ISSN:1059-910X    

DOI:10.1002/jemt.1070250102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn order to gain a further insight into the relationships of the complex process of replication of adenovirus genomes to the substructures which occur in the nuclei of adenovirus type 5 (Ad5) infected HeLa cells, we have visualized directly, at the electron microscopic level, viral double‐stranded DNA (dsDNA) in late infected nuclei by the use of a post‐embedding in situ hybridization technique with a biotinylated specific DNA probe. The procedure is based on the removal of single‐stranded (ss) nucleic acids by S1 nuclease. The highest levels of signal density for viral dsDNA were detected over the fibrils of the large, centrally located viral genome storage site and over the viral nucleoids of both clustered and isolated viruses. Lower but significant signals were observed over the fibrillo‐granular network of the peripheral replicative zones, where both transcription and replication of viral DNA occur. On the other hand, the labeling of the enclosed viral ssDNA accumulation sites, also involved in viral replication but not transcription, was negligible, which suggests that, in the latter, the newly synthesized viral dsDNA immediately extends into the adjacent peripheral replicative zone to be transcribed and/or rep

ISSN:1059-910X    

DOI:10.1002/jemt.1070250103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe technique of in situ hybridization has been used to evaluate the expression of an ovulation hormone mRNA (caudodorsal cell hormone; CDCH) in the central nervous system (CNS) of the molluscLymnaea stagnalis. Hybridization with radioactive as well as with nonradioactive labeled oligonucleotide and plasmid probes revealed a specific labeling on cell bodies of caudodorsal cells (CDCs), which are known to produce CDCH, on the light microscopical level. In addition, specific labeling was observed outside the cell bodies, as far as the cerebral commissure, where CDCH is released in the haemolymph. To investigate whether these signals represent an axonal localization of the CDCH mRNA, we performed in situ hybridization at the electron microscopical (EM) level. The results showed an intraaxonal localization of CDCH mRNA with digoxigenin labeled oligonucleotide and plasmid probes. Gold labeling was observed in secretion granules, and double labeling experiments showed that these granules also contain CDCH. This specific intragranular localization suggest that CDCH mRNA is transported through the axon and released by exocytosis in the haemolymph. © 1993 Wiley‐Liss, I

ISSN:1059-910X    

DOI:10.1002/jemt.1070250104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe secondary and tertiary structure of RNA, in situ, is thought to be involved in distinct functions such as directing association of the RNA with the cytoskeleton, enzymatic activity of some RNAs, and the control of translation. In situ transcription (IST), a procedure by which cDNA is synthesized in situ, has been used to assess mRNA structure in situ using fixed cells or tissues. Distinct banding patterns were noted for mouse and rat POMC. Unique IST banding patterns were observed when an oligonucleotide complementary to a putative POMC stem‐loop structure was used to prime IST. Indeed local changes in banding patterns could be elicited by pharmacological agents which modulate POMC translation. Inhibition of POMC synthesis with NaF or dexamethasone decreased the number of POMC mRNAs in the polysome fractions and increased the intensity of high molecular weight IST‐derived bands. Forskolin, a stimulator of POMC synthesis, had the opposite effect. One mechanism by which translational control is thought to occur is by regulation of ribosome movement down the mRNA by specific binding of cytosolic proteins to RNA structure. Cytosolic protein fractions from AtT20 pituitary cells have been shown to specifically bind to the IST‐predicted RNA structure. These findings suggest that 1) mRNA structure can be assessed in situ, 2) translation may be altered by the secondary and tertiary structure of mRNAs, and 3) a predicted stem‐loop structure exists in situ in the 5′‐end of POMC mRNA. © 1993 Wil

ISSN:1059-910X    

DOI:10.1002/jemt.1070250105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn situ hybridization has been used to localize erythropoietin (EPO)‐producing cells in murine kidney and liver. Peritubular interstitial cells were the only cell type that produced EPO in the kidney. The EPO‐producing cells were primarily concentrated in the inner cortex but were also seen in the outer medulla and outer cortex. EPO‐producing cells represented less than 10% of the total interstitial cell population. The number of EPO‐producing cells per square centimeter of cortex directly correlated with the amount of renal EPO mRNA and varied in an inverse exponential manner with hematocrit. These results suggest that EPO is expressed in an all‐or‐none fashion in peritubular interstitial cells and that the oxygen carrying capacity of blood is the major regulator of renal EPO production. Peritubular interstitial cells were also identified as the renal source of human EPO in transgenic mice that expressed human EPO mRNA in a regulated fashion in the kidney. Transgenic mice exhibiting inducible supranormal liver expression of human EPO were used to identify EPO‐producing cells in the liver. Hepatocytes surrounding central veins produced human EPO in these mice. Individual hepatocytes were able to modulate their production of human EPO depending upon the severity of anemia to which they were subjected. Two types of widely scattered cells produced EPO in severely anemic nontransgenic mice. Eighty percent of EPO‐producing cells were hepatocytes and 20% were classified as being nonepithelial based on their nuclear morphology and location in venous sinusoids. © 1993

ISSN:1059-910X    

DOI:10.1002/jemt.1070250106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have developed an assay that allows one to monitor gene expression in and peptide secretion from individual cells. By combining the reverse hemolytic plaque with in situ hybridization, investigators can quantitate simultaneously the level of gene expression and the level of secretion of a peptide. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available. It can be used to monitor the level of mRNA and secretion of the peptide product, or expression of one gene and the secretion of another peptide. In this paper we will describe the major steps of the method. We have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we believe that this method may be an important approach to answer many questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level. © 1993 Wiley‐Liss, I

ISSN:1059-910X    

DOI:10.1002/jemt.1070250107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractSuprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18–39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP‐containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA‐immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker. Published 1993 Wiley‐Li

ISSN:1059-910X    

DOI:10.1002/jemt.1070250108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractOne serviceable feature of in situ hybridization is its potential for assessing relative levels of mRNA in specific regions of tissues and organs. To determine its efficacy as a quantitative technique, we applied a nested factorial design to a multifactorial experiment. Estimates of the magnitude of variance components then allowed an assessment of variation over samples of sections from the same tissue source, variation in label over 2 anatomical sites within the same section of tissue, as well as experiment‐to‐experiment variation. We found ∼ 51% of the total variance arose from experiment‐to‐experiment variation, while ∼ 21% of the total variance was due to variation in autoradiography grain density over neurons in the same brain region. Rat‐to‐rat variation accounted for approximately 11%. About 10% of the variance was due to variationbetweensections of tissue that were derived from the same tissue source and were hybridized in the same hybridization experiment. Variation between 2 homologous, bilaterally located brain regions located on the same tissue section (the right and left supraoptic nucleus), accounted for ∼ 5% of the total variance. The remaining unaccounted error variance was ∼ 2% of the total variance. Since an expected change in cellular content of a particular mRNA was observed as a function of experimental treatment, results suggest in situ hybridization is a useful quantitative method. Findings also indicate, however, the importance of experimental design: the use of multiple samples of tissue from the same tissue source, a sufficient number of tissue sources (animals, batches of cultured cells) to account for variations in sample sources, and the need to assess experiment‐to‐experiment and rat‐to‐rat variations. Results suggest the utility of analyzing the data of in situ hybridization experiments from the perspective of experimental des

ISSN:1059-910X    

DOI:10.1002/jemt.1070250109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIt is a widely held belief that the inactive X‐chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X‐chromosome (Xa). We have reconsidered this problem and painted X‐chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46, XX and 46, XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X‐chromosome specific library DNA. FITC‐conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two‐dimensional (2D) and three‐dimensional (3D) analyses. 2D analyses of Xi‐ and Xa‐domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI‐stained nuclei prior to CISS‐hybridization was confirmed by its colocalization with one of the two painted X‐domains. In 31 of the 34 nuclei the area AXifor the inactive X‐domain was smaller than the area AXafor the active domain (mean ratio AXa/AXi= 1.9 ± 0.8 SD, range 1.0–4.3). The signed rank test showed a highly significant (Pr(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI‐image, FITC‐image) were registered with equal spacings (approx. 0.4 μm). A thresholding procedure was applied to determine the PI‐labeled nuclear and FITC‐labeled X‐domain areas in each section. Estimated slice volumes were used to compute total nuclear and X‐domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X‐chromosome domains comprised (3.7 ± 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 ± 0.2 SD (range 1.1–2.3) was found for the volumes of the larger and the smaller X‐domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X‐chromosome domain volume comprised (4.0 ± 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X‐chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa‐ as compared to Xi‐domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitations of nuclear and chromosome domain volume measurements using confocal las

ISSN:1059-910X    

DOI:10.1002/jemt.1070250110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn situ hybridization (ISH) for HIV is an arduous, demanding means of detecting viral genetic material in cells and tissues. Good ISH requires broad technical skills and devotion to controls for every step of the process as well as a critical eye when interpreting results. ISH may be used to detect HIV in three ways: by hybridizing to viral RNA, by hybridizing to proviral mRNA being produced for virion packaging, and by hybridizing to proviral DNA in the cytoplasm or integrated in the nucleus of an infected cell. Here we discuss the technical considerations involved and the problems encountered in using ISH to study the pathobiology of HIV infection. Published 1993 Wiley‐Liss, In

ISSN:1059-910X    

DOI:10.1002/jemt.1070250111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY