Cellular location of thymus-leukemia (TL) antigen as shown by immuno-cryoultramicrotomy

Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL⁺ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitochondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.     

Comparison of transsynaptic viral labeling of central nervous system structures from the uterine horn in virgin, pregnant, and lactating rats

Using the transneuronal viral tracing method, the central nervous system (CNS) connections of the uterine horn were studied in virgin, pregnant, and in lactating rats. The frequency of viral labeling in the brain and the distribution of virus-infected neurons from the uterine horn were compared among groups. There was a marked difference in the frequency of viral labeling in the brain stem. In virgin rats more than half of the brain stems (5 out of 9) were labeled. In contrast, in pregnant animals viral-labeled neurons were detected in only a few cases (3 out of 16) and almost each brain stem of the lactating group was labeled (12 out of 13). A similar, less marked difference was observed in the hypothalamus. The pattern of distribution of infected neurons was similar in each group. In the brain stem, the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, gigantocellular and paragigantocellular nucleus, ventrolateral medulla, A5 cell group, and caudal raphe nuclei were the most frequently labeled structures. In the diencephalon, viral-infected neurons were detected primarily in the hypothalamic paraventricular nucleus. The telencephalon was devoid of infected cells. Data suggest that the CNS control of the uterine horn varies depending on reproductive status. The low frequency of brain labeling in pregnant rats may be related to the almost complete lack of sympathetic fibers in the uterus prior to parturition and the very high frequency of labeling in lactating animals to the postpartum hyperinnervation of the uterus.     

Action of the chemical agent fipronil (active ingredient of acaricide Frontline®) on the liver of mice: an ultrastructural analysis

Fipronil, active ingredient of the acaricide Frontiline®, is a phenyl‐pyrazolic derivative, and its efficacy in the elimination of several plagues, even in low concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the objective of this study was to evaluate the effects of different dosages of fipronil on the liver of mice in artificial conditions. Results showed that the animals exposed to fipronil present significant ultrastrucutural changes in hepatic cells with evident cellular and cytoplasm disorganization in hepatocytes characterized by an increase in the number of organelles, mainly mitochondria and rough endoplasmic reticulum, organelles that, in the case of the exposed animals, were probably responsible for the enzymes' synthesis that have the function of inactivating the toxic metabolites. A fat accumulation in the hepatocytes' cytoplasm (steatosis) was observed, in addition to extended vacuolated areas, mainly in regions next to the cell nucleus. Alterations observed in the nuclei of the hepatocytes pointed out cell death processes. Moreover, Kupffer cells increased in number (hyperplasia) suggesting an increase in the phagocytic activity of the liver in the exposed animals. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Ketamine effects on the urogenital system--changes in the urinary bladder and sperm motility

Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long-term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3-month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3-month and 6-month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6-month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well.     

一种改进的可扩展标记语言素数编码方法

在可扩展标记语言的发展过程中,如何有效地查询可扩展标记语言数据已成为一个研究热点.其中如何快速有效地判断两元素是否具有祖先/后裔关系最为关键.目前已有的素数编码方法需要大量素数和大素数,为此提出一种改进的素数编码方法--最多亲兄弟素数,该方法不必每个节点都使用新素数.理论分析和实验表明,最多亲兄弟素数编码方法大大减少了素数的使用量,减小了所用的大素数,加快了可扩展标记语言查询的速度.     

Impact of aging and life-long calorie restriction on expression of apoptosis-related genes in male F344 rat liver

Aging enhances apoptosis of hepatocytes under normal physiological conditions and increases the susceptibility to apoptosis of hepatocytes, whereas chronic calorie restriction (CR) suppresses the age-enhanced susceptibility to apoptosis. To clarify the subcellular mechanisms of age-associated dysregulation of apoptosis and the effects of CR, we analyzed the expression of genes promoting apoptosis (p53, Fas receptor, Fas ligand, TNF receptor 1, TNFalpha, Bax, TGF beta 1) and genes preventing apoptosis (Bcl-2 and Bcl-XL) in the livers of 3-, 6-, 15-, and 24-month-old male F344 rats that were either fed ad libitum or subjected to a 30% reduction in food intake (CR). After the age of 6 months, expression of p53, Fas receptor, Fas ligand, and TNFalpha mRNAs was up-regulated with aging. CR suppressed this age-enhanced p53 and Fas receptor mRNA expression, but expression of the other genes was not altered significantly by aging or CR. Expression of Fas receptor in hepatocytes, as detected immunohistochemically, increased with age, but CR suppressed age-accelerated Fas receptor expression. Our findings suggest that TNF ligand/TNF receptor family signaling, particularly Fas receptor expression, is important in age- and CR-modulated apoptosis of hepatocytes. Hepatocytes that were immunoreactive for p53 had slightly increased with aging, suggesting that p53 may mediate the age-enhanced up-regulation of Fas receptor in hepatocytes.     

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both nonencapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.     

Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro

The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as (1) undifferentiated multilayers in glucose-containing culture media and (2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; “pre-differentiated” columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.     

Histochemical and ultrahistochemical localization of heavy metals in calf organs

Cadmium, zinc, selenium, and copper were administered, singly or in combination, orally or subcutaneously. Experiment I included 32 calves of both sexes; six received Cd (two groups), Zn, Cd, and Zn, and Cd and Se (two groups) and one group was a control. In Experiment II (21 bulls), three were given Cd, Cd, and Cu, and Cd and Zn, respectively, and one group was a control. For light microscopy, in Experiment I the highest amounts of silver granules were present in the samples of liver, small intestine, and vesicular gland of all the exposed groups; in Experiment II the most affected organs were liver, kidney, and small intestine. For electron microscopy, in Experiment I, after administration of Cd and Zn, the highest amounts of granules were seen in the cytoplasm of hepatocytes and cells of the proximal and distal renal tubules and the lowest amounts were found in glandular cells of the pancreas. Administration of Cd and Se resulted in the presence of large numbers of granules in the nuclei and nucleoli of spermatogonies. In Experiment II, ingestion of Cd and Zn in feed led to the appearance of highest amounts of granules in the nucleoli, nuclei, and cytoplasm of cells in testes, kidneys, and pancreas. Following Cd intake, the highest accumulation of granules was observed in the nucleoli of hepatocytes and cells of the proximal and distal renal tubules. Combined Cd and Cu produced the highest number of granules in cells of the proximal and distal renal tubules and in the nucleoli and nuclei of germinal epithelium.     

Synthesis and turnover of DNA in hepatocytes of neonatal rats

The incorporation of ³H-thymidine has been studied in the hepatocytes nuclei of rats during the first 3 days of life. The synthesis of DNA which occurs in 18% of nuclei after a single injection of the labelled precursors 2 h after birth, cannot be considered as premitotic since:

• (i) The hepatic cell number is constant during the first 3 days of life.
• (ii) The mitotic index is less than 0·1%, 20 times lower than that expected on the basis of the labelling index and of the length of cellular cycle.
• (iii) The percentage of labelled nuclei does not increase in the following 3 days.
• (iv) The average number of grains per nucleus is unchanged for 18 h after the injection, a period longer than an entire cellular cycle (13 h).

This synthesis is better explained as due to an increase of DNA content per nucleus, previously described, in the hepatocytes during the first 3 days of life. After 20 h of life (18 h after label injection) a decrease of label of about 30% takes place without variation of DNA-Feulgen dye content per nucleus. It is concluded that synthesis and turnover of DNA are present in the hepatocytes nuclei and that these processes are probably related to the functional maturation of the cells.     

机床铸铁件时效处理方法的绿色性综合评价及抉择

针对铸铁件的各种时效处理方法,从绿色制造的角度出发,对包括环境、资源、时间、质量和成本5项因素在内的各种时效处理方法,采用模糊综合层次评价方法(Fuzzy-AHP)对其进行了绿色性综合评价.得到三种时效处理方法的绿色性综合评分值分别为:振动时效66.8分、热时效56.8分、自然时效53分.评价结果证明,机床铸铁件的各种时效处理方法中,振动时效方法是唯一的绿色制造方法.     

Ki67 and doublecortin positive cells in the human prefrontal cortices of normal aging and vascular dementia

Immunohistochemical localizing of the proliferation of Ki67 nuclei and doublecortin positive cells were performed in the prefrontal cortex of normal aged and vascular dementia (multiple infarct dementia) patients. Positive Ki67 nuclei and doublecortin positive cells were observed in both groups, with slightly higher density in the prefrontal cortex of vascular dementia. When the Ki67 sites were superimposed with the neuronal specific enolase localizations, only about 5% of the cells was doubly labeled, indicating few proliferating cells were neurons. This percentage did not vary between specimens of normal aging and those of vascular dementia.     

Immunohistological evidences of Ginkgo biloba extract altering Bax to Bcl-2 expression ratio in the hippocampus and motor cortex of senescence accelerated mice

Ginkgo biloba extract 761 appears to display neuroprotective effect in many nervous diseases and aging. Deterioration of mental functions during aging is always accompanied by loss of neurons, presumably through apoptosis. Here, we studied the effect of G. biloba extract in the expression of Bax/Bcl-2 ratio, an important apoptotic index, in the hippocampus and motor cortex of the aging brain. Bax and Bcl-2 expressions were examined with immunohistochemical methods. Senescence Accelerated Mice Prone Strain 8 was used because the aging process was accelerated with neuropathological alterations similar to those found in the aging human brain. The mice were fed with either G. biloba extract or sucrose from the age of 3 weeks until sacrifice at 3 or 9 months old. In the hippocampus of G. biloba fed 9-month-old mice, the ratio of Bax positive cell to Bcl-2 positive cell (Bax/Bcl-2 expression ratio) was 11.43 +/- 3.11 (mean +/- SD); significantly lower (P < 0.05) than the Bax/Bcl-2 expression ratio of 20.99 +/- 5.34 in the sucrose fed mice. The Bax/Bcl-2 expression cell ratios, however, in the motor cortex were not significantly different between the two groups (2.22 +/- 1.35 versus 2.27 +/- 2.02 for the G. biloba and the sucrose fed mice, respectively). The decrease in the Bax/Bcl-2 expression cell ratio following G. biloba treatment might hence be able to protect the aging hippocampus from moving further down the apoptotic pathway. Western blotting confirmed the decrease of Bax in the brain even after a short term and high dose Ginkgo treatment. It is speculated that the G. biloba extract may be a potential neuroprotective agent against apoptosis through the differential expressions of the Bax and Bcl-2 in the hippocampus.     

Caveolin-1 and mitochondrial alterations in regenerating rat liver

The liver has a remarkable ability to regenerate after partial hepatectomy (PH), although the factors governing such ability are still poorly understood. During the prereplicative phase of the regeneration, ultrastructural alterations of periportal hepatocytes were seen, including mitochondrial swelling, abnormal accumulation of lipids, and myelin figures which could lead to the formation of lipid droplets. As it has been hypothesized that caveolin-1 is involved in lipidogenesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveolin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and electron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of caveolin-1 was assessed by immunohistochemical and immunocytochemical techniques. Following PH, at 24 h, membranes of altered mitochondria of periportal hepatocytes exhibited significant decrease of caveolin-1 expression compared with control. Myelin figures showing high expression of caveolin-1 were also seen. At 96 h, hepatocytes became ultrastructurally similar to the control liver, and the expression of caveolin-1 on mitochondria showed a moderate increase compared with 24 h after PH. Decrease of expression of caveolin-1 in the altered liver mitochondrial membranes at 24 h following PH, and the high expression of caveolin-1 observed on myelin figures, suggests involvement of caveolin-1 is in both mitochondrial homeostasis and lipidogenesis. Addressing the role played by caveolin-1 during liver regeneration might disclose additional features of mitochondrial homeostasis and lipidogenesis during frequent metabolic liver diseases.     

Imaging of immunolabeled membrane receptors in uncoated sem specimens

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V_o). At V_o=2kV, the SE and BSE signals were too weak to identify all markers, while at V_o=3–7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the V_o to above 7 kV sometimes provokes instability of the specimens. A V_o of ? 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.     

Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways

Biotinylated dextran amine (BDA) has been used for neural pathway tracing in the central nervous system for many decades, in which high molecular weight BDA appeared to be transported predominantly in the anterograde direction and less in the retrograde direction. In the current study, we reexamined the properties of neural labeling with high molecular weight BDA through a reciprocal neural pathway between thalamus and somatosensory cortex. After injection of BDA into the ventral posteromedial nucleus of thalamus (VPM) in the rat, the BDA labeling was sequentially examined on somatosensory cortex at 3, 5, 7, 10, and 14 survival days. Both of anterogradely labeled axonal terminals and retrogradely labeled neuronal cell bodies were observed simultaneously on the somatosensory cortex. With the increasing of survival times after injection, morphological changes occurred on the labeled axonal arbors and neuronal dendrites, in which the high quality of BDA labeling appeared on the tenth survival day. These results indicate that high molecular weight BDA is not only a sensitive anterograde tracer but also an excellent retrograde marker to be used for tracing through thalamocortical and corticothalamic pathways. And the detailed structure of neural labeling with BDA similar to Golgi‐like resolution can be obtained at optimal survival times of animals after the injection of high molecular weight BDA.