Hybridization of a nucleic acid probe to nucleic acids within cytological preparations permits a high degree of spatial localization of sequences complementary to that probe. This localization can be useful in a number of ways for answering biological questions. For example, in situ hybridization to the DNA of condensed chromosomes can be used to map the sites of particular sequences. Hybridization to the DNA of interphase nuclei can be used to study the functional organization of specific sequences within the diffuse chromatin that characterizes this stage of the cell cycle. In situ hybridization to cellular RNA allows a very precise analysis of the tissue distribution, as well as the temporal distribution, of any RNA species of interest. In some cases, the distribution of RNA within single cells has interesting biological implications. In addition, in situ hybridization makes it possible to study the RNA of individual cells without interference from the RNA of other cells in the same tissue. Thus it is possible to use the technique to detect RNAs that are present in only a small subset of cells. Such RNAs might never be detected in RNA extracted from a whole tissue because of dilution by other RNA species from the many cells which do not contain the RNA of interest. Because it depends on the concentration of RNA within a cell, rather than within a tissue, in situ hybridization is useful not only to show where an RNA is localized but, in some cases, it may be the best way to show that an RNA exists at all.
Since lysosomes play an important role in maintaining cellular homeostasis by degrading exogenous and endogenous macromolecules with a variety of lysosomal hydrolytic enzymes, wide attention from various aspects has been paid in detail to cellular phenomenon associated with lysosomal activity. Recently, it became evident that lysosomes are mobile, dynamic organelles; they move actively through the cytoplasm to carry out their functions in intracellular digestion, i. e. in heterophagy and autophagy. However, little is known about the regulatory mechanism of intracellular lysosomal movement. In the first part of this paper, we demonstrate that cytoskeletal elements such as actin filaments and microtubules are necessary when lysosomes reveal polymorphic behavior in their shape and size during the heterophagic process by detecting the effects of cytoskeleton-affecting drugs. This is further supported by the morphological evidence that the direct interaction of lysosomes and cytoskeletal elements can be observed in vivo and in vitro. In the second half of this paper, by using cryo-ultrathin section labeling technique, the nature of membranes and matrices of autophagic vacuoles and lysosomes in hepatocytes was examined with respect to the distribution of electric charges and the lectin-binding pattern.
Recently, attention has been focused on a distinct type of secondary lysosome, the tubular lysosome. These lysosomes have been reported in a number of different cell types. In most instances, the tubular lysosomes have been identified as a distinct population of lysosomes solely on the basis of their morphology. Thus, the question remains whether or not the tubular lysosomes are a separate population. In order to address this question, we have examined the tubular lysosomes in exocrine acinar cells. These cells are highly polarized, with the tubular lysosomes being concentrated near the basal surface of the cells and the more typical secondary lysosomes located near the Golgi apparatus. The tubular lysosomes often form an anastomosing network, are intercalated between cisternae of rough endoplasmic reticulum and are frequently associated with mitochondria and microtubules. Cytochemically, these lysosomes are unique. In contrast to the typical secondary lysosomes, they appear to lack acid phosphatase, but they do react for trimetaphosphatase, aryl sulfatase B, and non-specific esterase. The tubular lysosomes in the exocrine acinar cells can also be distinguished on the basis of their participation in the endocytic process. When exocrine acinar cells are exposed in vivo to intravenously injected horseradish peroxidase, the tracer is sequestered in the tubular lysosomes within 5-10 min after injection, while the peroxidase does not appear in the secondary lysosomes for 2-3 hr. In addition, the tubular lysosomes can be separated from the secondary lysosomes on Percoll density gradients. The presence of the tubular lysosomes is also related to the state of differentiation of the acinar cells. In undifferentiated AR42J cells, a pancreatic acinar cell line, tubular lysosomes are extremely rare, but when the cells are stimulated to differentiate with dexamethasone, tubular lysosomes are seen adjacent to the plasma membrane. These results confirm the fact that, at least in exocrine acinar cells, the tubular lysosomes are distinct from the typical spherical secondary lysosomes.
Precursor forms of lysosomal cathepsins B, H and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39-, 41-, and 39-kDa, respectively, by immunoblotting analysis. The proenzymes were then concentrated by applying the microsomal contents to a concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B, H and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased. Immunoblotting analysis showed that after 36 hr incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increased of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin which is a potent inhibitor of aspartic proteinases. The results strongly suggested that a processing proteinase for procathepsins B, H, and L might be cathepsin D, a major lysosomal aspartic proteinase. Indeed, lysosomal cathepsin D could convert the immunoaffinitypurified microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B, H, and L seem to be firstly synthesized as the enzymatically inactive forms in endoplasmic reticulum and may successively be converted into the active forms by cathepsin D in lysosomal compartments.
Lysosomal storage diseases are usually classified into three categories: accumulation of sphingolipids (e. g. Gaucher disease and GM1-gangliosidosis, etc.), accumulation of mucopolysaccharides (e. g. Hurler disease and Hunter disease, etc.) and glycogen storage disorder, as in Pompe disease. All three categories showed a variation of severity and time of onset, etc. These variations have been studied biochemically and genetically (1, 4, 21, 24, 25, 28, 29, 33, 35, 37, 40). The transgenic mouse is considered to be a useful animal model for lysosomal storage diseases. Additionally, the enzymology of lysosomal enzymes has also advanced recently, including knowledge of the intracellular processing mechanism of the enzyme and of the cDNA sequence (5, 7, 9, 10-12, 14, 21-25, 30-34, 36, 38, 41-43). Referring to these recent advances, and based on our previously reported results, we would like to discuss the pathological aspects of the lysosomal storage diseases. In this report, we classified the lysosomal storage diseases into two groups, A and B, according to the types of cells containing the storage substances: In group A, the storage substances are mainly found in the parenchymal cells; in group B, they are observed mainly in the macrophages and other cells of the mononuclear phagocyte system. Pompe disease, in which there is a deficiency of acid a-glucosidase and Hurler disease, in which there is a deficiency of α-L-iduronidase were investigated in groups A and B respectively.
Ultrastructural examinations of skin and/or rectal biopsy specimens were performed in 45 cases of various lysosomal storage diseases, including Pompe's disease, mucopolysaccharidoses I-IV, GM1 & GM2 gangliosidoses, Niemann-Pick disease, Gaucher's disease, metachromatic leukodystrophy, Fabry's disease, sialidosis, I-cell disease, mucolipidosis III, mannosidosis, fucosidosis, and ceroidlipofuscinosis. We found characteristic storage inclusions in all disorders except for Gaucher's disease, probably indicating the absence of storage outside the reticuloendothelial system. In some cases of adult-onset neurolipidosis and ceroid-lipofuscinosis, the examination of the rectal submucous plexus neurons was important to detect characteristic inclusions, suggesting some superiority of the rectal biopsy to the skin biopsy in these conditions. On the contrary, because of the absence of the myelinated fibers in the rectal mucosa, the skin biopsy seems to be superior to the rectal one in diseases affecting the myelin sheath primarily. These morphological studies about skin and/or rectal biopsy specimens are useful to support the diagnosis obtained by the biochemical methods, to be a clue to diagnosis of cases in which biochemical defects of the disease are still unclear, and to make a probable diagnosis of some lysosomal storage diseases in atypical cases showing neurodegeneration.
Three-dimensional organization of synaptic glomeruli in the rat substantia gelatinosa was investigated using high voltage electron microscopy. In the present study, thiamine monophosphatase activity was used as a marker enzyme for synaptic glomeruli because it has been selectively localized in synaptic glomeruli in the spinal cord. The synaptic region of dense sinusoid axon terminals forming the central part of synaptic glomeruli displayed a rounded appearance. The thiamine monophosphatase activity was irregularly presented on the surface of terminal knobs and preterminals, and weak reaction products were observed in the interior of cross-cut terminal knobs. This study suggested that the primary afferent fiber formed the complicated synapses which are pre- and postsynaptically surrounded by a large number of processes of intradorsal neurons.
The phenotype and distribution of mesenchymal cells were studied immunohistochemically in thioacetamide-induced hepatic fibrosis in rats, using monoclonal antibodies specific to smooth muscle actins, muscle actins, desmin and vimentin, respectively. All studies were performed in tissues fixed with methanol-Carnoy's fixative and embedded in paraffin. In normal liver, scattered desmin-positive cells were recognized in the periportal areas. In the early stages of septal fibrosis, desmin-positive cells appeared around the central veins first and increased in the fibrous strands. With progression of fibrosis, desmin-positive cells decreased, while muscle actin-positive cells increased predominantly within the fibrous septa. Muscle-actin positive cells surrounded each parenchymal nodule in a circular pattern, suggesting their important role in the formation of nodularity of fibrotic liver. Examining the change in phenotype of mesenchymal cells during progressive liver fibrosis has given us new insight regarding cirrhosis pathogenesis.
We improved the in situ hybridization method using biotinylatyed probes for the detection of human papillomavirus (HPV) 16 and 18 DNA in formalin-fixed and paraffin-embedded tissue sections. Our method included HCl treatment, digestion with 30μg/ml proteinase K, post-fixation with 70% ethanol and incubation with a streptavidin-biotinylated alkaline phosphatase conjugate for visualization of biotin molecules on DNA probes. The sensitivity of this method using biotinylated DNA probes was comparable to using 35S labeled RNA probes. Using this method, 72 cases of cervical dysplasia and 51 cases of squamous cell carcinoma of cervix were examined for the presence of HPV 16 and 18 DNA. HPV 16 DNA was detected by in situ hybridization in 19 (26%) cases: 8 of 17 (47%) with mild dysplasia, 4 of 24 (16%) with moderate dysplasia and 7 of 30 (23%) with severe dysplasia. In 25 cases of carcinoma in situ and 26 cases of invasive carcinoma, HPV 16 or 18 DNA was detected in 8 cases of which 4 cases had an associated nonmalignant lesion. All four cases showed that the intensity in the malignant lesions was less than the dysplasia or benign lesions in the same patients. These findings suggest that replication of HPV DNA might be suppressed in the transformed cells.
Thiamine mono- and pyrophosphatase (TMPase, TPPase) activities were demonstrated in the rat small ganglion cell under high-voltage electron microscopy. From a series of micrographs of the same field tilted in increments, a trans tubular network (TTN) consisting of looping and anastomosing tubules was found; this network showed relatively higher intense TMPase activity, and exhibited connections with TMPase-positive vesicle-like bodies. TPPase activity was localized in the trans Golgi saccules, and the TPPase-positive transmost saccule had a structural resemblance to the TTN. These results indicate that the TTN plays an important role in thiamine metabolism.