Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Electrochemical immunosensor based on Pd–Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd–Au nanoparticles (CNTs/PDDA/Pd–Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd–Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.     

The mercapturic acid pathway metabolites of a glutathione conjugate of aflatoxin B1

A glutathione conjugate of aflatoxin B1 (AFB1) which has previously been identified as 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy aflatoxin B1 (AFB1-GSH) (E.J. Moss, D.J. Judah, M. Przybylski and G.E. Neal, Biochem. J., 210 (1983) 227-233) has been degraded in vitro to all of the intermediates of the mercapturic acid pathway (MAP) and the chromatographic and spectral characteristics of each of these compounds investigated. The cysteinylglycyl conjugate (AFB1-Cys.Gly) was prepared by incubating the AFB1-GSH conjugate with a rat hepatoma cell line rich in gamma-glutamyl-transpeptidase (GGT). Incubations of the AFB1-Cys.Gly conjugate with dipeptidase produced a metabolite, which was purified and characterized by 1H-NMR spectroscopy as 8,9-dihydro-8-(S-cysteinyl)-9-hydroxy aflatoxin B1 (AFB1-Cys). The N-acetyl derivative of the AFB1-Cys conjugate resulted from the incubation of the AFB1-GSH conjugate in vitro with isolated rat kidney cells. Mass spectral data were consistent with the compound being 8,9-dihydro-8-(S-cysteinyl-(N-acetyl))-9-hydroxy aflatoxin B1 (AFB1-Nac.Cys). A chromatographically identical compound was obtained by the chemical acetylation of AFB1-Cys.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis.

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.     

黄曲霉毒素B_1抗独特型抗体的制备和应用研究II.抗独特型抗体应用

以抗独特型抗体(anti-idiotypeantibody,Ab2)的酶切片段Fab2替代黄曲霉毒素B1(AFB1)建立一种不需要使用AFB1的无毒酶联免疫吸附(Enzyme-LinkedImmunosorbentAssay,ELISA)试剂盒,研究该试剂盒特异性、稳定性和AFB1的加标回收,并将该试剂盒用于农产品和饲料中AFB1的检测。结果表明,该试剂盒具有和常规ELISA试剂盒一样的特异性和加标回收能力,无毒试剂盒和常规试剂盒一样都可以用于农产品和饲料中AFB1的检测,并且两种试剂盒的检测结果并无显著差异,无毒ELISA试剂盒在适当处理后,40C放置3个月和-200C放置5个月,以间接非竞争ELISA测定的吸光值分别是起始时的85%和87%。     

Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.     

Aflatoxin B1-induced Hprt mutations in splenic lymphocytes of Fischer 344 rats. Results of an intermittent feeding trial

In a previous study, we found an increase in the mutant frequency at the Hypoxanthine phosphoribosyl transferase (Hprt) locus in the splenic lymphocytes of Fischer 344 rats acutely exposed to aflatoxin B1 (AFB1). Because an acute exposure may not reflect the exposure pattern of individuals whose diet may contain AFB1-contaminated foodstuffs, we sought to determine if the feeding regimen affected the induction of Hprt mutations in the rat splenic lymphocyte. Thus, Fischer 344 rats were fed either (A) a control diet, (B) various doses of AFB1 for three four-week periods interspersed with two four-week periods of the control diet, or (C) continuously fed 1.6 ppm of AFB1. Not only was a significant increase in the mutant frequency detected in the lymphocytes of rats fed a dose as low as 0. 01 ppm of AFB1, but the increase in the mutant frequency at the end of the 20-week experimental period was consistent with an accumulation of damage induced by AFB1. These results indicate that the rat lymphocyte/Hprt assay is useful for detecting chronic low level exposures. Further, these data suggest that an intermittent, low-level exposure to AFB1 may present a human health risk.     

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.     

A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

Summary We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.Abbreviations AFB1 aflatoxin B1 - AE aldrin epoxidase - AHH aryl hydrocarbon hydroxylase - PAH polycyclic aromatic hydrocarbons - PB phenobarbital     

Ultrastructure of the seminal vesicle of Phlebotomus perniciosus Newstead (Diptera, Psychodidae)

The seminal vesicles of Phlebotomus perniciosus were investigated by light microscopy, confocal scanning laser microscopy and by scanning and transmission electron microscopy. They have a complex structure, and three different morphological compartments called A, B and C are distinguished on the basis of their position and fine structure. Compartment A is continuous with the vasa deferentia and consists of a cylindrical wall limiting a lumen in which the spermatozoa are stored. Compartment B is hemispherical and surrounds compartment A like a muff. Compartment C constitutes an external coat surrounding A and B. The epithelial cells of each compartment are characterized by morphologically different secretory granules. The ultrastructural features of these cells are described and their role in sandfly reproductive biology is discussed.     

Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells

The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed.     

Interaction of aflatoxin with L-ascorbic acid: a kinetic and mechanistic approach.

Aflatoxins containing B(1), B(2), G(1) and G(2) obtained by growing Aspergillus parasiticus on SMKY liquid medium were tested for cytotoxicity (hemolysis) on RBC suspension in the presence and absence of L-ascorbic acid (AA). The results revealed that hemolysis was significantly increased on increasing the concentration of aflatoxin (0.5-3 microg ml(-1)). It was also found that pretreatment with AA (5-100 microg ml(-1)) significantly decreased aflatoxin-induced hemolysis. The solution chemistry of the interaction of aflatoxin with AA in aqueous solutions showed enhanced conversion of AFB(1) and AFG(1) to AFB(2) and AFG(2), respectively. Hemolytic, kinetic and mechanistic aspects of the interactions of aflatoxins and AA are discussed.     

Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

甲基苯丙胺中毒小鼠脑组织神经元超微结构变化的研究

目的：通过观察甲基苯丙胺中毒后小鼠脑组织超微结构的改变,探讨脑组织超微结构改变与甲基苯丙胺神经毒性机制的关系。方法：将40只小鼠随机分为对照组和3组实验组（A,B,C）。A组给予MA（20mg/kg,ip,single）、B组给予MA（20mg/kg,8am,8pm,ip×2d）、C组给予MA（20mg/kg,8am,8pm,ip×4d）,对照组给予等量生理盐水。用电镜观察前额叶皮质、海马、纹状体三个部位组织神经元胞体超微结构改变,并与空白对照组比较,结果进行统计学处理。结果：给予MA后,小鼠各脑区神经元胞体出现神经元固缩、变性、凋亡、坏死等超微病变。结论：MA可诱导神经细胞发生神经元固缩、变性、凋亡、坏死等超微病变,其变化程度随时间和药物蓄积有逐渐增加的趋势。     

不同温度下黄曲霉菌菌丝超微形态的比较研究

为探究黄曲霉菌的毒素合成是否影响菌丝超微形态,本研究结合扫描和透射电镜技术比较观察产毒（28℃和30℃）和不产毒（37℃和40℃）温度下培养的不同发育阶段的黄曲霉菌菌丝形态和超微结构。扫描电镜结果显示:28℃下,在24h和44h菌丝体表面有丝状粘性分泌物附着,48-72h之间菌丝体逐渐出现皱缩、塌陷和扭曲现象,而37℃下的菌丝体24-72h期间保持粗壮饱满。透射电镜结果显示:28℃下,培养24h后菌丝细胞内部出现大囊泡,44-72h期间内部细胞器模糊不清;而37℃下的菌丝细胞内部细胞器清晰,在44-48h期间线粒体和脂质体数量最多,72h后观察到隔膜结构。随后比较观察30℃和40℃下培养的黄曲霉菌丝超微结构验证了上述发现。综上,28℃和30℃促进黄曲霉菌产毒却导致菌丝体更早更快衰老,37℃和40℃不利于黄曲霉毒素合成但菌丝生长旺盛,且菌丝衰老进程相对减缓。本研究表明黄曲霉菌的毒素合成与菌丝内部形态变化密切相关。