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Background: Despite its clear advantages over immunoassay-based testing, the measurement of serum thyroglobulin by mass spectrometry remains limited to a handful of institutions. Slow adoption by clinical laboratories could reflect limited accessibility to existing methods that have sensitivity comparable to modern immunoassays, as well as a lack of tools for calibration and assay harmonization., Methods: We developed and validated a liquid chromatography-tandem mass spectrometry-based assay for the quantification of serum thyroglobulin. The protocol combined peptide immunoaffinity purification using a commercially available, well-characterized monoclonal antibody and mobile phase modification with dimethylsulfoxide (DMSO) for enhanced sensitivity. To facilitate harmonization with other laboratories, we developed a novel, serum-based 5-point distributable reference material (Husky Ref)., Results: The assay demonstrated a lower limit of quantification of 0.15 ng/mL (<20 %CV). Mobile phase DMSO increased signal intensity of the target peptide at least 3-fold, improving quantification at low concentrations. Calibration traceable to Husky Ref enabled harmonization between laboratories in an interlaboratory study., Conclusions: Sensitive mass spectrometry-based thyroglobulin measurement can be achieved using a monoclonal antibody during peptide immunoaffinity purification and the addition of mobile phase DMSO. Laboratories interested in deploying this assay can utilize the provided standard operating procedure and freely-available Husky Ref reference material., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 THE AUTHORS.)

Abstract: Nutrient availability is one of the major non-genetic factors determining embryonic growth and larval or fetal size. Due to the high human consumption of blood lipid regulators, fibrates have recently been reported as pollutants in rivers. Our study investigated the developmental toxicity of fibrates in zebrafish. Treatment with micromolar concentrations of clofibrate or gemfibrozil induced an embryonic malabsorption syndrome (EMS) with very little yolk consumption, resulting in small-sized larvae. This effect was reversible on removing the drug from the water. Clofibrate delayed hatching time and decreased the amount of oil red O lipid staining in the vasculature. It also induced higher density, round-shaped neuromuscular junctions associated with disorganization and less striation of muscular fibers, and pericardial edema, as well as impairing thyroid gland morphogenesis. acox1, apoa1 and mtp hybridization transcript signals were not affected in the yolk syncytial layer (YSL) after clofibrate exposure. Di-(2-ethylhexyl)-phthalate did not slow down yolk resorption, whereas brefeldin A induced EMS. These findings suggest that the inhibition of yolk sac resorption on exposure to fibrate is not at a pre-translational level or peroxisome proliferator-activated receptor alpha dependent and may be due to an inhibition of the YSL constitutive cell secretion. The effects of fibrates and the potential bioconcentration in eggs as well as the additive action of structurally related toxicants warrant an evaluation of the developmental impact of these compounds after long-term exposure at environmentally relevant concentrations. Fibrate-induced EMS in zebrafish seems useful for studying the morphogenetic consequences of impaired nutrient availability during the early stages of vertebrate development. [Copyright &y& Elsevier]

Abstract: The thermal behaviors of three chemically modified starches (cross-linked waxy corn, hydroxypropylated regular corn, and hydroxypropylated and oxidized waxy corn) were studied using light microscopy, SEM, DSC, XRD, and HPSEC. During the gelatinization process, molecular and crystalline order losses occurred independently from each other. Oxidation treatment altered the effects of hydroxypropylation on starch gelatinization. Both cross-linking and hydroxypropylation tended to preserve granular crystalline order during initial stages of gelatinization. The crystallinities and X-ray patterns of each starch remained essentially unchanged prior to phase transition. [Copyright &y& Elsevier]

Abstract: Biphalin is a potent opioid peptide agonist, with a palandromic structure, composed of two enkephalin-like active fragments connected tail to tail by a hydrazine linker (Tyr-D-Ala-Gly-Phe-NH-NH&#60;-Phe&#60;-Gly&#60;-D-Ala&#60;-Tyr). This study presents the synthesis and in vitro bioassays of six new biphalin analogues with three different non-hydrazine linkers, some of which have higher binding affinity and bioactivity than biphalin. [Copyright &y& Elsevier]

Abstract: CYP1A is a subfamily of cytochrome P450 enzymes involved in the metabolism of numerous therapeutic drugs and in the bioactivation of procarcinogens to mutagens. Because of their diverse metabolic capacities, differences in expression of CYP1A enzymes may profoundly influence drug–drug interactions and drug or carcinogen activation and detoxification. Here, we demonstrate that cell-based bioassays are capable of identifying xenobiotics that either alter aryl hydrocarbon receptor (AhR)-mediated CYP1A levels or produce inhibition of enzyme activity. To assess induction, a stable cell line harboring a luciferase reporter driven by multiple dioxin response elements (DREs) was developed. Using this cell line, AhR agonists and antagonists were identified among drugs, dietary agents, and environmental compounds. Of the chemicals examined, the therapeutic agent omeprazole induced reporter gene activity 12.5±0.41 fold above control, whereas the phytochemical, chrysin and environmental pollutant, benzanthracene enhanced luciferase activity 3.3±0.03 and 28.7±1.7 fold above control, respectively. Several natural products, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) prevented TCDD-mediated increases in luciferase expression. For example, the botanical kava inhibited TCDD-mediated induction by 88%. Northern blot analyses of CYP1A1 in HepG2 cells treated with similar agents validated results generated in the stable cell line. The stable cells were further used to identify inhibitors of CYP1A-mediated metabolism. Resveratrol and furafylline exhibited dose-dependent decreases in CYP1A1 and CYP1A2 enzyme activities with IC50 values of 1.89 and 0.79μM, respectively. In summary, chemicals that possess the ability to alter CYP1A expression or inhibit CYP1A enzyme activities can be rapidly identified with the cell-based bioassays described here. [Copyright &y& Elsevier]

Abstract: This study examined the effects of the organochlorine (OC) insecticides chlordane, o,p′-DDT, dieldrin, endosulfan, kepone, methoxychlor, and toxaphene on human HepG2 cytochrome P450 (1A-EROD and 2B-PROD) activities and glutathione (GSH) levels. Cells were exposed for 24h at high concentrations (1, 5 or 10mM) and for 48h at lower concentrations ranging from 0.01 to 1mM to evaluate dose responses. Our results show that after 48h all but dieldrin significantly induced both P4501A and 2B. P4502B responses were greater at all exposure concentrations and times. Mixed responses in GSH levels were observed. All OCs except dieldrin and MXC significantly depleted GSH after 24h. At 48h, chlordane, endosulfan and toxaphene significantly increased GSH at low levels and decreased GSH at high levels, while kepone and methoxychlor produced significant declines in GSH at all concentrations. These results support findings of OC insecticides inducing CYP1A, 2B in rats, with CYP2B responses more important. GSH levels declined when P4502B activity was significantly elevated and were significantly increased in the absence of significant P450 activity, suggesting that GSH levels influence the catalytic activity of the cytochrome P450s and the cytochrome P450s influence the cell’s ability to regulate GSH. [Copyright &y& Elsevier]

Abstract: Neuropeptide Y inhibits neuronal excitability and seizures in various experimental models. This peptide delays kindling epileptogenesis but the receptors involved in this action are unknown. We have studied the role of Y5 receptors in kindling using the selective antagonist GW438014A (IC50=210 nM), a small heterocycle molecule that crosses the blood–brain barrier, and the selective peptide agonist Ala31Aib34 NPY (IC50=6.0 nM). Intraperitoneal injection of GW438014A (10 mg/kg), 30 min before the beginning of a rapid-kindling protocol, significantly accelerated the rate of kindling acquisition as compared to vehicle-injected rats. Thus, the number of electrical stimuli required to reach stages 3 and 4–5 of kindling were reduced by 50% and 25%, respectively. The average afterdischarge duration in the stimulated hippocampus was prolonged by 2-fold. Conversely, kindling rate was delayed by intracerebroventricular administration of 24 nmol Ala31Aib32 NPY. Thus, the number of stimuli necessary to reach stages 2 and 3 of kindling was increased by 3- and 4-fold, respectively. During the stimulation protocol (40 stimuli) none of the rats treated with the Y5 agonist showed stages 4–5 seizures. Twenty-four hours after the last kindling stimulation, thus during the re-test session, Y5 agonist- or antagonist-treated rats had stages 4–5 seizures as their controls. In rats treated with both the antagonist and the agonist, kindling rate was similar to vehicle-injected rats. These data indicate that Y5 receptors mediate inhibitory effects of NPY in kindling and display anticonvulsant rather then antiepileptogenic effects upon agonist stimulation. [Copyright &y& Elsevier]

Abstract: Precision-cut rat liver slices are a widely accepted in vitro tool for the examination of drug metabolism, enzyme induction or hepatotoxic effects of xenobiotics. After prolonged incubation, however, distinct histopathological changes and increasing losses in function are seen with liver slices from adult animals. Since tissue from neonatal animals is expected to be less vulnerable, in the present study liver slices from 1-day-old rats were examined for morphological changes and for the expression of different cytochrome P450 (CYP) isoforms after incubation for up to 24h and after a 24h in vitro exposure to β-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or pregnenolone 16α-carbonitrile (PCN). In parallel, CYP activities were assessed by different model reactions in slice homogenates and in intact slices. Histopathological changes were less pronounced in liver slices from 1-day-old rats than in those from adult animals. During the 24h of incubation even a maturation of the tissue occurred, since the proportion of haemopoietic stem cells declined and the glycogen content of the hepatocytes increased. The CYP expression pattern after 2 and 24h of incubation was similar to that of normal liver specimens from neonatal rats showing a moderate CYP1A1, 2B1 and 3A2 expression. The immunostaining for CYP1A1 and 2B1 was elevated after incubation with BNF. PB enhanced CYP2B1 and 3A2 expression, and DEX and PCN increased CYP3A2 immunostaining. This induction pattern was paralleled by respective effects on the corresponding model reactions. Thus, besides increased viability, slices from neonatal rats are excellently suited for the evaluation of an in vitro induction of CYP enzymes as well. [Copyright &y& Elsevier]

Abstract: Estradiol (E2) and insulin-like growth factor-I (IGF-I) can act independently or in concert to promote neurite outgrowth in vivo and in cultured neurons. This study examined the role of crosstalk between estrogen receptor (ER)α and the IGF-I receptor as a critical mediator of hormone- and growth factor-dependent neurite outgrowth in a homogenous cell system. We used control PC12 cells and PC12 cells stably transfected with ERα, both of which express IGF-I receptor. Cells were treated for 1 week with vehicle, 1 nM E2 or 100 ng/ml IGF-I alone or with E2 or IGF-I in the presence of either the IGF-I receptor antagonist JB1 or the ER antagonist ICI 182,780. IGF-I significantly increased neurite outgrowth, as measured by the percentage of process-bearing cells, and absolute neurite length per cell in both control and ERα-transfected PC12 cells. In contrast, E2 increased process formation and extension only in PC12 cells that were stably transfected with ERα. ICI 182,780 and JB1 blocked the IGF-I-induced increases in neurite length in both cell types. The efficacy of ICI 182,780 in control PC12 cells may have been due to the upregulation of ERα in these cells by the 7-day treatment with IGF-I. The ER and IGF-I receptor antagonists similarly blocked the E2-induced increase in neurite lengths in ERα-transfected cells. Immunofluorescent analysis of the cellular distribution of an axonal marker, phospho-neurofilament, verified that the processes extended by PC12 cells were neurites. These data suggest that receptor crosstalk between IGF-I receptors and ERα has an important role in neurite formation and extension even in a single-cell system. [Copyright &y& Elsevier]

Aloe–emodin (1,8-dihydroy-3-[hydroxymethyl]-anthraquione) purified from Aloe vera leaves has been reported to have antitumor activity. The objectives of our research were to determine how aloe–emodin regulates the cell cycle, cell proliferation and protein kinase C (PKC) during glioma growth and development. To establish the cell cycle effects of aloe–emodin on brain cells [transformed glia cell line (SVG) and human glioma U-373MG cell line (U-373MG)], cells were treated with either dimethylsulfoxide (DMSO; control) or aloe–emodin (40 μM). Results from flow cytometry demonstrated that aloe–emodin delayed the number of cells entering and exiting DNA synthesis (S) phase in both SVG and U-373MG cells indicating that aloe–emodin may inhibit S phase progression. Assessment of cell viability demonstrated that SVG and U-373MG glioma cell were highly sensitive to aloe–emodin. The aloe–emodin-induced decreased proliferation was sustained at 48–96 h. A PKC activity assay was quantified to establish the role of PKC in aloe–emodin''s mode of action. Exposure of SVG and U-373MG glioma cells to aloe–emodin suppressed PKC activity and reduced the protein content of most of the PKC isozymes. We determined that cancer growth inhibition by aloe–emodin was due to apoptosis (i.e., programmed cell death). Taken together, these results support the hypothesis that aloe–emodin represents a novel antitumor chemotherapeutic drug. [Copyright &y& Elsevier]

Abstract: FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia–reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3h with immunosuppressant drugs. A significant reduction (ca. 30–40%) of caspase-3 activation by 5μM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia–reperfusion injury following liver transplantation. [Copyright &y& Elsevier]

Abstract: The outer mantle epithelium of the freshwater bivalve, Anodonta cygnea, is responsible for the mineralisation of the shell. Under short circuit conditions, it generates a current that is due to the operation of an electrogenic proton pump located in the apical barrier of that epithelium. Bafilomycin A1 and Concanamycin A inhibited the short circuit current. The IC50 and maximum inhibition dose were 0.17 and 0.5 μM for Bafilomycin A1, and 0.7 and 5 μM for Concanamycin A. The present work was undertaken to further characterise V-type ATPase of the outer mantle cells. The V-ATPase enzymatic activity of crude homogenate, measured as the amount of inorganic phosphorous released, due to ATP hydrolysis, in the presence of Na2SO3 (200 mM) was found to be 4.6±1.1 μmol Pi/mg protein/h, at 27 °C, pH 7.0–7.4 and ATP 4.5–6.0 μM. Bafilomycin A1 and Concanamycin A inhibit the V-ATPase activity with an IC50 of 14 and 8 nmol mg−1, respectively. Dicyclohexylcarbodiimide (DCCD; 100 mM) and NaNO3 (100 μM) inhibited the V-type ATPase in what it seems a non-specific manner and NEM (100 mM) was unable to do it. Bafilomycin A1 (10 μM) and Concanamycin A (10 μM), inhibited 50–60% of the total activity. [Copyright &y& Elsevier]

The effects on cell proliferation of arylhydrocarbon receptor (AhR) agonists in estrogen-responsive T47D and ZR-75-1 cells were investigated. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the non-ortho-substituted polychlorinated biphenyl (PCB) congeners, PCB 77, PCB 81, PCB 126, and PCB 169 each inhibited 17β-estradiol (E2)-stimulated cell proliferation in a dose–responsive manner. In the absence of added E2, TCDD, PCB 77, PCB 81, and PCB 169 had no significant effect on cell proliferation, while PCB 126 at high concentrations caused slight elevations. The order of effective inhibition of E2-stimulated cell proliferation by the PCB congeners was: PCB 81>PCB 126≃PCB 169>PCB 77. In the comparative literature, mammalian TEFs for these congeners toxic potency are in the order: PCB 126>PCB 169>PCB 81≃PCB 77 [Organohalogen Compd. 34 (1997) 237]. Our results thus show an unexpected different pattern for the inhibitory effects of PCBs congeners on E2-mediated cell proliferation. [Copyright &y& Elsevier]

We summarize and compare recent molecular dynamics simulations on the interactions of dipalmitoylphosphatidylcholine (DPPC) bilayers in the liquid crystalline phase with a number of small molecules including trehalose, a disaccharide of glucose, alcohols, and dimethylsulfoxide (DMSO). The sugar molecules tend to stabilize the structure of the bilayer as they bridge adjacent lipid headgroups. They do not strongly change the structure of the bilayer. Alcohols and DMSO destabilize the bilayer as they increase its area per molecule in the bilayer plane and decrease the order parameter. Alcohols have a stronger detrimental effect than DMSO. The observables which we compare are the area per molecule in the plane of the bilayer, the membrane thickness, and the nuclear magnetic resonance (NMR) order parameter of DPPC hydrocarbon tails. The area per molecule and the order parameter are very well correlated whereas the bilayer thickness is not necessarily correlated with them. [Copyright &y& Elsevier]

Abstract: The intentional and accidental discharges of toxic pollutants into the lithosphere results in soil contamination. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil constitutes a genotoxic hazard. This work is a comprehensive review of published information on soil mutagenicity. In total, 1312 assessments of genotoxic activity from 118 works were examined. The majority of the assessments (37.6%) employed the Salmonella mutagenicity test with strains TA98 and/or TA100. An additional 37.6% of the assessments employed a variety of plant species (e.g., Tradescantia clone 4430, Vicia faba, Zea mays, Allium cepa) to assess mutagenic activity. The compiled data on Salmonella mutagenicity indicates significant differences (p<0.0001) in mean potency (revertents per gram dry weight) between industrial, urban, and rural/agricultural sites. Additional analyses showed significant empirical relationships between S9-activated TA98 mutagenicity and soil polycyclic aromatic hydrocarbon (PAH) concentration (r2=0.19 to 0.25, p<0.0001), and between direct-acting TA98 mutagenicity and soil dinitropyrene (DNP) concentration (r2=0.87, p<0.0001). The plant assay data revealed excellent response ranges and significant differences between heavily contaminated, industrial, rural/agricultural, and reference sites, for the anaphase aberration in Allium cepa (direct soil contact) and the waxy locus mutation assay in Zea mays (direct soil contact). The Tradescantia assays appeared to be less responsive, particularly for exposures to aqueous soil leachates. Additional data analyses showed empirical relationships between anaphase aberrations in Allium, or mutations in Arabidopsis, and the 137Cs contamination of soils. Induction of micronuclei in Tradescantia is significantly related to the soil concentration of several metals (e.g., Sb, Cu, Cr, As, Pb, Cd, Ni, Zn). Review of published remediation exercises showed effective removal of genotoxic petrochemical wastes within one year. Remediation of more refractory genotoxic material (e.g., explosives, creosote) frequently showed increases in mutagenic hazard that remained for extended periods. Despite substantial contamination and mutagenic hazards, the risk of adverse effect (e.g., mutation, cancer) in humans or terrestrial biota is difficult to quantify. [Copyright &y& Elsevier]

Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca2+, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation.The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool. [Copyright &y& Elsevier]

A sequence critical for phenobarbital (PB) induction, the PB response unit (PBRU), situated upstream of the rat CYP2B1 and CYP2B2 genes, includes two nuclear receptor binding sites, NR1 and NR2. When NR1 and NR2 are mutated PB responsiveness is abolished. While no nuclear receptor for which PB is an agonist ligand has yet been identified, PB is a ligand of GABAA receptors and it can displace [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) from its binding site on the peripheral benzodiazepine receptor (PBR). We assessed CYP2B levels in primary rat hepatocytes following treatment with 10 ligands of either or both of these receptors. All compounds tested were found to be CYP2B1/CYP2B2 inducers and most were CYP3A inducers. Five had not previously been described as CYP2B1/CYP2B2 inducers: bicuculline, flunitrazepam, 4′-chlorodiazepam (Ro5-4864), N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN 1–27) and 7-(dimethylcarbamoyloxy)-6-phenylpyrrolo-[2,1-d][1,5]benzothiazepine (DCPPBT). Reporter gene analysis demonstrated that CYP2B induction by these agents and other PBR or GABAA receptor ligands is mediated through the PBRU and the NR1/NR2 sites, suggesting a molecular mechanism similar to that for PB induction. The potencies for PBRU-dependent induction by 11 ligands of PBR or the GABAA receptor was evaluated. FGIN-127, DCPPBT and PK 11195 exhibited EC50 values for PBRU-dependent transcription activation about three orders of magnitude higher than the reported affinities of the PBR for these agents, arguing against the involvement of the PBR in PB induction. However the EC50 values found for the agents tested encourage further investigation on the possible involvement of the GABAA receptor in PB induction. [Copyright &y& Elsevier]

Colorectal cancer is one of the most common cancers in the world, poorly responding to available chemotherapeutic agents. To investigate whether natural molecules can inhibit colon cancer progression, we investigated a principle phytoestrogen found in soybean known as daidzein, and determined its effects on the human colon cancer cell line LoVo. LoVo cells were treated with 0.1, 1, 5, 10, 50 and 100 μM daidzein for 2, 3, 4 or 5 d. The results indicated that daidzein stimulated the growth of LoVo cells at 0.1 and 1 μM whereas at higher concentrations (10, 50 and 100 μM) cell growth was inhibited in a dose-dependent manner. Treatment of daidzein at 10, 50 and 100 μM resulted in cell cycle arrest at G0/G1 phase, DNA fragmentation and increases in caspase-3 activity. There were no changes in alkaline phosphatase activity (ALP), an indicator of cell differentiation, upon treatment with daidzein when compared to controls. These results indicate that daidzein has a biphasic effect on LoVo cell growth and its tumor suppressive effect is by means of cell cycle arrest and apoptosis but not through cell differentiation. [Copyright &y& Elsevier]

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase α in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase α inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 μM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors. [Copyright &y& Elsevier]

Abstract: Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A2 (D-PLA2) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE1) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways. [Copyright &y& Elsevier]

Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies. [Copyright &y& Elsevier]

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1β (IL-1β). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). We previously demonstrated that 15d-PGJ2 was more potent than troglitazone to counteract IL-1β effects on chondrocytes. Here, we studied the action of 15d-PGJ2 on intracellular targets in nuclear factor-κB (NF-κB) signalling pathway in IL-1β treated rat chondrocytes. We found that 15d-PGJ2 decreased inhibitor κBα (IκBα) degradation but not its phosphorylation by specifically inhibiting IκB kinase β (IKKβ), but not IKKα, enzymatic activity. We further evaluated the involvement of PPARγ in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARγ protein, 15d-PGJ2 pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE2 production, p65 translocation and NF-κB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ2 exhibited the same effect in chondrocytes overexpressing mutated PPARγ protein. These results suggest that 15d-PGJ2 exerts its anti-inflammatory effect in rat chondrocytes by a PPARγ-independent mechanism, which can be conferred to a partial inhibition of IκBα degradation. [Copyright &y& Elsevier]

Betel quid (BQ) chewing is strongly associated with the occurrence of oral leukoplakia, oral submucous fibrosis, and oral cancer. There are about 200–600 million BQ chewers in the world. Previous animal studies support the potential carcinogenicity of BQ in different test systems. However, little animal experiment has let hamsters or rats to chew BQ directly, similar to that in humans. In the present study, we established a hamster model of chewing BQ or areca nut (AN). A total of 81 2-week-old hamsters were randomly divided into three groups: 25 for control group, 28 for BQ-chewing group, and 28 for AN-chewing group. These animals were fed with powdered diet with/without BQ or AN for 18 months. Although the consumption of BQ or AN showed some variations, hamsters fed with powdered diet could chew and grind AN or BQ into small pieces of coarse fibers during the entire experimental period. The survival rate of AN-chewing hamsters decreased significantly after 6 months of exposure. The mean survival time was 15.6 ± 0.9 months for control animals, 13.6 ± 0.98 months for AN-chewing animals, and 15.7 ± 0.55 months for BQ-chewing animals. The body weight of BQ- or AN-chewing animals also decreased after 4–13 months. Hamsters fed with AN for 18 months showed hyperkeratosis in 80% and acanthosis in 50% of cheek pouches. Animals fed with BQ for 18 months also showed hyperkeratosis in 93% and acanthosis in 14% of cheek pouches. These results indicate that AN and BQ components may induce alterations in proliferation and differentiation of oral epithelial cells. Animal model of chewing BQ or AN can be useful for future tumor initiation, promotion and chemoprevention experiments simulating the condition of BQ chewing in humans. [Copyright &y& Elsevier]

The mystery of how a protein sequence specifies a unique structure has intrigued chemists, leading to the design and study of foldamers, non-natural oligomeric molecules that adopt well-defined structures. Recently, the sequence specificity of the various regular repeating structures has been revealed for bioinspired foldamers and such foldamers have been created to adopt helical bundle tertiary structures. One major strategy for the generation of abiotic foldamers has involved molecular design of the monomer geometry. These advances in foldamer research may lead to future applications in biomedical and materials science. [Copyright &y& Elsevier]

Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis. Overall, serine proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain. The soluble trypsin-like proteinase activity of the resistant strain was approximately half that of the susceptible strain. The number and relative molecular masses of soluble and membrane serine proteinases were different. However, there were no significant differences in the activities of serine proteinases and aminopeptidases extracted from midgut membranes of the two strains. Cry1Ab protoxin hydrolysis by soluble proteinase extracts of the resistant strain was reduced approximately 20–30% relative to that of the susceptible strain. Reduced protoxin processing due to decreased activities of Bt protoxin activation proteinases may be associated with resistance to Bt toxin in this resistant strain of O. nubilalis. [Copyright &y& Elsevier]

GroEL can solubilize membrane proteins by binding them in its hydrophobic cavity when detergent is removed by dialysis. The best-studied example is bacteriorhodopsin, which can bind in the GroEL chaperonin at two molecules per tetradecamer. Applying this approach to the holin and antiholin proteins of phage λ, we find that both proteins are solubilized by GroEL, in an ATP-sensitive mode, but to vastly different extents. The antiholin product, S107, saturates the chaperonin at six molecules per tetradecameric complex, whereas the holin, S105, which is missing the two N-terminal residues of S107, forms a hyper-solubilization complex with up to 350 holin molecules per GroEL, or approximately 4 MDa of protein per 0.8 MDa tetradecamer. Gel filtration chromatography and immunoprecipitation experiments confirmed the existence of complexes of the predicted masses for both S105 and S107 solubilization. For S105, negatively stained electron microscopic images show structures consistent with protein shells of the holin assembled around the chaperonin tetradecamer. Importantly, S105 can be delivered rapidly and efficiently to artificial liposomes from these complexes. In these delivery experiments, the holin exhibits efficient membrane-permeabilizing activity. The S107 antiholin can block formation of the hypersolubilization complexes, suggesting that their formation is related to an oligomerization step intrinsic to holin function. [ABSTRACT FROM AUTHOR]

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PRV82A and PRI84V that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10–1.53 A˚. The crystal structures of PR and PRI84V with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PRV82A complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PRV82A showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10 A˚ resolution structure of PRV82A with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism. [Copyright &y& Elsevier]

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified MoVI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo MoVI form of the enzyme and prevents the formation of a dioxo pentacoordinate MoVI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. [Copyright &y& Elsevier]

Influenza A and B viruses are still a major worldwide threat. We demonstrate that influenza B virus infection induces signaling via the Raf/MEK/ERK cascade, a process required for efficient virus production. Expression of dominant-negative Raf and ERK mutants or treatment with a MEK inhibitor (U0126) strongly impaired viral propagation, while selective activation of the pathway resulted in increased virus titers. MEK inhibition appears to interfere with a distinct viral nuclear export process. Most importantly, no resistant virus variants emerged in the presence of U0126 demonstrating that influenza viruses cannot easily adapt to the missing cellular function. [Copyright &y& Elsevier]

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions. [ABSTRACT FROM AUTHOR]

The effects of the L-type Ca2+ channel blocker nimodipine on the spectrum of the spontaneous neural noise (SNN) and the waveform of the gross sound-evoked compound action potential (CAP) were investigated by perilymphatic perfusion in the guinea pig cochlea. Both the SNN and the CAP were reversibly suppressed by nimodipine. The percentage reduction in SNN was dose-dependent in a manner very similar to the results obtained with the measures of CAP threshold changes. The reduction in the peak SNN caused by 10 μM nimodipine was the same as that caused by 500 μM kainic acid, which totally eliminated any neural responses. For 1 μM nimodipine there was an apparent dissociation between the SNN and CAP changes such that the SNN could be markedly suppressed with only very small changes in CAP thresholds. These results imply that spontaneous release of neurotransmitter from the inner hair cell is more sensitive to block of calcium channels than evoked release. There was no evidence for any marked shift caused by nimodipine, in the position of the main (900 Hz) spectral peak in the SNN. Comparison of the CAP waveform before and after nimodipine perfusion showed that the CAP waveforms were unchanged despite the change in sensitivity. These data do not support the notion of any significant postsynaptic site of action of nimodipine. The data hence provide further support for an exclusively presynaptic role for L-type Ca2+ channels in the regulation of both evoked and spontaneous neurotransmitter release from inner hair cells. [Copyright &y& Elsevier]

A series of four cationic lipids derived from cholesterol was synthesised and their efficiencies to vectorise nucleic acids were compared. The investigation concerns the effects of systematic chemical modifications in the polar head and in the spacer. The cationic lipid molecules used are in the same family of 3β[N-(N′,N′,N′-trimethylaminoethane)-carbamoyl] cholesterol iodide (TMAEC-Chol), presenting a spacer of two or three carbons and a quaternary ammonium polar head ramified with methyl or ethyl groups. These lipids formed stable liposomes sizing from 100 to 200 nm when prepared with the colipid dioleoyl phosphatidylethanolamine (DOPE). The goal of this work was to investigate the effect of the chemical structure of these cationic lipids on lipofection. Their ability to form complexes with DNA, their cytotoxicity and their transfection efficiency in vitro and in vivo were studied. Results were compared with those obtained from the well known cholesterol-based cationic lipid DC-Chol. In a melanoma cell line (B16-F10), results showed that either the polar head or the spacer affected the cytotoxicity. Cationic lipids with three ethyl groups in the head are more toxic than those with three methyl groups while cationic lipids with three carbons in the spacer are less toxic than those with two carbons in the spacer. The best transfection level was obtained in vitro and in vivo with cationic lipids having 3C in the spacer. Data indicated that among these lipids, in vivo gene transfer is advantaged by the methylated polar head while in vitro the best level was obtained with the ethylated one. Finally, it was observed that the chemical structure influences the transfection in the presence of serum while the complex charge and the DOPE ratios in liposomes preferentially affect the interaction with erythrocytes. Argumentations are proposed to explain the discrepancies between in vitro and in vivo transfection results concerning the optimal charge ratio and the chemical nature of the cationic lipid head group. [ABSTRACT FROM AUTHOR]

Background/Aims: Free radical-mediated oxidative stress has been implicated in the pathogenesis of acute liver injury. The aim of our study was to investigate whether melatonin, a potent free radical scavenger could prevent fulminant hepatic failure in rats.Methods: Liver damage was induced by two consecutive injections of thioacetamide (TAA, 300 mg/kg/i.p.) at 24 h intervals. Treatment with melatonin (3 mg/kg/daily, i.p) was initiated 24 h prior to TAA.Results: Twenty-four h after the second TAA injection, serum liver enzymes and blood ammonia were lower in rats treated with TAA+melatonin compared to TAA (P<0.001). Liver histology was significantly improved and the mortality in the melatonin-treated rats was decreased (P<0.001). The increased nuclear binding of nuclear factor κB in the livers of the TAA-treated rats, was inhibited by melatonin. The hepatic levels of thiobarbituric acid reactive substances, protein carbonyls and inducible nitric oxide synthase were lower in the TAA+melatonin-treated group (P<0.01), indicating decreased oxidative stress and inflammation.Conclusions: In a rat model of TAA-induced fulminant hepatic failure, melatonin improves survival and reduces liver damage and oxidative stress. The results suggest a causative role of oxidative stress in TAA-induced hepatic damage and suggest that melatonin may be utilized to reduce liver injury associated with oxidative stress. [Copyright &y& Elsevier]

This paper evaluates the ability of organoselenium compounds [ebselen, selenocystine N-ethyl-carbamate (SeCis), bis-4-isopropyl-2-oxazolinyl phenyl diselenide (AASe)] to prevent HgCl2 toxicity. Rats were injected with HgCl2 (0 or 17 μmol/kg, sc) 6 h after organoselenium compounds had been injected (0 or 50 μmol/kg, sc). In vivo, HgCl2 inhibited renal ALA-D activity (∼48%), increased TBARS level in kidney (∼52%) and reduced the hepatic content of non-protein thiol groups (∼40%), but organoselenium compounds did not prevent such effects. SeCis, per se, increased renal TBARS level (∼42%), while AASe increased hepatic content of ascorbic acid (∼38%). In vitro, renal and hepatic ALA-D activity was inhibited by HgCl2 (&ges;25 μm), ebselen (&ges;12 μm) and SeCis (&ges;4 μm). HgCl2 (400 μm) significantly increased TBARS production in renal and hepatic tissue preparations in vitro, and this effect was completely or partially prevented by organoselenium compounds. Ebselen exhibited thiol peroxidase activity in our assay conditions, while SeCis exhibited thiol-oxidizing properties regardless of the presence of peroxide. AASe had no effect on thiol oxidation. Results suggest that organoselenium compounds could not prevent mercury toxicity in vivo. The protective effect of these compounds against mercury-induced increase of TBARS production in vitro is probably related to an antioxidant action rather than to mercury binding. [Copyright &y& Elsevier]