Effect of the auxin, 2-(2-methyl-4-chloro-phenoxy)propionic acid, on cell cycle regulation in maize embryonic tissues

During seed maturation, cells from embryonic tissues stop division at different phases of the cell cycle. In maize, neither these phases nor the effect of exogenous auxin on them are known. Disinfected whole maize ( Zea mays L. Mexican commercial hybrid H30) seeds or sectioned embryonic axes were incubated in Murashige and Skoog medium, with or without 2-(2-methyl-4-chlorophenoxy)propionic acid (MCPP), a synthetic auxin. For some in vitro experiments, radioactive [³H]-thymidine was also added. After the stated incubation period, meristems of mesocotyl, primary and seminal roots from embryonic axes were dissected, fixed, and analyzed under a microscope. The percentage of mitotic indices was recorded. In the labeling experiments, labeled and non-labeled percentage of mitotic figures (MI %) were determined. It was found that cell division is a programmed event in the meristematic tissues of maize embryonic axes. Populations of cells entering cell division were obseved during the germination process. The mesocotyl was the first tissue to divide, followed by seminal and primary roots.
Meristematic cells from dry embryos are arrested during the G₂ and G₁ phases of the cell cycle. MCPP has a differential effect, stimulating G₂ cells to enter cell division. It is concluded that MCPP might regulate the cell cycle at specific points.     

The antiinflammatory effects of adenosine receptor agonists on the carrageenan-induced pleural inflammatory response in rats

Adenosine and adenosine receptor agonists have a variety of inhibitory effects on the generation of inflammatory mediators by neutrophils and other cell types. In human neutrophils stimulated with the chemotactic peptide FMLP, adenosine agonists inhibit O2- generation and degranulation. Because these findings suggest that the agonists may have potential as antiinflammatory agents, several compounds were evaluated for effects on the exudative and cellular phases of carrageenan-induced pleural inflammation in rats. All of the agonists tested inhibited both parameters of the inflammatory response. Inhibition appeared to correlate better with binding to the A1 than to the A2 receptor and was reversible by a known adenosine receptor antagonist, 8-phenyltheophylline. In mechanistic studies, R-N-(1-methyl-2-phenylethyl)adenosine, a standard A1 selective agonist, reversed the drop in circulating neutrophil count that occurs after injection of carrageenan. These results suggest that the agonists may prevent cell emigration by inhibiting adhesion to the endothelium or diapedesis. In addition (R)-N-(1-methyl-2-phenylethyl)adenosine had weak inhibitory effects on superoxide production by FMLP-stimulated rat neutrophils. Control studies showed that the effects of the agonists were not the result of agonist-induced hypotension nor corticosterone production by the adrenal glands. These findings indicate that adenosine receptor agonists are effective new pharmacologic tools for the study of inflammatory processes.     

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488 M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1_G465R) and CMAC(hOCT1_R488M). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [³H] MMP⁺ (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.     

Three Distinct Phases of Isoprene Formation during Growth and Sporulation of Bacillus subtilis

During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.     

Identification of oviposition attractants for the sandfly Lutzomyia longipalpis (Diptera: Psychodidae) in volatiles of faeces from vertebrates

Abstract. Extracts of volatiles from rabbit and chicken faeces preferentially attracted gravid sandflies, Lutzomyia longipalpis (Lutz and Neiva), in an oviposition bioassay. In electrophysiology experiments, the same extracts selectively stimulated two olfactory cells while inhibiting another in ascoid sensilla on the antennae of these flies. Analysis of faeces volatiles by gas chromatography linked to ascoid sensillum recording revealed two early eluting electrophysiologically active components of rabbit faeces. These active compounds were identified in both rabbit and chicken faeces volatile extracts by gas chromatography -mass spectrometry as hexanal and 2-methyl-2-butanol. Hexanal stimulated one cell type and inhibited another, whereas 2-methyl-2-butanol stimulated a third cell type. A 1:l mixture of synthetic hexanal and 2-methyl-2-butanol elicited the same targeted oviposition response from gravid females on the treatment septum of the bioassay as did the total volatile extract of rabbit or chicken faeces.
The monoterpenes α(+)-pinene (plus some optical and positional isomers) and a-terpinene activated a separate cell type, whereas benzaldehyde stimulated the same receptor as hexanal, but with a higher threshold. Furthermore, an olfactory cell selectively tuned to the perception of the male sex pheromone of this species was also found in the ascoid sensillum.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

Biological Effects of Cytokinin Antagonists 7-(Pentylamino) and 7-(Benzylamino)-3-Methylpyrazolo(4,3-d)Pyrimidines on Suspension-cultured Tobacco Cells

We have studied the biological effects of two structural analogs of cytokinins, 3-methyl-7-(pentylamino)pyrazolo(4,3-d)pyrimidine and 3-methyl-7-(benzylamino)pyrazolo(4,3-d)pyrimidine, on tobacco cell suspension cultures. These two cytokinin analogs are highly inhibitory to cytokinin-autonomous and cytokinin-requiring tobacco cells. The growth inhibitory effect is markedly antagonized by benzyladenine, but not by adenine. Cell suspensions of tobacco cells of a cytokinin-autonomous strain become cytokinin-dependent in the presence of 0.1 micromolar 3-methyl-7-(benzylamino)pyrazolo(4,3-d)pyrimidine. It is also demonstrated that growth inhibition of cell suspension cultures is accompanied by cell death and that only dividing cells are sensitive to this cytotoxic effect.     

Formation of edulinine and furoquinoline alkaloids from quinoline derivatives by cell suspension cultures of Ruta graveolens

The formation of furoquinoline alkaloids and of edulinine, elaborated by cell suspension cultures of Ruta graveolens, was found to occur by way of 4-hydroxy-2-quinolone. Other substrates transformed to furoquinolines included 4-hydroxy- and 4-methoxy-3-(3-methyl-2-butenyl)-2-quinolone, known earlier as natural precursors in studies with whole plants. Involvement of dictamnine as a natural precursor of 8-methoxydictamnine (γ-fagarine) and skimmianine was proved using ¹⁴C-labelled compounds. Edulinine in the cell suspensions was formed from such substrates as 4-hydroxy-N-methyl-2-quinolone, 4-hydroxy-3-(3-methyl-2-butenyl)-N-methyl-2-quinolone and its 4- methyl ether; this is probably the natural biosynthetic sequence. Changes in alkaloid yields were noted upon prolonged subculturing.     

传粉榕小蜂与非传粉小蜂间寄主识别行为的趋同进化

在高度专性传粉的榕树-榕小蜂互惠共生系统中普遍存在着一些非传粉小蜂,它们中的一些种类进入果腔后也能为榕树传粉,且在形态和物候上已与传粉榕小蜂发生了趋同进化。但其寄主识别行为是否也与传粉榕小蜂发生了趋同进化还不得而知。我们在西双版纳选择了钝叶榕(Ficuscurtipes)及其3种进果繁殖小蜂开展了相关的行为实验。3种小蜂中,1种是钝叶榕的专性传粉榕小蜂(Eupristina sp.),另外2种是寄居性非传粉小蜂(杨氏金小蜂Diaziellayangi和Lipothymus sp.),这2种非传粉小蜂进入果腔后也像传粉榕小蜂那样为钝叶榕传粉。我们以钝叶榕不同发育时期的榕果及这3种小蜂为材料,采用Y型嗅觉仪观察了这3种小蜂对各发育时期榕果和信息化学物质6-甲基-5-庚烯-2-醇、6-甲基-5-庚烯-2-酮及这2种化合物的混合物的选择行为。结果表明,当提供雌花期榕果与其他发育时期榕果和空气对照供这3种小蜂选择时,它们均显著地偏向于选择雌花期榕果;当提供雄花期榕果与其他发育时期榕果和空气对照供这3种小蜂选择时,它们均显著地偏向于选择其他发育时期榕果和空气对照,即都会避开雄花期榕果;此外,这3种小蜂均对钝叶榕雌花期果释放的一种主要化合物6-甲基-5-庚烯-2-醇的同一剂量(1μL)表现出显著的偏好。这一结果为传粉榕小蜂与非传粉小蜂间的寄主识别行为趋同进化的假说提供了证据。     

Morphology of the bovine Sertoli cell during the spermatogenetic cycle

Summary The morphology of the bovine Sertoli cell was studied during 6 different phases of the spermatogenetic cycle. Tubular dimensions do not vary significantly during the phases. Sertoli cells occupy 27.0% (phase 4) to 38.4% (phase 8) of the tubular epithelium. Sertoli cells of phase 1 are approximately 20% larger than during the other phases. 30–35% of Sertoli cell volume consists of organelles. Mitochondrial (about 5.0%) and nuclear (about 5.7%) volume densities remain remarkably stable during the cycle, irrespective of changes in Sertoli cell size. Phagocytic capacity of bovine Sertoli cells is only moderate. Elimination of excess spermatid cytoplasm occurs to a large extent prior to spermiation. The majority of spermatid residual bodies undergoes autolytic decay while attached to the Sertoli cell apical surface. Aggregates of densely packed cisternae of the smooth endoplasmic reticulum (ER) located in a basal position and associated with the acrosome-phase and maturation-phase spermatids contribute between 14 and 17% to Sertoli cell volume. During phase 3 the ER pinches off a large number of small, smooth-walled vesicles filled with flocculent content. The contact area between Sertoli cells and other tubular constituents changes considerably during the different phases. It is concluded that the blood-testis barrier is particularly impassable during phases 1 and 8. A lipid cycle does not exist in the bovine testicular tubular epithelium.     

Branched alkanes and other apolar compounds produced by the cyanobacterium Microcoleus vaginatus from the Negev desert

Gas chromatography-mass spectrometry on serially coupled capillary columns with different polarity of stationary phases showed that the soil cyanobacterium Microcoleus vaginatus from the Negev desert produces an unusual mixture of 4 normal and more than 60 branched alkanes, as well as a number of fatty acids, cyclic and unsaturated hydrocarbons, aldehydes, alcohols, and ketones. The dominant compounds were heptadecane (12%), 7-methylheptadecane (7.8%), hexadecanoic acid (6.5%), (Z)-9-hexadecenoic acid (5.6%), 4-ethyl-2,2,6,6-tetramethylheptane (2.8%), (Z)-9-octadecenoic acid (2.8%), and 4-methyl-5-propylnonane (2.7%).     

In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa.

In the present paper we show that extracts from Aegle marmelos Correa are able to inhibit the in vitro proliferation of human tumor cell lines, including the leukemic K562, T-lymphoid Jurkat, B-lymphoid Raji, erythroleukemic HEL, melanoma Colo38, and breast cancer MCF7 and MDA-MB-231 cell lines. Molecules present within the studied Aegle marmelos C. extracts were identified by gas-chromatography/mass-spectrometry analysis; three derivatives (butyl p-tolyl sulfide, 6-methyl-4-chromanone and butylated hydroxyanisole) were found to exhibit strong activity in inhibiting in vitro cell growth of human K562 cells. The antiproliferative activity of these compounds was found to be comparable to that of known antitumor agents, including cisplatin, chromomycin, cytosine arabinoside and 5-fluorouracil. In addition, the antiproliferative activity of butyl-p-tolyl sulfide, 6-methyl-4-chromanone and 5-methoxypsolaren was associated to activation of the differentiation pattern of K562 cells.     

Synthesis and structure-activity relationship of a novel sulfone series of TNF-alpha converting enzyme inhibitors

Replacement of the amide functionality in IM491 (N-hydroxy-(5S,6S)-1-methyl-6-[[4-(2-methyl-4-quinolinylmethoxy)anilinyl]carbonyl]5-piperidinecarboxamide) with a sulfonyl group led to a new series of alpha,beta-cyclic and beta,beta-cyclic gamma-sulfonyl hydroxamic acids, which were potent TNF-alpha converting enzyme (TACE) inhibitors. Among them, inhibitor 4b (N-hydroxy-(4S,5S)-1-methyl-5-[[4-(2-methyl-4-quinolinylmethoxy)phenyl]sulfonylmethyl]-4-pyrrolidinecarboxamide) exhibited IC50 values of < 1 nM and 180 nM in porcine TACE (pTACE) and cell assays, respectively, with excellent selectivity over MMP-1, -2, -9 and -13 and was orally bioavailable with an F value of 46% in mice.     

Synchronization of the cell cycle using Lovastatin

Synchronization by Lovastatin arrests many cell types reversibly in the G1 phase of the cell cycle. Here we show that Lovastatin (10 µM) mediates cell cycle arrest in human breast cancer cells, MCF-7 and MDA-MB-231, where 85% of cells accumulate in the G1 phase of the cell cycle. Addition of mevalonate (at 100X the Lovastatin concentration) releases the cells from the G1 arrest and allows for synchronous entry into late G1, S and G2/M phases of the cell cycle. The expressions of different cyclins as a marker for different phases of the cell cycle are detected by western blot analysis and indicative of synchronous transition into each of cell cycle phases following the initial G1 arrest. Due to its level of synchrony and high yield of synchronous populations of cells, Lovastatin method of cell synchronization can be used for examining gene expression patterns in a variety of different cell lines.     

Estimation of the levels of calcium and other electrolytes during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum

A method is described for measuring calcium and other electrolyte levels during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum. Muscles are immersed in a solution of 160 mM Tris-Cl containing 10 mM lanthanum for 30 min to reduce the contribution of extracellularly bound cations, while the temperature is reduced to 4°C to prevent active cation fluxes. The muscle cells gained calcium and lost magnesium during the phasic and tonic phases of contractions by the muscarinic agent, CD (cis-2-methyl-4-dimethylaminomethyl-1, 3-dioxolane methiodide) and during the tonic phase of contractions by KCl. Muscles lost potassium during contractions to CD, but gained potassium during contractions to 60 mM KCl. The potassium lost during contractions to CD was regained slowly over a period of 30 min when the CD was washed out. This recovery corresponded to the rate of recovery of spontaneous activity and normal responsiveness of the muscle to further doses of stimulants. Calcium levels were reduced rapidly to below normal levels on washout of the CD and were regained in a similar time course to that of potassium. Sodium levels were not significantly changed with either stimulant.     

Inhibitory effect of the compounds from the water extract of Curcuma longa on the production of PGE2 and NO in a macrophage cell line stimulated by LPS

ABSTRACT

We wished to search for the compounds contributing to the anti-inflammatory effects of the water extract of Curcuma longa (WEC). WEC was fractioned and the fractions were evaluated with regard to their inhibitory effect on the production of nitric oxide (NO) from the macrophage cell line stimulated by lipopolysaccharide. Compounds in the active fractions were isolated and identified. One isolated compound was identified as new: (6S)-2-methyl-5-hydroxy-6-(3-hydroxy-4-methylphenyl)-2-heptene-4-one (1). Four isolated compounds were identified as known: (6S)-2-methyl-6-(4-hydroxyphenyl)-2-heptene-4-one (4), bisabolone-4-one (5), curcumenone (6), and turmeronol A (8). Three isolated compounds were not identified their stereostructures but their planar structures: 2-methyl-6-(4-hydroxymethyl-phenyl)-2-heptene-4-one (2), 2-methyl-6-(2,3-epoxy-4-methyl-4-cyclohexene)-2-heptene (3), and 4-methylene-5-hydroxybisabola-2,10-diene-9-one (7). Compounds 1, 4, 7 and 8 inhibited production of prostaglandin E2 and NO. Others inhibited NO production only. These results (at least in part) show the active compounds contributing to the anti-inflammatory effects of WEC, and may be useful for elucidating its various beneficial physiologic effects.     

Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP+) cytotoxicity

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.     

Uptake of 1-Methyl-4-Phenylpyridinium and Dopamine in the Mouse Brain Cell Nuclei

Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP⁺), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [³H]-MPP⁺ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [³H]MPP⁺. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP⁺ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.     

Induction of cell arrest in G2: structural specificity of trigonelline

Synthetic analogues of N-methyl nicotinic acid, trigonelline, were prepared to test the structural features necessary for the induction of cellular arrest in G2 in Pisum sativum. Analogues that (1) were regioisomers of trigonelline, (2) possessed different 1,3-substituents, and (3) contained additional substituents on the pyridine ring were tested for their ability to induce cell arrest in G2 and to antagonize trigonelline induced arrest in G2. Only N-methyi-3-quinoline-carboxylic acid and 1-methyl nicotinamide induced cell arrest in G2, and 1-methyl-4-pyridine carboxylic acid and 1-methyl-2-pyridine carboxylic acid were effective trigonelline antagonists. These data further support a specific role for trigonelline in the induction of cell arrest in G2.     

Preparative enantiomeric separation of new aromatase inhibitors by HPLC on polysaccharide-based chiral stationary phases: Determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis

The development of high-performance liquid chromatography methods on polysaccharide-based stationary phases (cellulose or amylose derivatives) has permitted preparative enantioseparations of various 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzoxazol-2(3H)-one and 6-[1(imidazol-1-yl)-1-phenylmethyl]-3-methyl-1,3-benzothiazol-2(3H)-one, aromatase inhibitors, with satisfactory yields. Analytical enantioseparation methods using both UV and evaporative light-scattering detection (ELSD) were validated to determine the enantiomeric purity of these compounds. Using UV detection, linear calibration curves in the range from 4 x 10(-6) to 4.8 x 10(-4) M range were obtained; repeatability, limits of detection (LD), and quantification (LQ) were determined: LD varied, for the various solutes, from 1 to 80 microg/l and from 2.05 to 10.05 mg/l with UV detection and ELSD, respectively. Single-crystal X-ray analysis was successful in determining the absolute configuration of the individual enantiomers. The relationship between retention order and absolute configuration of the enantiomers was established.