Concentration In Dose-dependent Manner Research Articles

The Inhibitory Effects of Ganoderma lucium on Cell Proliferation, Apoptosis, and TNF‐α‐Induced CCL2 Release in Genetically Different Triple‐Negative Breast Cancer Cells

Breast cancer is the second most lethal form of cancer among American women, presenting strong evidence of health disparities between Whites and African Americans. Triple-negative breast cancer (TNBC), in particular, is the most aggressive subtype, affecting African American women more aggressively. Several mushrooms have been shown to have nutritional value and contain bioactive compounds with a diversity of medicinal properties. The current study investigated the effects of Ganoderma lucidum, a mushroom that has been used across Asia as a nutraceutical and herbal remedy, on TNF-α stimulated MDA-MB-231 (White) and MDA-MB-468 (African American) TNBC cells. The extract's ability in causing cell toxicity, cell proliferation, and apoptosis was examined, and its modulatory effect on the mRNA expression of genes involved in cancer progression. The results showed that G. lucidum decreased cell viability in a dose-dependent manner in concentrations of 0.75 to 4.25 mg/mL, showing similar IC50s of 1.5 and 1.4 mg/mL MDA-MB-231 and MDA-MB-468 cells, respectively. After a 96-hr of treatment, the cell proliferation rate was reduced in both cell lines. However, the extract had a higher potency in MDA-MB-468, exhibiting more anti-proliferative effects in concentrations as low as 0.125 mg/mL. Apoptosis assays demonstrated that G. lucidum extract (2.75 mg/mL) increased the number of apoptotic cells in MDA MB-231, showing 90% of the apoptotic phase's analyzed, compared to 70% in MDA-MB-468 cells. Moreover, the mushroom extract downregulated mRNA expression of the proinflammatory cytokine, CCL2, reducing expression levels to less than 10%, compared to the cells treated with TNF-α only. This study demonstrates that the extract of G. lucidum may have an anti-cancer potential by inducing cytotoxicity, reducing cell proliferation, inducing apoptotic effects, and suppressing CCL2 expression in MDA-MB-231 and MDA-MB-468 TNBC cells. Furthermore, the results reveal that the genetically diverse TNBC cells used in this study responded differently to G. lucidum treatment. These results provide more evidence that genetically different cells may respond differently to treatments, explaining the inadequate therapeutic response in African American patients with advanced TNBC compared to other races.

Haemopoietic Potential of Tannin Fractionates of Vitex doniana Leaf on Nitrosobis (2-Oxopropyl) Amine Comobidity in Docetaxel-Induced Myelosuppression in Wistar Rat

Aim: This study investigated the haemopoietic potential of tannin fractionate of Vitex doniana leaf against nitosobis (2-oxopropyl) amine comorbidity in docetaxel-induced bone marrow suppression.
 Study Design: This is an experimental research.
 Place of Research: University of Nigeria, Enugu campus.
 Methodology: The male Wistar rats used in this experiment were twenty-eight in number, and they were grouped into 7, with each group having four rats. Group 1 served as control, and received 1ml of normal saline, while groups 2-7 were treated with Nitrosobis (2-oxopropyl) amine 5 mg/kg daily for 2 weeks. Then groups 3-7 were treated with 8 mg/Kg of docetaxel weekly for 2 weeks. And groups 4, 5 and 6 also received 250 mg/Kg and 500 mg/Kg and 1000 mg/kg of tannin, respectively, daily for 2 weeks. Group 7 received 40 mg/Kg of fesolate daily for 2 weeks.
 Results: The haemoglobin concentration and white blood cell count of rats in the groups treated with Nitrosobis (2-oxopropyl) amine alone (group 2) and Nitrosobis (2-oxopropyl) amine plus Docetaxel (group 3) showed statistically significant reduction (p=.05) in number when compared with the group treated with normal saline (group 1). The haemoglobin concentration and white blood cell count of the rats in the groups treated with 250 mg/kg, 500 mg/kg, and 1000 mg/kg of the tannin fractionate, in addition to the Nitrosobis (2-oxopropyl) amine and Docetxel (i.e. groups 4, 5, and 6, respectively) showed statistically significant dose dependent increase in number, with the group treated with 1000mg/kg showing the highest increment (p=.05). The cells in the bone marrow show significant reduction in number in the rats treated with Nitrosobis (2-oxopropyl) amine (group 2) and Nitrosobis (2-oxopropyl) amine plus docetaxel (group 3) when compared with the rats in group 1 (treated with normal saline). With the addition of graded doses of the tannin fractionate, 250 mg/kg, 500 mg/kg, and 1000mg/kg (i.e. groups 4, 5, and 6, respectively), the number of cells in the bone marrow showed statistically significant increase when compared to group 3 (treated with Nitrosobis (2-oxopropyl) amine plus docetxel), with the rats in the group treated with 1000 mg/kg of tannin fractionate (group 6) having the highest increment (p=.05).
 Conclusion: Tannin obtained from Vitex doniana leaf extract increases the haemoglobin concentration in dose dependent manner. It also increases the white blood cell count, and number of proliferating bone marrow cells, following suppression by Nitrosobis (2-oxopropyl) amine and Docetaxel. So, this tannin obtained from Vitex doniana leaf extract may be useful in clinical practice to cushion the myelosupression, anaemia and leukopenia that are associated with use of docetaxel in treatment of malignancies.

Eco-Friendly Synthesis of Silver Nanoparticles Through Economical Methods and Assessment of Toxicity Through Oxidative Stress Analysis in the Labeo Rohita.

The physicochemical and biological properties of metals change as the particles are reduced to nanoscale. This ability increases the application of nanoparticles in commercial and medical industry. Keeping in view this importance, Silver nanoparticles (Ag-NPs) were synthesized by reduction methods using formaldehyde as reducing agent in the chemical route and lemon extracts in the biological route. The scanning electron microscope (SEM) images of nanoparticles suggested that the particles were either agglomerated or spherical in shape with mean diameter of 16.59nm in the chemical route and 42.93nm in the biological route. The particles were between 5 and 80nm with maximum frequency between 5 and 20nm in the chemical route and between 5 and 100nm with maximum frequency between 15 and 50nm in the biological method. In the second phase of the study, the effect of Ag-NPs on the oxidative stress was studied. For this purpose, Labeo rohita (20 ± 2.5g in weight and 12 ± 1.4cm in length) were involved. Six treatments were applied in three replicates having five fishes in each replicate. The first treatment was used as control group, and the other five treatments were exposed to either 10 or 20 or 30 or 45 or 55mgL-1 of Ag-NPs for 28days. The treatment of Ag-NPs caused oxidative stress in the liver and gill tissues, which induced alterations in the activities of antioxidant enzymes. The level of catalase (CAT) was decreased in response to Ag-NPs concentration in dose-dependent manner. Ag-NPs treatment stimulated the liver and gill tissues to significantly increase the level of superoxide dismutase (SOD), which might be due to synthesis of SOD and addition in the pre-existing SOD level. The level decreases again due to depletion of SOD level. There was a sharp decline in the activities of glutathione S-transferase (GST) in both gills and liver tissues even at lower concentration, and this decrease in the GST activity was significantly different at each treatment after 28days of treatment except 20mgL-1. The malondialdehyde (MDA) levels of gills and liver tissues were increased with the increase in the concentration. The elevated levels of glutathione (GSH) showed that the liver started defensive mechanism against the oxyradicals. This study finds out the cheap eco-friendly and economical method of Ag-NP synthesis. It is further revealed that Ag-NPs caused oxidative stress in the aquatic animals if exposure occurs at high concentrations.

Antisecretory and antiulcerative effects of ethyl acetate fraction of <i>Nigella sativa </i>(L.) Seed extract in rats

The present work was carried out to investigate the possible effects of ethyl acetate seed fraction of Nigella sativa on gastric ulcers and basal gastric secretions using the Non-Steroidal Anti-inflammatory Drug-induced (NSAID) model. Phytochemical screening according to Trease and Evans, 2002 and acute toxicity tests using the Lorke’s Method, 1983 were conducted. For the mucosal integrity study, ulcer and preventive indices were analysed, while volume of gastric juice, titratable acidity, acid output and pepsin concentration were assessed for basal gastric secretion parameters. Phytochemical screening revealed the presence of flavonoids, alkaloids, saponins, glucocinolates amongst others, while the acute toxicity studies revealed a median lethal dose above 5000mg/kg. The rats were grouped into 9 (n = 5), with the extract fraction administered at 50, 100 and 200mg/kg subcutaneously, followed by pyloric ligation with cimetidine used as the standard drug. Five rats received normal saline 1ml/kg/rat subcutaneously (S.C) as Negative Control, Five rats received indomethacin (20 mg/kg S.C), Ten rats for the study of the effect of two different doses of cimetidine 50 and 100 mg/kg S.C (5 rats for each dose). Ten rats for the study of effect of two different doses of cimetidine (50 mg and 100 mg/kg) S.C, given 30 minutes prior to indomethacin administration (5 rats for each dose). The three experimental doses of the extract at 50,100 and 200mg/kg showed a dosedependent decrease in both ulcer and preventive indices with the 200mg/kg dose at 0.6mm and 94% respectively. It also showed a significant (p<0.05) decrease in volume of gastric juice, titratable acidity, acid output and pepsin concentration in dose-dependent manner with the three experimental doses administered with the highest reduction at the 200mg/kg dose. The results obtained suggest that this fraction down-regulated all those parameters which might be attributed to the presence of the phytoconstituents present in this fraction, particularly the flavonoids. Therefore, the extract fraction of this plant possesses gastroprotective activity further explaining the folkloric use of this plant in the therapy of peptic ulcer disease. Keywords : Antiulcerative, Antisecretory, Nigella sativa, Phytochemicals, Rats, Seed Extracts.

Attenuation effect of Abnormal Savda Munziq on liver and heart toxicity caused by chemotherapy in mice

Abnormal Savda Munziq (ASMq), an Uighur medicine formula commonly used in the treatment of cancer, has been speculated to possess antioxidative and antiproliferative effects, and to regulate immune activity. The present study was designed to systematically elucidate the toxicity-reducing activity of ASMq in mice undergoing combination chemotherapy with doxorubicin and 5-fluorouracil (5-FU). The mice were divided into normal (saline, 10 ml/kg) and doxorubicin + 5-FU groups (doxorubicin, 2.5 mg/kg; 5-FU, 10 mg/kg on alternate days). In addition, three groups received different doses of ASMq (2, 4 and 8 g/kg), in addition to doxorubicin (2.5 mg/kg) and 5-FU (10 mg/kg) treatment on alternate days. The histology of the heart and liver, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) concentrations in heart homogenate, and various biochemical parameters of the liver were evaluated. Compared with the normal control group, ASMq dose-dependently improved a number of variables, including body weight, liver index, transaminase and total protein, and partially normalized liver and cardiac pathology. ASMq restored activities of defense antioxidant enzymes SOD and GSH-Px towards normal levels, and decreased MDA concentration in dose-dependent manner. These results demonstrated that ASMq provides significant protection against doxorubicin + 5-FU combination induced hepatotoxicity and cardiotoxicity. Further studies are required to determine the effects of ASMq against doxorubicin + 5-FU-induced toxicity during chemotherapy in vivo.

Abstract 4518: The PI3K-δ inhibitor TGR-1202 induces cytotoxicity and inhibits phosphorylation of AKT in 17p deleted and non-17p deleted CLL cells in vitro

Abstract Background: The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL), with expression of the delta isoform of PI3K largely restricted to lymphocytes. Clinical evaluation of PI3K-δ inhibitors, such as idelalisib, in CLL patients has been promising. CLL patients with 17p deletion (17pdel) have displayed inferior responses to chemo-immunotherapy, and we hypothesized that inhibiting the PI3K/AKT pathway would be equally efficacious in patients with and without 17pdel. TGR-1202 is a novel PI3K-δ specific inhibitor with unique pharmacologic properties and demonstrated in-vitro activity. Herein, we evaluate the in vitro effect of TGR-1202 on CLL lymphocytes, specifically evaluating differences between 17pdel CLL and non-17pdel CLL samples. Methods: We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity. Primary CLL cells were incubated with serial dilutions of TGR-1202 tested for apoptosis by activated caspase-3 and 7AAD staining measured by flow cytometry. Cytotoxicity was measured using the colorimetric MTS reagent. Phosphorylated AKT (S473) was measured by flow cytometry after incubation with TGR-1202 followed by incubation with anti-IgM or anti-IgD. AKT phosphorylation was quantified by median fluorescent intensity (MFI). Results: We have evaluated TGR-1202 in 15 CLL lymphocyte samples, 10 with normal non-17pdel cytogenetics, and 5 with 17pdel. TGR-1202 induced cytotoxicity and apoptosis in a dose-dependent manner in concentrations between 4 nM and 25 μM with no significant difference in results observed between normal CLL lymphocytes those that had 17pdel cytogenetics. The mean ED50 in 17pdel CLL cells was 326 nM, whereas the mean ED50 in non-17pdel CLL cells was 887 nM, although due to the small sample size, this difference was not statistically significant (p = 0.16, t-test). We also found that TGR-1202 significantly suppressed AKT phosphorylation in CLL lymphocytes. Conclusions: TGR-1202 is a potent PI3K-δ inhibitor that suppresses AKT phosphorylation and induces apoptosis-dependent cytotoxicity in primary CLL lymphocytes, both with favorable and adverse cytogenetics. We observed that higher concentrations of TGR-1202 were required to induce cytotoxicity in non-17pdel CLL lymphocytes on average, than in 17pdel CLL. Evaluation of additional CLL cell samples with 17pdel is ongoing to confirm initial findings. A Phase I trial with once-daily TGR-1202 is currently ongoing in patients with hematologic malignancies, including CLL. To date, TGR-1202 has been well tolerated, with no drug-related hepatic toxicities observed. Our initial observations suggest that clinical evaluation of TGR-1202 enriched in CLL patients with 17pdel may be warranted. Citation Format: Daphne R. Friedman, Tiffany Simms, Sallie D. Allgood, Danielle M. Brander, Peter Sportelli, Hari P. Miskin, Swaroop Vakkalanka, Srikant Viswanadha, J. Brice Weinberg, Mark C. Lanasa. The PI3K-δ inhibitor TGR-1202 induces cytotoxicity and inhibits phosphorylation of AKT in 17p deleted and non-17p deleted CLL cells in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4518. doi:10.1158/1538-7445.AM2014-4518

Comparison of the PI3K-δ Inhibitors TGR1202 and GS-1101 in Inducing Cytotoxicity and Inhibiting Phosphorylation of Akt in CLL Cells in Vitro

AbstractAbstract 3914 Background:The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL). Expression of the delta isoform of PI3K is largely restricted to lymphocytes. Inhibition of PI3K activity in vitro induces CLL cell apoptosis and death. Clinical evaluation of PI3K-d inhibitors, such as GS-1101, has been promising, with responses seen in relapsed and/or refractory CLL patients. TGR-1202 is a novel PI3K-d specific inhibitor previously demonstrated to inhibit Akt phosphorylation and induce apoptosis in B-cell lymphoma cell lines. Given the efficacy of other PI3K-d inhibitors observed in CLL, we assessed the ability of TGR-1202 to induce cytotoxicity and apoptosis, and inhibit Akt phosphorylation in primary CLL lymphocytes. Methods:We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity of CLL lymphocytes. Primary CLL cells were incubated with serial dilutions of TGR-1202 or GS-1101 for 48 hours and tested for apoptosis by activated caspase-3 and 7AAD staining measured by flow cytometry. After 72 hours of incubation with either compound, CLL cells were evaluated for cytotoxicity using the colorimetric MTS reagent. Phosphorylated Akt (S473) was measured by flow cytometry after one hour of incubation of either compound and ten minutes of incubation with anti-IgM or anti-IgD. Akt phosphorylation was quantified by median fluorescent intensity (MFI). Results:We evaluated TGR-1202 and GS-1101 in CLL lymphocytes collected from seven patients. Five had mutated IGHV, five had 13q deletion or normal cytogenetics determined by fluorescent in situ hybridization, three were ZAP-70 negative, and seven were CD38 negative. IgM expression ranged between 13% and 90%, whereas IgD expression was uniformly elevated. Both TGR-1202 and GS-1101 significantly induced apoptosis (caspase-3+/7AAD+) and cytotoxicity in a dose-dependent manner in concentrations between 0.1 and 25.6 μM (p < 0.05 in pairwise Wilcoxon signed rank tests). There was no significant difference observed between the compounds in terms of induction of apoptosis or cytotoxicity at any of the tested concentrations tested (0.1 – 25.6 μM) except 0.4 μM, where GS-1101 induced more apoptosis and cytotoxicity (median of 18.6 vs. 13.7% caspase-3+/7AAD+; median of 55.2 vs. 48.6% cytotoxicity, p = 0.03 for both, Wilcoxon signed rank test). Incubation with anti-surface immunoglobulin significantly induced Akt phosphorylation compared to media alone (median MFI 1011.5 and 369, respectively, p = 0.03, Wilcoxon rank sum test). The addition of either TGR-1202 or GS-1101 significantly abrogated this effect (p = 0.03 for 0.1, 0.4, and 1.6 uM, Wilcoxon rank sum test) and returned Akt phosphorylation to baseline (p > 0.05 comparing drug treatment to media control, Wilcoxon rank sum test). Conclusions:TGR-1202 is a potent PI3K-δ inhibitor that suppresses Akt phosphorylation and induces apoptosis-dependent cytotoxicity in primary CLL lymphocytes. These effects are comparable to those seen with GS-1101, another PI3K-δ inhibitor that has demonstrated efficacy in CLL clinical trials. Differences seen at the 0.4 μM concentration were not observed at higher or lower concentrations. Evaluating additional CLL patients’ cells would help clarify these findings. Given the improved selectivity for the delta isoform of PI3K seen with TGR-1202 compared to GS-1101, these results suggest that TGR-1202 may have benefit in treating CLL, while inducing fewer off-target effects and toxicities. These findings provide the rationale for future clinical trials evaluating TGR-1202 in CLL. Disclosures:Friedman:Rhizen Pharmaceuticals: Research Funding. Miskin:TG Therapeutics: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals: Employment, Equity Ownership.

차가버섯 추출물의 MMPs 발현 저해 및 HAS-2 발현 증가효과

본 연구에서는 차가버섯 추출물의 주름개선 화장품 원료로의 가능성을 확인하기 위하여, 차가버섯의 70 % 에탄올 추출물을 제조하여, DPPH (1,1-diphenyl-2- picrylhydrazyl) radical 소거능, 엘라스타제 저해능, MMPs (matrix metalloproteinases) 발현 저해 및 HAS-2 (hyaluronan synthase-2) 발현 저해 효능을 측정하였다. 차가버섯 추출물 및 함유성분에 대한 DPPH radical 소거능과 엘라스타제 저해 효능을 측정한 결과, 차가버섯 추출물은 농도 의존적으로 DPPH radical 소거능 및 엘라스타제 저해 효능을 보였으며( $SC_{50}$ = 91 <TEX 91 ${\mu}g$ /mL, $IC_{50}$ = 124 <TE 124 ${\mu}g$ /mL), 함유성분 중 Gallic acid가 가장 우수한 결과를 나타내었다. MMPs 발현능 평가에서, 차가버섯 추출물은 5 ~ 25 ${\mu}g$ /mL의 농도에서 50 ~ 79 % MMP-1 mRNA 발현을 저해하였으며 MMP-2의 발현량을 측정한 결과, 10 ${\mu}g$ /mL에서 약 20 %를 감소시켰다. MMP-9의 발현에서도 5 ~ 25 ${\mu}g$ /mL의 농도에서 54 ~ 70 %의 저해 효과를 나타내었다. 또한 HAS-2의 mRNA 발현량을 측정한 결과, 차가버섯 추출물이 전 농도에서 농도 의존적으로 HAS-2 발현량을 증가시켰다. 상기 결과를 종합해 볼 때 차가버섯 추출물은 주름개선 화장품 소재로서 개발이 가능할 것으로 사료된다. 【In order to investigate the possibility of Inonotus obliquus extract as an active ingredient for wrinkle-care cosmetics, we prepared 70 % ethanolic extract of Inonotus obliquus and measured its DPPH radical scavenging activity and elastase inhibitory activity. We also evaluated the effect of Inonotus obliquus extract on expression of MMPs and HAS-2 in fibroblast and HaCaT cell, respectively. Inonotus Obliquus extract showed DPPH radical scavenging activity and elastase inhibitory activity in a dose-dependant manner ( $SC_{50}$ = 91 <TEX 91 ${\mu}g$ /mL, $IC_{50}$ =124 <TEX 124 ${\mu}g$ /mL). Inonotus obliquus extract inhibited the expression of MMP-1 mRNA 50 ~ 79 % at concentrations of 5 ~ 25 ${\mu}g$ /mL, and reduced the expression of MMP-2 around 20 % at a concentration of 10 ${\mu}g$ /mL. And it also reduced the expression of MMP-9 54 ~ 70 % in tconcentrations of 5 to 25 ${\mu}g$ /mL. Furthermore, Inonotus obliquus extract increased HAS-2 mRNA expression in a dose-dependent manner in concentrations of 5 to 25 ${\mu}g$ /mL without cytotoxicity. These results suggested that Inonotus Obliquus extract could be useful as an active ingredient for wrinkle-care cosmetics.】

일문전(Stephania delavayi Diels.) 메탄올, 부탄올, 에틸아세테이트 분획물의 항산화 및 세포증식억제 효과

본 연구에서는 중국에서 급성 위장염에 대한 약제로 사용되어 오던 일문전의 메탄올 추출물을 이용하여 물, 에틸 아세테이트, 부탄올 층으로 분획한 뒤 항산화 활성과 항암활성을 측정하였다. DPPH 라디칼 소거능은 1,000 ㎍/mL 농도에서 부탄올(75.23%)＞메탄올(68.11%)＞에틸아세테이(63.58%)＞물(50.13%) 순으로 나타났으며 환원력의 경우 용매 분획물의 농도가 증가함에 따라 환원력이 증가하는 경향을 나타내었다. DPPH 라디칼 소거능 및 환원력 모두 부탄올 분획층에서 좋은 활성을 나타내었다. 항암활성 측정결과 메탄올 추출물과 부탄올 분획물, 에틸 아세테이트 분획물에서 MDA-MB-231 cell과 MCF-7 cell에 대한 세포증식억제효과를 나타내었다. 이상의 결과에서 일문전의 용매 분획물은 항암 후보물질로의 가능성이 있을 것으로 사료된다.

Brominated Cyclodipeptides from the Marine SpongeGeodiabarrettias Selective 5-HT Ligands

The brominated cyclodipeptides barettin (cyclo[(6-bromo-8-entryptophan)arginine]) and 8,9-dihydrobarettin (cyclo[(6-bromotryptophan)arginine]) isolated from the marine sponge Geodia barretti have previously been shown to inhibit settlement of barnacle larvae in a dose-dependent manner in concentrations ranging from 0.5 to 25 microM. To further establish the molecular target and mode of action of these compounds, we investigated their affinity to human serotonin receptors. The tryptophan residue in the barettins resembles that of endogenous serotonin [5-hydroxytryptamine]. A selection of human serotonin receptors, including representatives from all subfamilies (1-7), were transfected into HEK-293 cells. Barettin selectively interacted with the serotonin receptors 5-HT2A, 5-HT2C, and 5-HT4 at concentrations close to that of endogenous serotonin, with the corresponding Ki values being 1.93, 0.34, and 1.91 microM, respectively. 8,9-Dihydrobarettin interacted exclusively with the 5-HT2C receptor with a Ki value of 4.63 microM; it failed to show affinity to 5-HT2A and 5-HT4, indicating that the double bond between the tryptophan and arginine residue plays an important role in the interaction with the receptor proteins.

Protective effect of melatonin and pinoline on nitric oxide-induced lipid and protein peroxidation in rat brain homogenates

Nitric oxide (NO) is a physiological neurotransmitter, a mediator of the excitatory neurotransmitter glutamate pathways that regulates several neuroendocrine functions, but excessive NO is toxic by itself and it interacts with superoxide radical (O 2 −) to form the peroxynitrite anion (ONOO −). Using rat brain homogenates, we investigated the effects of melatonin and pinoline in preventing the level of lipid peroxidation (LPO) and carbonyl contents in proteins induced by nitric oxide (NO) which was released by the addition of sodium nitroprusside (SNP). Lipid and protein peroxidation were estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA) concentrations and carbonyl contents, respectively. SNP increased MDA + 4-HDA and carbonyl contents production in brain homogenates in a time and concentration dependent manner. Both, melatonin and pinoline reduced NO-induced LPO and carbonyl contents in a dose-dependent manner in concentrations from 0.03 to 3 mM and 1 to 300 μM, respectively. Under the in vitro conditions of this experiment, both antioxidants were more efficient in limiting SNP protein oxidation than lipid damage.

Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacleBalanus improvisus

We describe the effect of eight different imidazoline/guanidinium compounds on the settlement and metamorphosis of larvae of the barnacle Balanus improvisus. These agents were chosen on the basis of their similar pharmacological classification in vertebrates and their chemical similarity to medetomidine and clonidine, previously described as highly potent settlement inhibitors (nanomolar range). Seven of the tested compounds were found to inhibit settlement in a dose-dependent manner in concentrations ranging from 100 nM to 10 microM without any significant lethal effects. In vertebrate systems these substances have overlapping functions and interact with both alpha-adrenoceptors as well as imidazoline binding sites. Antagonizing experiments using the highly specific alpha(2)-antagonist methoxy-idazoxan or agmatine (the putative endogenous ligand at imidazoline receptors) were performed to discriminate between putative pharmacological mechanisms involved in the inhibition of cyprid settlement. Agmatine was not able to reverse the effect of any of the tested compounds. However, methoxy-idazoxan almost completely abolished the settlement inhibition mediated by guanabenz (alpha(2)-agonist, I(2) ligand), moxonidine (alpha(2)-agonist, I(1) ligand) and tetrahydrozoline (alpha-agonist, I(2) ligand). The actions of cirazoline (alpha(1)-agonist, I(2) ligand) BU 224 (I(2) ligand) and metrazoline (I(2) ligand) were not reversed by treatment with methoxy-idazoxan. These results suggest that the settlement inhibition evoked by the I(2) ligands and alpha(2)-agonists used in this study of the neurologically simple but well-organized barnacle larva is mediated through different physiological targets important in the overall settlement process.

A Novel Specific Bioassay for Serum Human Growth Hormone

Human GH receptor (hGHR) was recently expressed on a Ba/F3 cell line, which is a mouse pro-B cell lymphoma that has been induced to become a cloned cell line (Ba/F3-hGHR). Using a Ba/F3-hGHR cell line, we have established a bioassay for serum hGH. hGH stimulated cell proliferation in a dose-dependent manner in concentrations ranging from 1 ng to 100 ng/mL. Cell proliferation was not influenced by other hormones or growth factors in the bioassay, with the exception of insulin-like growth factor I (IGF-I) and GH binding protein. Free IGF-I significantly stimulated the proliferation of Ba/F3-hGHR cells at concentrations over 25.85 ng/mL in this bioassay system, but serum IGF-I did not stimulate cell proliferation because the sensitivity of cell proliferation was insufficient for free IGF-I in serum. GH binding protein, however, did suppress cell proliferation at the highest concentration (100 ng/mL), but did not at the average concentration (20 ng/mL). Human serum stimulated cell proliferation, which was completely suppressed by anti-GH antibody. The GH bioactivity of serum samples from normal children and patients with non-GH deficient short stature correlated strongly with the serum hGH concentration determined by immunoradiometric assay (IRMA) (r = 0.967, r = 0.924, P < 0.0001, respectively). The ratio of bioactivity/IRMA was 1.01 ± 0.26 in sera from normal children and 1.18 ± 0.24 and 1.00 ± 0.29 at basal values and peak values in GH stimulation tests, respectively, in sera from patients with non-GH deficient short stature. The bioactivity/IRMA ratio for the serum GH bioactivity of a patient who had biologically inactive GH caused by an amino acid substitution was 0.333 ± 0.056 (mean ± sd). In conclusion, we established a new sensitive bioassay for hGH that is specific for hGH somatogenic action and is useful for screening of patients with short stature caused by biologically inactive hGH.

Glucocorticoids Inhibit CD40 Ligand Expression of Peripheral CD4+Lymphocytes

The ligand for CD40 (CD40L) is a type II transmembrane glycoprotein that belongs to the tumor necrosis factor superfamily. CD40L expression on peripheral CD4+cells is increased upon activation and delivers signals to B lymphocytes which constitutively express CD40. We show that dexamethasonein vitroinhibits CD40L expression in a dose-dependent manner in concentrations ranging from 0.1 to 1 mg/mL. Semiquantitative analysis of CD40L mRNA by RT-PCR revealed that this effect was due to inhibition of CD40L transcription. The inhibitory effect of dexamethasone on CD40L expression was reversible and not due to affection of cell viability. Lymphocytes which have been exposed to dexamethasonein vitroretained the ability to express CD40L after incubation in medium alone for 48 h. Dexamethasone also inhibited PMA/ionomycin induced IL-2 and IFN-γ production but not CD25 and CD69 expression. Glucocorticoids may exert their immunosuppressive effect in part by suppression of CD40L. Regulation of CD40L expression is steroid sensitive and may be similar or in part identical with IL-2 and IFN-γ regulation.

Melatonin and vitamin E limit nitric oxide-induced lipid peroxidation in rat brain homogenates

We have investigated the level of lipid peroxidation (LPO) in rat brain homogenates in the presence of nitric oxide (NO) which was released by the addition of sodium nitroprusside (SNP) and compared it with that induced by H 2O 2. We also examined the effect of melatonin and vitamin E on the NO-induced LPO. The concentration of malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) was used as an index of LPO. While both H 2O 2 and SNP increased MDA+4-HDA production in brain homogenates in a concentration-dependent manner, SNP was more potent than H 2O 2 at all concentrations tested. Both melatonin or vitamin E reduced NO-induced LPO in a dose-dependent manner in concentrations ranging from 10 μM to 10 mM. Under the in vitro conditions of this experiment, vitamin E was more efficient than melatonin in limiting NO-induced LPO in rat brain homogenates.

Growth suppression and astrocytic differentiation of glioma cells by interleukin-1

The effect of recombinant human interleukin-1 (rHuIL-1) derivatives on human glioma cell lines was examined in vitro. Five glioma cell lines, U-251 MG, U-373 MG, U-87 MG, A-172, and T98G, were incubated in medium containing 1% fetal calf serum and various concentrations of different type of rHuIL-1: OCT-43 (rHuIL-1 beta), OCT-7000 (rHuIL-1 alpha), and OCT-8000 (rHuIL-1 alpha). The high-affinity IL-1 receptors were expressed in the U-251 MG and U-373 MG cell lines, and rHuIL-1 was found to suppress cell growth and to induce morphological differentiation of these cell lines. Growth inhibition occurred in a dose-dependent manner in concentrations or rHuIL-1 ranging between 1 and 100 ng/ml. Interestingly, rHuIL-1 induced a transient growth of glioma cells shortly after administration, then suppressed cell growth with accompanying elongation of cytoplasmic processes. This unique process of transient growth stimulation followed by growth suppression was parallel to the efficacy of bromodeoxyuridine uptake in the rHuIL-1-treated cells. Concomitantly, accumulation of glial fibrillary acidic protein and cyclic adenosine monophosphate contents was observed in four glioma cell lines. Continuous rHuIL-1 treatment for longer than 30 days elicited irreversible astrocytic terminal differentiation. These results indicate that IL-1 is an effector on the growth regulation of glioma cells, resulting in astrocytic differentiation in vitro.

Interferon-alpha 2b inhibits proliferation of human Tenon's capsule fibroblasts.

To evaluate the potential of interferon-alpha 2b (IFN-alpha) as an antifibrotic treatment after glaucoma filtering surgery, we studied its ability to inhibit mitogen-induced proliferation of human Tenon's capsule fibroblasts in vitro, as shown by [3H]-thymidine incorporation. IFN-alpha inhibited fetal calf serum-induced proliferation in a dose-dependent manner in concentrations ranging from 10 to 10,000 IU/ml (P < 0.001). Inhibition was also found when IFN-alpha was added prior to the mitogen (P = 0.001); this inhibitory effect lasted at least 3 days. We suggest that IFN-alpha deserves further evaluation as a possible agent for the control of ocular fibrosis.

The Effect of Surfactants on Metabolism of Choline Phospholipids in Human Epidermal Keratinocytes in Culture

AbstractThe pathophysiology of surfactant-induced cutaneous primary irritancy most likely involves surfactant interaction with phospholipids of skin cell membranes. The effect of such agents on membrane choline (Ch) phospholipid metabolism was investigated in human epidermal keratinocytes in culture. Cells were prelabeled with [3H]Ch and then treated with four surfactants: linear alkyl benzene sulfonate (LAS), sodium lauryl sulfate (SLS), alkyl ethoxylate sulfate (AEOS-3EO). and TWEEN-20. Media were assayed for release of labeled Ch metabolites. All four surfactants stimulated release of label in a dose-dependent manner in concentrations between 5 × 10−3 and 5 × 10−5 M.

Excitatory junctional responses and glutamate responses at the crayfish neuromuscular junction in the presence of chlorisondamine

At the crayfish neuromuscular junction chlorisondamine reduced the amplitude of both the excitatory junctional potential and the glutamate current in a dose-dependent manner in concentrations above 3 μM, and it is suggested that the drug is a powerful non-competitive antagonist for glutamate. Chlorisondamine did not act presynaptically on the crayfish neuromuscular junction. A double exponential decay of excitatory synaptic currents was observed in the presence of chlorisondamine, suggesting that this drug is an open channel blocker for the excitatory neurotransmitter. The glutamate current tail was prolonged in the presence of chlorisondamine. This prolongation increased with increasing iontophoretic current of glutamate. The rate of recovery from the refractory form of the glutamate receptor to the free reactive one was hardly affected by chlorisondamine. The inhibitory action of chlorisondamine on glutamate responses was voltage-dependent and hyperpolarization reduced the drug action. Chlorisondamine depressed the glutamate current even in Na-free, Ca-rich solution.

Secretory effects of gastrins on isolated perfused porcine pancreas.

The effects of the four main forms of gastrin (component I, gastrin-34, gastrin-17, and gastrin-14) on insulin, glucagon, and exocrine secretion were measured on the isolated perfused porcine pancreas. All gastrins were studied in concentrations ranging from 10(-11) to 10(-8) M. Depending on the glucose concentration in the perfusate, all four gastrins increased insulin or glucagon secretion in a dose-dependent manner in concentrations above 10(-10) M. These concentrations are slightly above the arterial concentrations in normal pig and man, but they correspond to gastrin concentrations measured in patients with achlorhydria and gastrinomas. The exocrine secretion was stimulated by all gastrins in a dose-dependent manner. The lowest concentrations that stimulated flow rate significantly were within the physiologic range, 10(-11) and 10(-10) M. All gastrins induced maximal flow rate at a concentration of 10(-9) M. The sulfated form of gastrin-17 had the greatest efficacy. The results indicate that all gastrins may influence the exocrine secretion under normal conditions and the endocrine secretion in diseases with endogenous hypergastrinemia.