A new technique for the visualization of the nucleolus based on a variant of the previously reported critical electrolyte concentration method for nucleoprotein complexes is proposed. The method consists of treating toluidine blue-stained preparations with Mg2+ ions in a concentration such that ribonucleoprotein complexes are metachromatically highlighted, while DNA-protein complexes attain their critical electrolyte concentration point, thus losing metachromasy. The technique, which is applicable to plant and animal cells, is proposed for studies involving identification, morphometry and eventually changes in substructure of the nucleolus at the light microscopy level.
In LEC (Long-Evans, with a cinnamon-like coat color) rats, intense staining for copper and iron was limited to Kupffer cells in the liver, and no staining was seen in hepatocytes, even though they appeared to be in various stages of degeneration. In the kidney, copper and iron reaction products were localized in the epithelial cells of urinary tubules. Intense staining for metallothionein was observed in Kupffer cells and hepatocytes, and also in epithelial cells of urinary tubules. These results suggest that hepatitis in the LEC rat may be associated with abnormal deposition of both iron and copper.
The aim of this work was to look for the stress protein P33, during long-term growth of human EUE cells in a hypertonic medium, with a polyclonal antibody against P33 produced in rabbit, and detectable in cells by both light and electron microscopy, using fluorescein-, peroxidase-anti-peroxidase- and gold-indirect immunolabelling. P33 was found to be mostly located in the cytoplasm, without apparent association with subcellular organelles; a slight, but significant, labelling was also observed in the nucleus. Dual parameter flow cytometric measurements of fluorescein-immunolabelled P33 and DNA content (after propidium iodide staining) showed that the amount of P33 increased progressively with increasing times of growth in a hypertonic medium in all phases of the cycle. Therefore there is no direct relationship between the increase of the P33 and the exit of cells from the cycling compartment that occurs in a hypertonic environment.
We evaluated the possible association between the morphometrical findings of the microtumors with a diameter of less than 5mm and the expression of p53, using 61 depressed epithelial minute neoplasias of large bowel. The nucleus/cytoplasm area ratio was associated with the frequency of positive expression of p53 in these tumors. Also we examined the expression of p53 in early and advanced carcinomas of large bowel. Although 6 lesions of the 12 minute adenocarcinomas showed positive reaction for p53, the positive-stained carcinoma cells could be recognized only in a part of the lesion. On the other hand, in early (except minute carcinoma) and/or advanced carcinomas lesions 100% of adenocarcinoma cells showed positive for p53. These findings suggest that the expression of p53 in the coloretal neoplasia have a relationship to both the morphological atypia of these lesion and the development of large bowel carcinoma.
Using urea-unmasked ultrathin sections, a postembedding staining method in combination with nitrous acid procedure has been established for the efficient visualization of sulfated glycoconjugates in a series of ultrastructures of three salivary glands of the rat. Pieces of the parotid, submandibular and sublingual glands from adult rats were subjected to glutaraldehyde-paraformaldehyde fixation, rinsed, dehydrated and embedded in LR-White resin. Ultrathin sections were prepared, grid-mounted, incubated in urea (6M) aqueous solution, and then stained with an alcian blue (AB) pH 1.0-phosphotungstic acid (PTA) sequence with or without prior treatment with nitrous acid. The results obtained have shown that sulfated glycoconjugates are involved in such ultrastructures as mast cell granules, basal lamina of arteriolar endothelial cells and intercellular matrix of arteriolar smooth muscle cells.
This paper describes a rapid, nonradioactive in situ mRNA hybridization method through the use of a chemically biotinylated DNA probe for the detection of human β1-IFN mRNA. The chemically biotinylated DNA probe is characterized with respects to: (1) the percentage of modification in nucleic acids analyzed by HPLC, (2) the sensitivity of detection, determined by reacting a series of quantities of biotinylated DNA with streptavidin-alkaline phosphatase, and (3) the hybridization specificity determined by hybridizing the biotinylated DNA to the in vitro synthesized β1-IFN sense RNA dotted on the filter. The optimal conditions of in situ mRNA hybridization were determined by investigating its specificity to the β1-IFN mRNA in the uninduced, or RNase-treated induced, or induced cells.
A rapid, quantitative in situ mRNA hybridization method was developed to study human β1-interferon gene expression through the application of biotinylated DNA probes and computer-assisted microscopy. The β1-interferon DNA probe was chemically biotinylated and used for the hybridization to β1-interferon mRNA in fixed human HT1080 cells induced with poly (IC). For the purpose of quantitation, a computerized microphotometric system was employed for acquisition of signal intensity generated by streptavidin-alkaline phosphatase or streptavidin-FITC (fluorescein isothiocyanate), which were used to detect the hybrids formed after in situ hybridization. About 60% of the cells exhibited hybridization signals in induced cells. The speed, specificity and quantitation of this non-isotopic in situ hybridization method should be generally useful to measure gene expression at the single cell level.
The livers of fetal rats were dissociated by collagenase treatment. Twenty-four hr after the dissociation, dexamethasone (DEX) or epidermal growth factor (EGF) was added to the culture medium, and hepatocytes were cultured for 3 more days. After 3 days in culture, clear boundary lines were observed between adjacent hepatocytes in the DEX-added medium under a phase-contrast microscope. A high level of activity of gamma-glutamyltranspeptidase (GGT) was observed along these lines by enzyme histochemical staining. On the other hand, the number of hepatocytes showing a positive reaction to immunohistochemical staining of alpha-fetoprotein (AFP) was decreased in the DEX-added medium. Moreover, bundles of actin filaments were formed along the periphery of cells in hepatocytes cultured in the DEX-added medium. In addition, in the same medium, the quantity of 3H-thymidine incorporated into the hepatocytes was less than that in the pure medium or in the EGF-added medium. When hepatocytes, cultured in the DEX-added medium, were examined under a transmission electron microscope (TEM), an accumulation of glycogen granules was observed in the cytoplasm, and bile canaliculuslike structures, consisting of large intercellular spaces, were observed between adjacent hepatocytes. In this study, it was indicated in vitro that DEX suppressed the proliferation of rat fetal hepatocytes, induced activity of GGT, and depressed the expression of AFP.
The secretion of proteinases and their resynthesis in the granular convoluted tubule (GCT) cells of mouse submandibular glands were studied after in vivo methoxamine (Methox) treatment (15mg/kg, i. p.), using a histochemical reaction with D-Val-Leu-Arg-4-methoxy-2-naphthylamide. The amidase reaction product was confined to the secretory granules in GCT cells, in unstimulated and stimulated glands. Methox caused extensive secretion of the granules. An increased staining reaction by the proteinases seemed to occur in granules prior to secretion, and fusions had often taken place. Reaccumulation of proteinases in the newly forming granules was found to start between 6 to 12hr after injection and the process was still incomplete at 48hr, but the granules showed less staining than at the earlier times. The variations observed in staining intensity may relate to the extent of binding with other proteins, and an unbinding of proteinases appears to be a possibility prior to the secretion of granules.
To elucidate the role of superoxide dismutase (SOD) in the uterine cervix and cervical cancer, we examined the immunohistochemical distribution of copper, zinc-superoxide dismutase (CuZn-SOD) in the human uterine cervix and cervical cancer. In the squamous epithelium, the basal, and the superficial, layers showed no or weak SOD imunoreactivity. In comparison, the prickle cell layer showed moderate immunoreactivity. In the columnar epithelium and cervical gland, ciliated cells showed moderate SOD immunoreactivity, whereas secretory cells showed no or weak reactivity. Subcolumnar reserve cells showed marked SOD immunoreactivity. These staining patterns were found to be constant throughout the menstrual cycle. In the carcinoma in situ, strong SOD immunoreactivity was seen in the cancer cells. In the invasive non-keratinizing and keratinizing squamous cell carcinomas, moderate immunoreactivity was seen. In the invasive adenocarcinoma of uterine cervix, marked immunoreactivity was seen in the well differentiated adenocarcinoma cells. On the other hand, no or weak immunoreactivity was seen in the moderately differentiated adenocarcinoma cells. Collectively, these results suggest that CuZn-SOD in uterine cervix might play an important role in the local defence mechanism against oxygen radicals and in the differentiation activity or reserve cells. Marked variability in CuZu-SOD immunoreactivity with various types of cervical cancers may reflect differences in cell proliferation kinetics. In the present survey of malignant tumors, we see no obvious relationship between generally assumed resistance to radiation or radical producing drugs and cellular immunoreactivity of CuZu-SOD. Futher studies are required to determine the value of CuZu-SOD analysis of tumors for the prediction of the response to radiation and redicalproducing drugs.
Ultracytochemical microscopy was used to characterize lysosomal morphology in luteal cells of normal, pseudopregnant, pregnant and nursing rat ovaries. Based on acid phosphatase (ACPase) reaction products, there were fewer lysosomes in luteal cells of newly formed diestrus-1, 7-day pseudopregnant, and 10- day pregnant corpora lutea than in aging corpora lutea or those of 18-day nursing rats. A unique type of lysosome, the nematolysosome, is described. It is tubular, elongated and thread-like, and only observed in degenerating luteal cells of aging and nursing corpora lutes. They were rarely observed in newly formed diestrus-1, 7-day pseudopregnant or 10-day pregnant corpora lutea where there was active secretion of steroids. The conclusion is that nematolysosomes are not a specialized population of lysosomes, but appear to be an intermediate structure of mobile large lysosomes which are involved in degeneration of endocrine steroidogenic cells.