Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

Effects of chelating agents on the cadmium burden of cells in culture

The effects of some new chelating agents on the cadmium burden of CHO cells in culture were investigated. The chelators were sodium-N-(4-methoxybenzyl)-D-glucamine-dithiocarbamate (MeOBG-DTC), sodium-N-benzyl-D-glucaminedithiocarbamate (BG-DTC) and di-isopropylmeso-2,3-dimercapto succinate (DiP-DMSA). The results were compared with the effect of the well known dimercaptopropanol (BAL).The derivates of dithiocarbamate are much less toxic than DiP-DMSA and BAL. All chelators effectively prevent Cd uptake into the cells. Mobilization of intracellular Cd, however, is more effective by the DTC-derivatives than by DiP-DMSA or BAL. Within the cell the major fraction of Cd after 48 hours incubation is found in the nuclei and cytosol and very little in the peroxisomes. The chelating agents remove the metal mostly from nuclei and cytosol. Incubation of the cells with cadmium leads to the induction of a Cd binding protein of an apparent molecular weight of 12500 Da, presumably metallothionein. MeOBG-DTC is more effective in removing the metal from this protein than BG-DTC.Abbreviations MeOBG-DTC Na-N(4-methoxybenzyl)-D-glucaminedithiocarbamate - BG-DTC Na-N-benzyl-D-glucaminedithiocarbamate - DiP-DMSA di-isopropyl-2,3-dimercaptosuccinate - BAL 2,3-dimercaptopropane-1-o1 - Da dalton - MEM minimum essential medium - IU international units - FBS fetal bovine serum - CD unbroken cells and debris - N nuclei - ML mitochondria, and lysosomes - P peroxisomes - HMW high molecular weight - MT metallothionein     

Time course of color induction in the honeybee

Color induction in the honeybee is investigated in color discrimination experiments. An individual bee walks in a dark arena and is trained to a self-luminant stimulus presented from below. In the dual-choice tests the dark background is replaced by a colored induction stimulus. Choice behavior is recorded by TV camera and analyzed by computer. Successive color induction is separated from simultaneous induction by analysis of the walking paths. Only successive color induction occurs. Simultaneous effects are not observed. That is a stimulus acts as a color inducing stimulus only when the bee crosses this stimulus. Thus, the color perceived by a given eye region is found to be dependent on the viewing history, but not on the stimuli presented simultaneously on neighboring parts of the retina. Color induction in the honeybee described in terms of selective sensitivity decrease (adaptation) does not explain all behavioral effects induced by the stimulus. The time course of successive color induction is calculated from the exposure times to the induction stimulus and from the choice behavior. The data suggest that color induction is complete after a few seconds. Photoreceptor adaptation is sufficient to explain the observed time course.     

Role of fibroblast growth factors as inducing agents in early embryonic development

To assess the potential role of a molecule in development we need to know three things: 1) what are the biological activities of the molecule, 2) what is its expression pattern, and 3) what are the consequences of removing it from the embryo? In the case of the FGF family in Xenopus embryos we have quite a lot of information about all three questions. Most members of the family can induce mesoderm from isolated animal caps, thus mimicking the natural “ventral vegetal” inducing signal operative in the blastula. This activity can be exerted on isolated, disaggregated cells and does not involve a change in division rate. When overexpressed from injected mRNA, the activity of FGFs depends largely on whether or not they possess a signal sequence, showing the importance of secretion in the inductive process. In addition to the mesoderm-inducing activity, there are effects of overexpression on whole embryos which lead to a suppression of anterior structures. Three types of FGF have so far been cloned from Xenopus: direct homologs of each of the mammalian types FGF-2 and FGF-3, and eFGF (“embryonic FGF”), which is equidistant in sequence from mammalian FGF-4 and FGF-6. Attempts to find homologs of mammalian FGF-5 and FGF-7 in Xenopus have proved unsuccessful. All three types of Xenopus FGF are expressed in early development. FGF-2 and eFGF are present in the oocyte and fertilized egg, and are thus both available at the time of mesoderm induction. FGF-3 and eFGF are both expressed from the embryonic genome during gastrulation and concentrated in the forming mesoderm. FGF-2 is expressed from the embryonic genome during neurulation in the brain, and a little later in the branchial arch mesenchyme and in the forming myotomes. These expression patterns suggest that there are several functions for the FGFs. The most successful strategy for inhibition of the FGF system has been the use of a dominant negative receptor construct introduced by Kirschner and colleagues. Overexpression of this construct can abolish the FGF responsiveness of animal caps. In whole embryos, the absence of FGF signaling causes a reduction, although not a total ablation, of mesoderm formation. There is also a severe effect on axis formation in which formation of the posterior parts is reduced consequent on an inhibition of invagination and elongation of the dorsal mesoderm. Thus, the present evidence suggests that the FGF system contributes to, although is not solely responsible for, mesoderm induction in vivo. It is also necessary for normal gastrulation movements, particularly in the dorsal mesoderm, and is likely to have several later functions, particularly in development of the central nervous system and the myotomes. © 1994 Wiley-Liss, Inc.     

HSP70 induction by bleomycin metal core analogs

Induction of heat shock protein 70 (HSP70) is known to be effective against various diseases. We are interested in HSP70 induction capability of an antitumor antibiotic bleomycin which produces oxidative stress by iron chelate formation and oxygen activation in a cell. The HSP70 induction activity of bleomycin and its six metal core analogs was examined, and a compound HPH-1Trt of 10 μM was found to induce this protein in a pheochromocytoma cell line and some T cell and monocytic cell lines. Its mechanism is increase of HSP70 mRNA, but higher concentration of this compound showed toxicity. Two new derivatives were then synthesized, and one of them named DHPH-1Trt was shown to have less toxicity and higher HSP70 induction activity. This study would lead to a clue for new HSP70 inducer clinically used in near future.     

Ethylene and IAA interactions in the inhibition of photoperiodic flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis>

It has been shown that both IAA and ethylene application inhibit flower induction in the short-day plant Pharbitis nil. However application of IAA has elevated ethylene production in this plant, as well. Strong enhancement of ethylene production is also correlated with the night-break effect, which completely inhibits flowering. In order to determine what the role of IAA and ethylene is in the photoperiodic flower induction in Pharbitis nil, we measured changes in their levels during inductive and non-inductive photoperiods, and the effects of ethylene biosynthesis and action inhibitors on inhibition of flowering by IAA. Our results have shown that the inhibitory effect of IAA on Pharbitis nil flowering is not physiological but is connected with its effect on ethylene biosynthesis.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

Design,synthesis and molecular modeling study of certain VEGFR-2 inhibitors based on thienopyrimidne scaffold as cancer targeting agents

Different series of novel thieno [2,3-d]pyrimidine derivative (9a-d,10a-f,l,m and 15a-m) were designed, synthesized and evaluated for their ability to in vitro inhibit VEGFR-2 enzyme. Also, the cytotoxicity of the final compounds was tested against a panel of 60 different human cancer cell lines by NCI. The VEGFR-2 enzyme inhibitory results revealed that compounds 10d, 15d and 15 g are among the most active inhibitors with IC₅₀ values of 2.5, 5.48 and 2.27 µM respectively, while compound 10a remarkably showed the highest cell growth inhibition with mean growth inhibition (GI) percent of 31.57%. It exhibited broad spectrum anti-proliferative activity against several NCI cell lines specifically on human breast cancer (T7-47D) and renal cancer (A498) cell lines of 85.5% and 77.65% inhibition respectively. To investigate the mechanistic aspects underlying the activity, further biological studies like flow cytometry cell cycle together with caspase-3 colorimetric assays were carried on compound 10a. Flow cytometric analysis on both MCV-7 and PC-3 cancer cells revealed that it induced cell-cycle arrest in the G0-G1phase and reinforced apoptosis via activation of caspase-3. Furthermore, molecular modeling studies have been carried out to gain further understanding of the binding mode in the active site of VEGFR-2 enzyme and predict pharmacokinetic properties of all the synthesized inhibitors.     

Delayed fluorescence induction in chloroplasts. Irradiance dependence

We have studied the delayed fluorescence in spinach chloroplasts produced 0.5 ms after each of a pair of (sub)-microsecond flashes. We observe an increase in the delayed fluorescence from the second flash relative to that produced by the first. This increase is proportional to the product of the first and second flash irradiances, appearing as an I² dependence if both flashes are increased together. The enhancement is observable at very weak flash levels (roughly 1 photon absorbed/100 PS II centers). If the irradiance of the first flash is increased, but the irradiance of the second held constant, the delayed fluorescence from the second flash is observed to increase, but then to saturate well below the first flash irradiance at which the delayed fluorescence from the first flash itself saturates. For most experiments, the dark time between flashes was 30 ms. If the dark time is varied, the enhancement changes, reaching a half-maximal value for a dark time of approx. 300 μs. The enhancement is stopped by hydroxylamine, but not by gramicidin, valinomycin, DCMU, or mild heating. These experiments are consistent with the notion that there are two different types of Photosystem II centers if we assume that only one type is responsible for the induction we see and has an optical cross-section about 4-times the size of the other type of center.