravenous acetylcysteine for prevention of radiocontrast-induced nephropathy (RCIN). BackgroundOral acetylcysteine (NAC) may provide better prophylaxis against RCIN than intravenous (IV) hydration alone. Current protocols preclude prophylaxis of same-day or emergency patients owing to the need for prolonged pretreatment. MethodsWe prospectively randomized 80 patients with stable renal dysfunction undergoing cardiac catheterization/intervention to a rapid protocol of IV NAC (150 mg/kg in 500 ml N/saline over 30 min immediately before contrast followed by 50 mg/kg in 500 ml N/saline over 4 h, n = 41, 67 ± 10 years, 90% men) or IV hydration (1 ml/kg/h N/saline for 12 h pre- and post-contrast, n = 39, 71 ± 8.8 years, 85% men). ResultsRadiocontrast-induced nephropathy occurred in 2 of the 41 patients in the NAC group (5%) and in 8 of the 39 patients in the hydration group (21%; p = 0.045; relative risk: 0.28; 95% confidence interval 0.08 to 0.98). In the NAC group, mean serum creatinine fellfrom 1.85 ± 0.59 to 1.77 ± 0.73 and 1.79 ± 0.73 mg/dl 48 h and four days post-contrast (p = 0.02 and 0.023 vs. baseline, respectively). In the hydration group, serum creatinine increased from 1.75 ± 0.41 to 1.81 ± 0.6 48 h and 1.80 ± 0.50 mg/dl four days post-contrast (p = 0.99 and 0.23, respectively). NAC infusion was ceased after the bolus in three patients (7%) due to flushing, itching, or a transient rash. ConclusionsAdministration of IV NAC should be considered in all patients at risk of RCIN before contrast exposure when time constraints preclude adequate oral prophylaxis, provided the patient is able to tolerate this degree of volume loading.

New Aspects of Neurotransmitter Release and Exocytosis: Rho-Kinase-Dependent Myristoylated Alanine-Rich C-Kinase Substrate Phosphorylation and Regulation of Neurofilament Structure in Neuronal Cells

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein whose function may be regulated by the phosphorylation of multiple sites, in which the phosphorylation site domain (PSD) is recognized to have three or four PKC-dependent sites. Recently, it is considered that MARCKS is implicated in some neuronal functions, such as synaptic vesicle trafficking and neurotransmitter release, through regulation of the actin-containing cytoskeletal structure; this is based on the experimental results with short-term or prolonged pretreatment with phorbol esters and treatment by protein kinase C (PKC) inhibitor. However, the precise molecular mechanism is yet obscure. Recently, we have demonstrated that MARCKS is phosphorylated at Ser159 in PSD by Rho-kinase in vitro and that the phosphorylation occurred in neuronal cells upon stimulation with lysophosphatidic acid (LPA), and its phosphorylation was inhibited by a novel and specific Rho-kinase inhibitor, H-1152. Our results allow us to speculate that a preinflammatory substance, such as LPA, interleukin 1-beta, and bradykinin, augments MARCKS phosphorylation in a novel signal transduction pathway besides the PKC-involved one, and thereby induces the release of a neurotransmitter through a reorganization of actin-containing microfilaments at the cell periphery, the so-called "active zone". In this section, I address a novel mechanism for MARCKS phosphorylation and its related cellular function.

Vascular endothelial growth factor induces protein kinase C (PKC)-dependent Akt/PKB activation and phosphatidylinositol 3'-kinase-mediates PKC delta phosphorylation: role of PKC in angiogenesis.

Activation of the protein kinase Akt/PKB mediates VEGF-dependent endothelial cell survival and eNOS activation. Here we examined the role of PKC in mediating VEGF-induced Akt activation. The PKC inhibitors GF109203X and calphostin C inhibited VEGF-induced Akt activation. Rottlerin and Go6976, inhibitors with specificities for PKC delta and alpha, respectively, also strongly inhibited VEGF-induced Akt activation. VEGF-induced Akt activation was prevented by down-regulation of PKC induced by prolonged pretreatment with the phorbol ester, PMA. VEGF induced phosphorylation of PKC delta at Thr 505 in the activation loop, and this phosphorylation was inhibited by LY294002, suggesting that modulation of PKC delta activation by VEGF occurs distal to phosphatidylinositol 3'-kinase. PKC and PI3K inhibitors both strongly reduced the stimulation of branching tubulogenesis by VEGF in vitro. The finding that PKC mediates VEGF-induced Akt activation identifies a novel signal transduction pathway through which Akt can be regulated by growth factors acting through receptor protein tyrosine kinases, and indicates that PKC-mediated Akt activity may play an essential role in VEGF-stimulated angiogenesis.

Preexposure of murine macrophages to CpG-containing oligonucleotides results in nuclear factor κB p50 homodimer-associated hyporesponsiveness

Background. DNA containing the CpG motif is associated with immunomodulation of the innate immune response. Preexposure of macrophages to CpG DNA elicits a hyporesponsiveness to subsequent lipopolysaccharide (LPS) stimulation. We tested the hypothesis that this effect is due to decreased nuclear translocation of nuclear factor κB (NF-κB). Methods. Murine macrophage-like RAW 264.7 cells were incubated with 1.5 μg/mL CpG-containing oligonucleotides (CpG ODN) for 0.5 to 9 hours followed by restimulation with 1 μg/mL LPS for 20 minutes. Some cells were cotransfected with an NF-κB sensitive luciferase reporter construct and a control β-gal plasmid. Cytoplasmic and nuclear extracts were assayed for NF-κB by electrophoretic mobility shift assay and supershift assays, for NF-κB, IκB and phospho-IκB by Western blot, for luciferase activity, and for p38, c-Jun NH2-terminal kinase, and extracellular signal-related kinase activity assay. Results. NF-κB functional activity was decreased as demonstrated by luciferase activity assay in the prolonged CpG ODN pretreatment groups. Unlike endotoxin tolerance, CpG ODN preexposure increased cytoplasmic phospho-IκB-α and did not abrogate mitogen-activated protein kinase activity. Conclusions. In macrophages, exposure to CpG DNA increases expression of the inhibitory p50 NF-κB homodimer and decreases NF-κB activity without inhibition of IκB kinases. Mitogen-activated protein kinase activity remains intact. Understanding these interactions between different toll receptor ligands may provide insight into novel therapeutic modalities. (Surgery 2002;132:245-51.)

Effect of Low Temperature Pretreatment of Buds or Inflorescence on Isolated Microspore Culture in Brassica rapa(syn. B.campestris).

The effect of low temperature pretreatment of buds or inflorescence on microspore culture for the production of haploids of Brassica rapa (syn. B.campestris) was examined. Incubation of the buds or the inflorescence at 4°C for 3 or 10 days before the culture of microspores induced efficient microspore embryogenesis. Pretreatment of flower buds was more effective than that of the inflorescence. Prolonged pretreatment up to 20 days promoted embryo induction. Microspores in the buds were examined for developmental stage before and after the pretreatment. Buds with a petal length to anther length ratio of about 0.5-0.7, which had microspores were at the late unicellular stage, were collected and were used. All microspores were at the late unicellular stage before the pretreatment. The percentage of the microspores at the stage of late unicellular decreased during the pretreatment while the percentage of bicellular stage microspores with two unequal size nuclei increased. At the same time, a small but notable number of bicellular stage microspores with equal size nuclei, which is the first step of microspore embryogenesis, was observed after the pretreatment.

Effectiveness and mechanism of potassium ferrate(VI) preoxidation for algae removal by coagulation

Jar tests were conducted to evaluate the effectiveness of potassium ferrate preoxidation on algae removal by coagulation. Laboratory studies demonstrated that pretreatment with potassium ferrate obviously enhanced the algae removal by coagulation with alum [Al 2(SO 4) 3·18H 2O]. Algae removal efficiency increased remarkably when the water was pretreated with ferrate. A very short time of preoxidation was enough to achieve substantial algae removal efficiency, and the effectiveness was further increased at a prolonged pretreatment time. Pretreatment with ferrate resulted in a reduction of alum dosage required to cause an efficient coagulation for algae removal. The obvious impact of cell architecture by potassium ferrate was found through scanning electron microscopy. Upon oxidation with ferrate, the cells were inactivated and some intracellular and extracelluar components were released into the water, which may be helpful to the coagulation by their bridging effect. Efficient removal of algae by potassium ferrate preoxidation is believed to be a consequence of several process mechanisms. Ferrate preoxidation inactivated algae, induced the formation of coagulant aid, which are the cellular components secreted by algal cells. The coagulation was also improved by increasing particle concentration in water, because of the formation of the intermediate forms of precipitant iron species during preoxidation. In addition, it was also observed that ferrate preoxidation caused algae agglomerate formation before the addition of coagulant, the subsequent application of alum resulted in further coagulation.

A multiple schedule model of limited access drinking in the cynomolgus macaque.

The present study reports on the development of a multiple schedule procedure of oral ethanol self-administration in cynomolgus macaques. Six adult cynomolgus macaques (four female, two male) were trained to self-administer ethanol and water under a 60 min, four-component multiple schedule of ethanol and water access with 1 g food pellets presented every 900 s (fixed-time 900 s). Water was available for the first and third 15 min components, ethanol in the second and fourth components. Total ethanol dose was stable at between 1-1.25 g/kg at ethanol concentration of 4%, 6% and 8%. Subsequently water was replaced with a sweetened drink (sugar-free Tang powder, General Foods). Ethanol and Tang were self-administered in similar volumes and both served as reinforcers compared with water. Acute pretreatment with 0.25 to 1.5 g/kg of intragastrically gavaged (i.g.) ethanol failed to alter ethanol or Tang self-administration significantly despite producing mean blood ethanol levels of up to 199 mg/dl when combined with self-administered ethanol. However, 1.0 g/kg i.g. ethanol administered for 15 consecutive days significantly decreased ethanol self-administration by 23%. The results suggest that ethanol self-administration under a multiple schedule is insensitive to alteration by acute ethanol pretreatment, but can be decreased by more prolonged chronic ethanol pretreatment.