石菖蒲根茎甲醇提取物对谷蠹的生物活性

采用冷浸法和溶剂萃取法,研究石菖蒲(Acorus gramineus Soland)根茎甲醇提取物对谷蠹Rhizopertha dominica(Fabricius)成虫的生物活性。结果表明,石菖蒲甲醇提取物以石油醚萃取物的生物活性最强,水层次之。石油醚萃取物对谷蠹具有较高的触杀、熏蒸、种群抑制活性和防治效果,并具有一定的驱避活性。药膜法触杀处理72h后对谷蠹的LC50和LC95分别为8.30μg/cm2,51.75μg/cm2。药纸熏蒸法6.25μL/L处理120h后对谷蠹的校正死亡率为94.25%。饲料拌药法250mg/kg处理对谷蠹的防效与5mg/kg马拉硫磷效果相当,且对F1代的种群抑制效果显著高于5mg/kg马拉硫磷处理。     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

Functions of milk protein gene 5′ flanking regions on human growth hormone gene

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S₁ casein (bαS₁CN), beta casein (bβCN), kappa casein (b_kCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS₁CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS₁CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS₁CN was shown most suitable, because the bαS₁CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.     

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.     

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were −/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3–5 μg/kg and 0.08 μg/kg and 0.20–0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02–12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71–8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.     

Isolation and Characterisation of Two Quercetin Glucosides with Potent Anti-Reactive Oxygen Species (ROS) Activity and an Olean-12-en Triterpene Glucoside from the Fruit of Abelmoschus esculentus (L.) Moench

Abelmoschus esculentus (Okra) is used in the traditional treatment of cancer, hyperlipidaemia and hyperglycaemia. We, therefore, investigated its composition and potential cytotoxic or antioxidant properties that might underlie its phytotherapeutic applications. Its methanolic fruit extract yielded compounds 1 , 2 and 3 , identified through NMR, UV and MS analyses as olean-12-en-3-O-β-d -glucopyranoside, isoquercitrin (quercetin glucoside) and 5,7,3′,4′-tetrahydroxy-flavonol-3-O-[β-d -glucopyranosyl-(1→6)]-β-d -glucopyranoside (quercetin diglucoside), respectively. Following 48 h exposure, oleanene glucoside was mildly toxic to the HeLa and the MRC5-SV2 cancer cells, isoquercitrin was not toxic except at 100 μg/ml in HeLa, and quercetin diglucoside elicited no toxicity. In a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay of intracellular levels of reactive oxygen species (ROS), hydrogen peroxide increased ROS levels, an effect not affected by oleanene glucoside but protected against by isoquercitrin and quercetin diglucoside, with IC₅₀ values, respectively, of 2.7±0.5 μg/ml and 1.9±0.2 μg/ml (3 h post-treatment) and 2.0±0.8 μg/ml and 1.5±0.4 μg/ml (24 h post-treatment.) This is the first report of this oleanene skeleton triterpenoid in the plant. The work provides some insight into why the plant is included in remedies for cancers, cardiovascular complications and diabetes, and reveals it as a potential source of novel therapeutics.     

Cytogenetic effect of malathion in in vitro culture of human peripheral blood

Chromosomal aberrations, sister-chromatid exchanges and mitotic indices were observed in human peripheral leukocytes, treated with four different concentrations of malathion (0.02, 0.2, 2 and 20 μg/ml), an organophosphate pesticide, added to the culture medium at 0, 24 and 48 h after culture initiation. These cultures showed a dose-dependent increase in the frequency of chromosomal aberration as well as sister-chromatid exchanges. There was a significant decreases in mitotic index at all concentrations.     

Dynamics of Free Pisatin Elicitor Accumulation in Infection-droplets of Monilinia fructicola on Pea Endocarp*

Rapid elicitor accumulation in the infection-droplet of the pea-M. fructicola interaction began between 2 and 3 h after inoculation. Rapid accumulation of pisatin began between 2 and 3 h, however, low levels (0.06–0.1 μg/ml) were detected in the infection-droplet as early as 1–2 h following inoculation with the fungus. The pisatin concentration reached levels inhibitory to the fungus between 6 and 12 h (ca. 1–5 μg/ml) and the ED₅₀ value of 10 μg pisatin/ml for mycelial growth of M. fructicola was attained after 14 h. Elicitor activity in infection-droplets after 4 and 18 h was a function of inoculum concentration and pisatin accumulation in diffusate after 40 h was a function of elicitor concentration (linear doseresponse curve). However, when elicitor was applied at zero time, the rate of pisatin accumulation over the first 12 h was indistinguishable from that which occurred when M. fructicola conidia were applied to the endocarp and elicitor accumulated with time. The initial rate of pisatin accumulation therefore appears to be dependent on the pea pod and independent of any time delays associated with conidial germination and elicitor accumulation. However, the final pisatin concentration which accumulated in the infection-droplet was dependent on the, dose' of elicitor irrespective of the nature and timing of the elicitor treatment. The presence of elicitor activity was demonstrated in the interaction in space and time where and when these molecules could function as elicitors of pisatin in vivo.     

In vitro lipolytic effect of bovine pituitary diabetogenic protein

Bovine diabetogenic protein has been further purified by gel filtration yielding a fraction (M_r 25 000–28 000) having increased diabetogenic and in vitro lipolytic activity. Using rat epididymal fat pads, this fraction was shown to be lipolytic at concentrations as low as 1–10 μg/ml. The in vitro lipolytic effect was unaffected by the nutritional state of the animals, was not potentiated by dexamethasone, could be demonstrated in the presence and absence of glucose and was not mediated by α- and β-adrenergic receptors. A lag phase of > 1 h was observed before diabetogenic protein induced lipolysis occurred, suggesting that protein synthesis might be involved. Cycloheximide (10 μg/ml), added initially, prevented the diabetogenic protein-induced lipolysis. This direct effect of the purified protein on adipose tissue helps explain the elevation of free fatty acids seen when bovine diabetogenic hormone is administered in vivo and suggests that this anterior pituitary protein may be a new lipid-mobilizing hormone.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Confirmation of spectinomycin in milk using ion-pair solid-phase extraction and liquid chromatography–electrospray ion trap mass spectrometry

A confirmation procedure is described for residues of spectinomycin in bovine milk. Spectinomycin is extracted from raw milk using ion-pair reversed-phase solid-phase extraction. The extracts are ion-pair chromatographed on a polymeric reversed-phase column and analyzed on a quadrupole ion trap mass spectrometer equipped with an electrospray interface. MS–MS data are acquired in the scan mode of product ions deriving from m/z 333, the protonated molecular ion. The estimated limit of confirmation is between 0.05 and 0.1 μg/ml. The procedure was validated with control milk, fortified milk (0.1–5.0 μg/ml), and milk from cows dosed with spectinomycin.     

Activation of Plasmin in Mastitic Milk

Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.     

Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3–63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 μg/l) by several orders of magnitude.     

Protective effect of HDL on endothelial NO production: the role of DDAH/ADMA pathway

Accumulating studies have demonstrated that the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine (DDAH/ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of high density lipoprotein (HDL) on endothelial NO production was related to its effect on DDAH/ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were prior exposed to HDL (10, 50, or 100 μg/ml) for 1 h, and then incubated with oxidized low density lipoprotein (ox-LDL) (100 μg/ml) for 24 h. The cultured medium was collected for measuring the concentration of NO and ADMA. The cells were collected for measuring the mRNA and protein expression of DDAH-II as well as DDAH activity. HUVECs treated with ox-LDL (100 μg/ml) for 24 h significantly decreased the concentration of NO, the mRNA and protein expression of DDAH-II as well as DDAH activity and increased the level of ADMA. Pretreatment with HDL (10, 50, or 100 μg/ml) could counteract these changes induced by ox-LDL (100 μg/ml). HDL significantly increased the attenuated endothelial cell NO production induced by ox-LDL, which was attributed to its effect on DDAH/ADMA pathway.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

Threshold level for theophylline in doping analysis

HPLC in the reversed-phase mode is used to assay methylxanthines including theobromine, paraxanthine, theophylline and caffeine in urine. The calibration graphs show good linearity in the concentration range 0–10 μg/ml. The limit for accurate quantitation of theophylline was 0.25 μg/ml. Between 6 and 20% of the parent drug is recovered in urine (0–12 h) after the oral administration of sustained release preparations containing 150 and 250 mg theophylline to four volunteers. Theophylline levels above 0.25 μg/ml were found in 1539 out of 3885 urine samples collected from athletes during unannounced doping control in Flanders. Statistical evaluation of the results gives a far outside value [75th percentile + (3× interquartile range)] of 2.25 μg/ml. The ratio theophylline paraxanthine (TP/PX) as an indicator for the non-dietary intake of theophylline seems to be more reliable. The far outside ratio was 0.20. To ensure with the greatest possible degree of certainty that no false-positive result is reported, decision limits of 5 μg/ml and 0.50, for theophylline and the ratio TP/PX respectively, are proposed.     

Hormone-induced desensitization of cultured rat granulosa cells to FSH

Monolayers of granulosa cells (GC) derived from immature hypophysectomized diethylstilbestrol-treated rats became refractory in terms of FSH-stimulable cyclic AMP production following exposure to the homologous hormone. In the presence of ovine FSH (5 μg/ml), maximal refractoriness was attained after 4 h of incubation. Upon removal of the FSH from the medium, the cells regained their full responsiveness within 24 h. The extent of desensitization was dependent upon the dose of FSH, and could not be overcome by increasing the dose of the hormone during the challenge period. Exposure of GC monolayers for 2–4 h to the protein synthesis inhibitors actinomycin D (8 μg/ml) and cycloheximide (5 μg/ml) on their own enhanced FSH-stimulable cyclic AMP production. When added together with FSH, the inhibitors did not prevent the process of desensitization to the hormone. The results suggest that the initial phases of FSH-induced desensitization do not require de novo protein synthesis.     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.