Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.     

Fluorescence quenching studies of Trp repressor-operator interaction

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern–Volmer quenching constant equal to about 2.0–2.3 M^–1. The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern–Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern–Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M^–1. The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.     

内源性一氧化氮在内毒素引起的肺动脉高压和肺损伤中的作用

本实验观察了家兔静脉内注入内毒素的主要成分脂多糖(LPS)后平均动脉血压(MAP)、肺动脉压(PAP)及入、出肺血NO含量的变化,并观察了静脉内预注入NO生成抑制剂Nω-硝基-L-精氨酸(L-NNA)及诱生型NO生成抑制剂氨基胍(AG)后PAP和肺损伤的变化.结果观察到:家兔LPS注入后,MAP均明显下降,LPS注入后0.5、1、1.5、2h PAP明显增高(P<0.05).LPS注入后PAP的高峰期(1h)入肺血NO含量明显降低,出肺血NO无明显变化.与对照组相比,LPS注入后3h出肺血NO含量和5h入、出肺血NO含量均明显增多.相关分析表明,兔LPS注入前和LPS注入后1h PAP与入肺血NO含量呈明显的负相关,而LPS注入后 3h和5h两者相关不明显.静脉预注入L-NNA后,LPS处理组的动物PAP明显增高,入、出肺血丙二醛(MDA)含量也明显增高,动物生存率明显降低.肺组织光镜下可见肺萎陷和小血管淤血加重,白细胞明显增加.静脉预注入AG后,LPS处理组的动物MAP在3～5h明显增高,此时PAP无明显改变,但5h时血中MDA含量明显减低,5h时与LPS组相比肺萎陷和小血管淤血减轻,白细胞也明显减少.以上结果提示,内毒素入血后较早期阶段可出现PAP的升高,此时入肺血NO的减少是参与肺动脉压增高(PAH)的机制之一.家兔内毒素进入血后较早期阶段NO对减轻内毒素引起的PAH和肺损伤起重要作用,而较晚的时期当诱生型NO合酶(iNOS)诱生后释放的NO则参与内毒素引起的肺组织炎症反应和肺损伤.     

Some Characteristics of Red Light-induced Substance(s) against Botrytis cinerea Produced in Broad Bean Leaflets

The red light-induced antifungal substance(s) produced in broad bean was of relatively high molecular weight, water soluble, heat stable and fungi specific. Cellulose thin layer chromatography (TLC) of infection droplets of Botrytis cinerea or water droplets without spores of B. cinerea, recovered from inoculated broad bean leaflets kept under red light for 48 h, displayed inhibition zones at approximate Rf values of 0.0 and 0.6. Inhibition zones observed in cellulose TLC of water droplets were relatively faint compared to those of infection droplets. In a time-course study of accumulation of the antifungal substance(s), antifungal activity in both water and infection droplets recovered from red light irradiated broad bean leaflets occurred after 24 h irradiation. However, the antifungal activity in infection droplets was significantly higher than in water droplets. The antifungal substance(s) was less active against Botrytis fabae than B. cinerea.     

Analysis of DNA polymorphism in a relict Uralian species,large-flowered foxglove (<Emphasis Type="Italic">Digitalis grandiflora</Emphasis> Mill.), using RAPD and ISSR markers

Genetic polymorphism of the Uralian relict plant species, large-flowered foxglove Digitalis grandiflora Mill. (family Scrophulariaceae), was examined using RAPD and ISSR techniques. A total of 149 RAPD and 74ISSR markers were tested. The indices characterizing polymorphism and genetic diversity were calculated. The data obtained pointed to a high level of genetic variation of D. grandiflora (P ₉₅ = 65%). The cenopopulation examined was weakly differentiated with most of genetic diversity accounted by within-population differentiation.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

Changes in Antioxidant Defense Enzymes after d-amphetamine Exposure: Implications as an Animal Model of Mania

Studies have demonstrated that oxidative stress is associated with amphetamine-induced neurotoxicity, but little is known about the adaptations of antioxidant enzymes in the brain after amphetamine exposure. We studied the effects of acute and chronic amphetamine administration on superoxide dismutase (SOD) and catalase (CAT) activity, in a rodent model of mania. Male Wistar rats received either a single IP injection of d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle (acute treatment). In the chronic treatment rats received a daily IP injection of either d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle for 7 days. Locomotor behavior was assessed using the open field test. SOD and CAT activities were measured in the prefrontal cortex, hippocampus, and striatum. Acute and to a greater extent chronic amphetamine treatment increased locomotor behavior and affected SOD and CAT activities in the prefrontal cortex, hippocampus and striatum. Our findings suggest that amphetamine exposure is associated with an imbalance between SOD and CAT activity in the prefrontal cortex, hippocampus and striatum.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models

Cytotechnology - Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity...     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.