Anticoccidial drugs: Effects on infectivity and survival intracellularly of Eimeria tenella sporozoites

The effects of various anticoccidial drugs on extracellular and intracellular sporozoites were studied in cell culture and in chickens. Treatment of freshly excysted, extracellular sporozoites of Eimeria tenella for 18 hr with monensin, decoquinate, or robenidine at 100 ppm had no effect on oocyst production 7–10 days after the sporozoites were rinsed free of drugs and fed to chickens. Treatment of cultures of E. tenella in chick kidney cell monolayers with monensin (0.001 μg/ml), decoquinate (0.01 μg/ml), zoalene (20.0 μg/ml), or robenidine (0.01 μg/ml) had no effect on intracellular sporozoites at 4 hr following introduction of sporozoites and drugs into the culture. A significant reduction of intracellular parasites occurred at 24 hr in the cultures treated with monensin or zoalene. Remaining intracellular sporozoites in monensin-treated cultures were morphologically abnormal or degenerate, while sporozoites in other cultures appeared normal. The number and condition of sporozoites in the nontreated cultures were unchanged at 24 hr postinoculation. These results indicate that sporozoites undergo changes subsequent to penetration of host cells that render them susceptible to drug action.     

The influence of prolactin (PRL) on progesterone secretion by porcine luteal cells in vitro

Porcine luteal cells were obtained from corpora lutea on the 5th, 13th and 17th days of the estrous cycle. The cells were suspended at a concentration of 5 × 10⁴ cells/ml in Eagle's medium with 2% human serum albumin. These cells were incubated with or without 0.01, 0.1, 1 or 10 μg/ml porcine prolactin. The amount of progesterone in cultures was estimated by a radio-immunological method after 30 min, 3 h and 6 h of culturing.Luteal cells obtained on the 5th day of the estrous cycle and incubated without prolactin secreted 71.24 ± 21.91 ng progesterone/ml of medium, whereas under the influence of prolactin at 0.01, 0.1, 1 and 10 μg/ml, 39.06 ± 13.33, 44.31 ± 12.69, 44.88 ± 16.85 and 51.62 ± 15.01 ng progesterone/ml (P<0.01) were secreted. Luteal cells from the 13th day of the estrous cycle incubated without prolactin secreted on average 70.72 ± 9.21 ng progesterone/ml of medium, whereas under the influence of different prolactin doses 50.75 ± 8.52, 46.54 ± 7.13, 43.30 ± 6.78 and 41.68 ± 7.21 ng progesterone/ml (P<0.01) were secreted.Prolactin did not change progesterone secretion by luteal cells obtained on the 17th day of the estrous cycle. An influence of the incubation time on progesterone secretion by these cells was observed: after 30 min of incubation the cells secreted 8.83 ± 2.95 ng/ml, after 3 h 8.12 ± 2.57 ng/ml and after 6 h 6.86 ± 1.91 ng/ml, irrespective of the amount of PRL added.The results suggest that prolactin plays a role in the luteolysis of the corpus luteum.     

Rat adipocyte lipoprotein lipase activity is inhibited by theophylline and stimulated by inosine through adenosine-independent mechanisms

Incubation of rat adipose tissue or isolated rat adipocytes with high (50 mM) but not with low concentrations (0.5 mM) of theophylline results in a decrease of lipoprotein lipase (LPL) activity. This effect is not altered by the addition of adenosine deaminase, indicating that the decrease of adipose LPL activity by theophylline is not due to the competition of theophylline with adenosine. On the contrary, incubation of isolated fat cells with adenosine (0.1 – 100 μM) results in an increase of the intracellular form of LPL activity. As this effect is also observed in cells incubated with adenosine deaminase (40 mU/ml) or with inosine (0.1 – 100 μM) but not in cells incubated with the adenosine analog N⁶-phenylisopropyladenosine, it is concluded that the increase in the intracellular form of LPL found after incubation with adenosine is not due to adenosine $\text{per se}$ but to inosine generated from the breakdown of endogenous adenosine by adenosine deaminase.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。     

Phosphocreatine in Ehrlich ascites tumor cells detected by noninvasive 31P NMR spectroscopy

³¹P NMR spectra of intact Ehrlich ascites tumor cells of high phosphorylation potential reveal a well-defined resonance peak assignable to phosphocreatine, corresponding to 2.3 μmoles/ml cell H₂O in adenosine-treated cells containing 5.2 μmoles ATP/ml. The NMR spectrum of Ehrlich cells incubated with iodoacetate and glucose indicates depletion of phosphocreatine and ATP to undetectable levels and substantial accumulation of fructose-1,6-bisphosphate. From the difference between the chemical shifts of internal P_i and phosphocreatine resonances, the intracellular pH was estimated to be 7.1 ± 0.1 in protein-synthesizing cells suspended in a medium of pH 7.4 at 10°C. Ehrlich cells are unable to transfer the labeled amidine group from L-(guanidino-¹⁴C)-arginine to the large intracellular glycine pool to form labeled guanidinoacetate, the demethylated precursor of creatine. These results imply that the synthesis of phosphocreatine in ATP-rich Ehrlich cells is limited primarily by the extracellular free creatine supply, the extent of which depends upon the degree of cachectic perturbation of energy and nitrogen metabolism of the tumor-bearing host.     

Eimeria tenella: accumulation and retention of anticoccidial ionophores by extracellular sporozoites

Extracellular sporozoites of Eimeria tenella were incubated at either 25 or 40 C in the presence of ¹⁴C-lasalocid or ¹⁴C-narasin, both anticoccidial ionophores. Liquid scintillation analysis shows that both ionophores are accumulated by the sporozoites outside their host cell. The relative degree of retention was significantly different for the two incubation temperatures and the concentration of lasalocid retained was consistently greater than that of narasin.     

桦木酸对人胃癌MGC-803细胞增殖的影响

目的:探究不同浓度桦木酸对人胃癌MGC-803细胞增殖的影响.方法:将人胃癌MGC-803细胞分成4组,每组设置3个复孔,对照组细胞为加入浓度为0 μg/ml的桦木酸实验组细胞分别加入终浓度为10、20、30 μg/ml的桦木酸,各组细胞在含5％的CO2培养箱中孵育48 h后,使用吉姆萨染色法和台盼蓝拒染法检测桦木酸对...     

The influence of the fatty acid composition of Acholeplasma laidlawii membranes on the growth inhibitory activity of narasin,a polyether ionophorous antibiotic

Narasin, a polyether ionophorous antibiotic capable of acting as a transmembrane carrier of cations, has a growth inhibitory effect on Acholeplasma laidlawii, permitting only 20% survival when present at 0.1 μg/ml in an undefined growth nutrient or fatty acid-deficient nutrient supplemented only with palmitic acid. When A. laidlawii is propagated in fatty acid-deficient nutrient supplemented with linoleic acid, however, the organisms become 40 times more sensitive to the growth inhibitory effect of this antibiotic. The actual fatty acid compositions of the membranes would indicate that a higher degree of unsaturation enhances ionophore activity.     

Enzymatic conversion of dehydrodivanillin to vanillin by an anaerobic recombinant FE7

Enzymatic degradation of dehydrodivanillin (DDV) was studied using high performance liquid chromatography (HPLC) with an anaerobic DDV-degrading recombinant FE7 under both aerobic and anaerobic conditions. When 200 mg of FE7 cells were mixed with 40 μg DDV in 1 ml phosphate buffer (0.01 M, pH 7.0) and 10 mM mercaptoethanol and incubated at 37°C for 24 h under an O₂-free CO₂ atmosphere, about 20 μg of DDV was decomposed. Only 12 μg DDV could be degraded when the same reaction was done under aerobic conditions, suggesting that the reaction occurs more easily under anaerobic than aerobic conditions. Enzymatic degradation of DDV was performed using a cell-free extract as a crude enzyme solution under aerobic conditions in a similar way. A reaction product detected and analysed by thin layer, high performance liquid and gas chromatographies and mass spectrometry was found to be vanillin from enzymatic reaction mixture. This enzymatic activity was not detected in either the culture supernatant or the heat-inactivated control. These results suggest that there may be an intracellular enzyme system which is involved in the conversion of DDV to vanillin. This is the first report to study the enzymatic degradation of DDV by anaerobes.     

Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro

The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined in vitro. Lung, liver, and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations were measured as indices of induced membrane peroxidative damage. Homogenates of brain, lung, and liver were incubated with LPS at concentrations of either 1, 10, 50, 200, or 400μg/ml for 1 h and, in another study, LPS at a concentration of 400 μg/ml for either 0, 15, 30, or 60 min. Melatonin at increasing concentrations from 0.01–3 mM either alone or together with LPS (400μg/ml) was used. Liver, brain, and lung MDA + 4-HDA levels increased after LPS at concentrations of 10, 50, 200 or 400 μg/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after tissues were incubated with an LPS concentration of 400 μg/ml for 60 min; in liver and lung this effect was totally suppressed by melatonin and partially suppressed in brain in a concentration-dependent manner. In addition, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM, and in liver at 0.1 to 3 mM; in all cases, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration of melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin.     

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium,pH and membrane integrity in mammalian cells

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD₅₀ 110 μg/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca²⁺ entry by toxin (0.1–10 μg/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37°C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 μg/ml. No progressive Ca²⁺ entry was observed on toxin addition, instead a rapid rise in intracellular Ca²⁺, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 μg/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.     

Effect of prostaglandin producing modulators on in vitro growth of buffalo uterine epithelial cells

Studies were conducted to examine the effect of seven prostaglandin producing modulators on the in vitro growth of uterine epithelial cells in buffalo. The uterine epithelial cells isolated from slaughtered buffaloes were cultured in media containing a) Lipopolysaccaride (LPS): 0, 0.01, 0.1, 1, 10 and 100 μg/ml, b) linoleic acid: 0, 0.01, 0.1, 1, 10 and 100 μg/ml, c) linolenic acid: 0, 0.01, 0.1, 1, 10 and 100 μg/ml, d) oxytocin: 0, 10, 100, 1,000, 10,000 and 100,000 nm, e) tumor necrosis factor-α (TNF-α): 0, 0.05, 0.5, 1, 2.5 and 5 nm, f) progesterone: 0.1, 10, 25, 50, 75 and 100 nM, and g) estradiol: 0, 2.5, 5, 10, 20 and 50 nM. The control medium consisted of RPMI-1640 plus 10% bovine fetal serum. The growth of uterine epithelial were measured in terms of viability, cell number increment and monolayer formation. Results suggested that the growth of uterine epithelial cells were significantly (P < 0.05) higher in media containing 10 μg/ml, 10 μg/ml, 1 nm and 10 μg/ml linoleic acid, linolenic acid, TNF-α and LPS, respectively compared to control and lower doses used. Progesterone, estradiol and oxytocin did not significantly (P > 0.05) increase the growth of uterine epithelial cells. In conclusion, the growth of uterine epithelial cells increased when exposed to modulators in the order of linoleic acid ≥ linolenic acid ≥ LPS ≥ TNF-α > progesterone > estrogen > oxytocin.     

DNA strand scission by the antitumor protein neocarzinostatin

The antibiotic protein, neocarzinostatin, induces the scission of DNA strands $\text{in vivo}$ and $\text{in vitro}$. HeLa cell DNA prelabelled with [¹⁴C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [³H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.     

Interferon-gamma inhibits the intrahepatocytic development of malaria parasites in vitro

In this study, we examined the activity of recombinant interferon (IFN)-gamma against Plasmodium berghei exoerythrocytic forms (EEF) grown in vitro within the highly differentiated human hepatoma cell line HEPG2. We assayed the effect of IFN-gamma on parasite growth by DNA hybridization using a P. berghei specific DNA probe. The specific activity of IFN-gamma against EEF is very high, and depends upon the time of lymphokine addition. When IFN-gamma is added to HEPG2 cells containing intracellular EEF, 6 hr after sporozoite invasion, parasite DNA replication is inhibited by approximately 75% at 10(3) U/ml and 50% at 1 U/ml. This treatment can either abolish or greatly reduce the infectivity of EEF for mice. When added earlier, 3 hr after completion of sporozoite invasion, IFN-gamma inhibits parasite replication to an even greater degree. The highest levels of inhibition were obtained when IFN-gamma was added 6 hr prior to sporozoite invasion (100% inhibition at 10(2) U/ml, approximately 55% inhibition at 0.1 U/ml, and 17% inhibition at 0.001 U/ml). We found that HEPG2 cells express approximately 44,000 surface receptors for IFN-gamma. These data are consistent with the view that IFN-gamma exerts its antimalarial activity by binding to surface receptors on hepatocytes and inducing intracellular changes unfavorable for parasite development. Tryptophan starvation does not appear to be involved in this process. These findings also support the idea that IFN-gamma, released from immune T cells upon encountering sporozoite antigen, may be an important effector mechanism in sterile immunity to sporozoite challenge.     

Determination of caffeic acid in rabbit plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method involving UV detection was developed for determination of caffeic acid in rabbit plasma. A Lichrosphere CN column (250 mm × 4 mm I.D., 5 μm) was used as the stationary phase and the mobile phase consisted of 2% acetic acid solution at a flow-rate of 1.0 ml/min. The UV absorbance was monitored at 320 nm. The plasma sample was acidified by the addition of 0.01 parts of concentrated phosphoric acid (85%) to maintain caffeic acid stability. After a simple clean-up procedure, the limit of quantitation achieved was 0.1 μg/ml, and the standard curve was found to be linear over the concentration ranges of 0.1–2.0 μg/ml and 0.1–40 μg/ml. The coefficient of variation for within- and between-run precision and accuracy was less than 10%, and the recovery was 82.3%.     

白花蛇舌草对人胃癌细胞MNK-45线粒体膜电位及凋亡相关基因表达的影响*

目的: 探讨白花蛇舌草(注射液)对人胃癌细胞MNK-45线粒体膜电位及凋亡相关基因表达的影响。方法: 将人胃癌细胞MNK-45分成4组,每组设置3个复孔,对照组为未加入白花蛇舌草的MNK-45细胞,3组实验组分别加入终浓度为20、30、40 μg/ml白花蛇舌草的MNK-45细胞,各组在5％的CO₂培养箱中孵育48 h后,利用激光共聚焦显微镜下观察细胞形态变化,流式细胞术检测线粒体膜电位,qRT-PCR检测Cytochrome C (Cyt c)、caspase3和caspase9基因的表达变化,Western blot检测Cytochrome C (Cyt c)、caspase3和caspase9蛋白的表达变化。结果: 与对照组相比,终浓度为20、30、40 μg/ml的各白花蛇舌草处理组,其MNK-45细胞的线粒体膜电位均明显降低(P＜0.01),Cyt c、caspase3和caspase9的基因表达均明显上调(P＜0.01)、蛋白表达也均显著升高(P＜0.05或 P＜0.01),40 μg/ml的白花蛇舌草处理组的表现最佳。结论: 在终浓度为20~40 μg/ml的范围内,白花蛇舌草能够降低人胃癌MNK-45细胞线粒体膜电位, 诱导细胞凋亡,并可上调Cyt c、caspase3和caspase9基因表达。     

Glutamine synthetase in cultured whole retinas from the embryonic chick: Enhancement of basal and induced activity by 4 °C storage

Storage of whole retinas from the embryonic chick for 24 h at 4 °C resulted in increased basal levels of glutamine synthetase (GS) during subsequent incubation at 37 °C in the absence of cortisol. GS levels in these retinas maintained initially at 4 °C (CS), in many cases, exceeded GS levels in cortisol-induced whole retinas incubated solely at 37 °C. The increase in basal GS activity is seen within 48 h of the transfer of the retinas from 4 to 37 °C. If cortisol (0.001 μg/ml = 2.8 nm or 0.01 μg/ml = 28 nm) is added during the last 24 h of culture to CS retinas subsequently transferred to 37 °C, levels of GS are attained that are higher than those in the corresponding retinas cultured continually at 37 °C. However, the activity ratios (GS specific activity in cortisol-treated retinas/GS specific activity in retinas not exposed to cortisol) are similar for CS retinas and those maintained at 37 °C throughout. Monolayers of retinal cells display similar basal and cortisol-induced levels of GS independent of treatment. Retinal monolayers maintained at 4 °C for 24 h and subsequently incubated at 37 °C do not exhibit increases in either basal or cortisol-induced levels of GS over those in monolayers maintained at 37 °C throughout. The CS-promoted increase in the basal and cortisol-induced GS activity of whole retinas is eliminated by enzymatic dispersion of the retina just prior to 37 °C culture of the cells as monolayers. Both basal and cortisol-induced GS levels in the latter monolayers resemble those in retinal cells kept as monolayers throughout.