The organic-inorganic relationship in calcified mitochondria.

Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl(4) intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca(2+)-binding sites in mitochondria and to the general problems of soft tissue calcification.

Mitochondrial inclusions in human cancer of the gastrointestinal tract.

Four cases of adenocarcinoma of the gastrointestinal tract have been examined. Large electron-dense mitochondrial inclusions were found in the mitochondria of many cells; after detailed histochemical tests they could be classified into two types. The first type of inclusion consisted of clusters of electron-dense, calcium-containing granules linked to a glycoproteic substrate. These inclusions were linked to a glycoproteic substrate. These inclusions were always associated with cristae and were found in the mitochondria of cells that showed clear signs of degeneration. The second type of inclusion was found much more frequently and consisted essentially of phospholipids of which electron density was strictly osmium dependent. Their structure was usually at least partly lamellar, but in some cases it was homogeneous throughout. It is hypothesized that inclusions of the second type may have the same biological role as the morphologically identical inclusions found in the mitochondria of brown fatty tissue in the perinatal rat and in yeasts during glucose repression or anaerobiosis. The resemblance between the homogeneous variety of inclusions within the second type and the mitochondrial inclusions recently described in human leukemic lymphoblasts and monoblasts has been stressed to bring out the need for a histochemical check on the supposedly viral nature of the latter inclusions.

An improved periodic acid-thiosemicarbazide-osmium technique to reveal glycoconjugates at the molecular level in situ.

The periodic acid-thiocarbohydrazide or thiosemicarbazide-OsO4 method (Seligman AM, Hanker JS, Wasserkrug H, Katzoff L: J Histochem Cytochem 13:629, 1965) has been modified in order to obtain a periodic acid-Schiff (PAS)-like reaction for electron microscopy capable of visualizing structures at the molecular level in situ. Thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) have been used dissolved in distilled water and bubbled with SO2. Treatment of previously oxidized thin sections with TCH (SO2) or TSC (SO2), followed by osmification, resulted in selective and very good staining of all the PAS-positive structures examined: glycogen, intestinal mucopolysaccharides, plasma membrane glycoproteins, basement membranes, Golgi apparatus, and collagen. The staining reaction was highly specific when TSC was used on thin sections from paraformaldehyde-fixed samples. The non-particulate end-reaction product made possible visualization of a periodic distribution of sugar residues in the 64-nm unit of collagen and the structural organization of the PAS-positive glycoconjugate components in the glomerular basement membrane.

Modifications of interphasic NORs as a diagnostic parameter of atypical lesions of the female breast.

Seventy-five breast samples including normal tissue, hyperplastic, metaplastic, atypical and neoplastic lesions were employed for the determination of interphasic Nucleolar Organizer Regions (NORs) modifications and Proliferating Cell Nuclear Antigen (PCNA) immunoreactivity. Interphase NORs were quantitatively and qualitatively modified in atypical lesions and breast carcinomas, whereas only modifications in the Ag-NORs count were found in benign samples. Our results investigated the nature of interphase NORs in the hope of finding a use for their evaluation in the diagnosis and biological clarification of breast epithelial atypia.

Silver staining of interphase nucleolar organizer regions in cytologic smears previously stained by the Papanicolaou and May-Grünwald-Giemsa techniques.

Malignant cells are known to display a distinctive argyrophilic nucleolar organizer region (Ag-NOR) protein distribution compared with distribution in their benign counterparts. In this study, Ag-NOR staining was applied to smears from reactive and malignant serous effusions. The smears were either unstained or had been previously stained with the Papanicolaou or May-Grünwald-Giemsa (MGG) technique. The purpose of this study was to assess the feasibility and usefulness of Ag-NOR staining in diagnostic cytopathology and to set up a procedure that could be used on prestained smears in retrospective studies. The one-step silver staining method was applied to prestained smears after a weak destaining step in trichloracetic acid and to unstained smears after a postfixation step in Carnoy's fluid. Positive results were observed on both destained and unstained preparations, MGG-stained smears showing a better visualization of the silver deposits than Papanicolaou-stained smears. Major differences in size, shape, and distribution patterns of the Ag-NOR-positive granules were observed between neoplastic and reactive cells. Furthermore, the mean number of silver-stained dots per cell was significantly higher in malignant cells (33.32 +/- 2.23) than in reactive mesothelial cells (9.71 +/- 0.66). These data indicate that this Ag-NOR technique can be profitably applied to prestained cytologic smears to assist in the diagnosis of malignancy and that the technique has the advantage that cellular morphology and silver staining can be evaluated on the same slide.

Ultrastructural localization of sulphate glycoconjugates in the human glomerular capillary wall using the high iron diamine method.

The distribution of fixed anionic sites within glomerular capillary walls has been studied in man by applying two ultrastructural histochemical methods--the high iron diamine and dialysed colloidal iron methods--to tissue chopper sections and to isolated glomeruli obtained from surgical fragments of renal tissue. By using the high iron diamine method we have been able to demonstrate that in man, too, there are sulphate (possibly heparan sulphate proteoglycan) sites preferentially located in the lamina rara esterna of the basement membrane and in the cell coat of the urinary surface of podocytes. Non-sulphate (high iron diamine-negative, dialysed colloidal iron-positive) anionic sites have been identified not only in the glycocalyx of the epithelial and endothelial cells but also in the laminae rarae of the basement membranes, where they show a more extensive distribution pattern than sulphate sites. The proposed methods seem particularly suitable for the study of human renal tissue; they could, in fact, provide useful information about the behaviour of the various anionic components of the glomerular capillary wall in pathological conditions.

Telephonic transmission of cardiotocographic recordings from a remote facility to an obstetric unit.

The Authors describe their experience in telephonic transmission of cardiotocographic recordings. With a Corometrics 116 and 410 unit installed in a remote facility, 61 CTG recordings were tele-transmitted and received by a Toshiba personal computer. The concordance between original and transmitted CTG record was excellent and a very low percentage of signal loss during transmission (3.2%) was found. The advantages of telephone transmission of CTG in peripheral areas are stressed.

[A case of nodular pseudosarcomatous fasciitis treated with "presuture" technique]. / Su di un caso di fascite nodulare pseudosarcomatosa trattato con tecnica di "presutura".

This paper reviews a case of nodular (pseudosarcomatous) fasciitis. It consists of a rare benign lesion of the soft tissues. The rapidity of its growth and the cellular features represent a diagnostic challenge when compared with that of a sarcoma. The patient, a 36 year old female, 20 days before surgical treatment, noticed the growth of a nodular lesion on the left forearm skin. After echotomography and ultrasound guided needle biopsy diagnosis of nodular pseudosarcomatous fasciitis was established and the patient underwent surgical treatment. The nodular lesion, noncapsulated, (diameter 2.5 x 1.2 cm) greyish-white colored, with slimy consistency, and myxoid character, was microscopically composed by atypical fibroblasts, with myxoid ground substance. The nodular lesion was removed using the "pressure" technique by Serge Baux of Rothschild Hospital in Paris using two Kirschner threads pulling the skin surrounding the biopsy wound. By this technique was possible to achieve a rapid skin expansion. The nodular pseudosarcomatous fasciitis remains a very difficult lesion to recognise unless the aid of a biopsy.

Critical analysis of the use of the acrolein-Schiff method as a possible DNA reaction.

Histophotometric examination was carried out on nuclei of lymphocytes in human peripheral blood, which were subjected to various tests in order to assess the acrolein-Schiff method as a possible DNA specific reaction, in comparison with the traditional Feulgen reaction. Special attention was paid to the degree of difference between responses attributable to a direct Schiff reaction obtained in the fraction of nuclear proteins after treatment with acrolein. From the results obtained it appears that an acrolein-Schiff reaction, following extraction of proteins, may be considered a qualitative reaction for DNA. Our findings also show that there is no relationship between the degree of response to the acrolein-Schiff reaction and that the Feulgen reaction, which is to be expected in view of the different mechanisms of the two reactions.

Effects of alpha-amanitine on chromatin in regenerating rat hepatocytes: a biochemical and morphologic study.

Almost all the chromatin in nuclei of regenerating rat liver hepatocytes 15 h and particularly 24 h after partial hepatectomy appeared as decondensed chromatin. Treatment with alpha-amanitine induced a clear condensation of decondensed chromatin as early as 30 min after toxin injection. The degree of condensation increased further until, 2 h after poisoning, almost all the chromatin was found to be in condensed form. Because the synthesis of DNA, after toxin injection, was not modified during the first 2h and 30 min, our results indicate that the chromatin condensation did not affect the synthesis of DNA. In the present paper the relationships between the inhibition of RNA synthesis and the chromatin ultrastructural changes are also discussed.

Congenital extracardial malformations accompanying congenital heart disease.

A statistical review of 1,354 autopsies performed in cases of congenital heart disease in three major Italian hospitals was carried out. In 446 cases one or more extracardial malformations were found; those most commonly encountered were malformations of the urinary tract, anomalies of the respiratory tract, malformations of the intestine and spleen and trisomy 21. Ventricular septal defects and persistent common atrioventricular canals occurred in a higher percentage of cases with extracardial malformations than in those where congenital heart defects were the only malformations. The reverse was true of endocardial fibroelastosis and transposition of the great vessels. Ventricular septal defects are particularly common in the Klippel-Feil syndrome, in trisomy 18 and, together with a persistent common atrioventricular canal and atrial septal defects, in trisomy 21. The typical picture of so-called "congenital heart disease associated with asplenia" was found in cases with splenic agenesis, either with or without situs inversus viscerum, but also in cases with situs inversus viscerum without asplenia.

Presence of an alpha-galactolipid on the cell surfaces of endothelial cells of human kidney.

The presence of an alpha-galactolipid was investigated with a peroxidase-labelled lectin from Griffonia simplicifolia (GSA-I) with specific binding for terminal alpha-D-galactose residues. Normal kidney tissue was obtained from patients undergoing nephrectomy for renal neoplasms. For light microscopy, tissue was snap-frozen; 4 microns-thick sections were briefly fixed in paraformaldehyde and incubated with GSA (0.025 mg ml-1). The peroxidase activity was developed with 3-amino-9-ethylcarbazole. Adjacent sections were stained at the same time after lipid extraction with 3:1 (v/v) chloroform/methanol. For electron microscopy, 0.2-0.5 mm-thick paraformaldehyde-fixed blocks, with or without lipid extraction, were stained with peroxidase-labelled GSA. The label was developed with diaminobenzidine and osmium tetroxide. Some structures, such as tubular epithelia, stained both in lipid-extracted and non-extracted tissues, suggesting that glycoproteins were most likely involved. In addition, tissue stained immediately after fixation showed GSA reactivity on endothelial cell surfaces of intertubular capillaries and larger vessels. In lipid-extracted tissues, however, tubular epithelium was still positive for GSA but endothelial cells failed to stain. These findings suggest that a glycolipid, bearing a terminal alpha-galactose residue, is present on the endothelial cells in human kidney and possibly on tubular epithelia. Our data may explain the preferential storage of alpha-galactolipid in endothelial cells of patients with Fabry's disease and other biological phenomena such as Escherichia coli adhesion.

Carbohydrate histochemistry of human Brunner's glands.

Brunner's glands are known to produce neutral mucins. In order to achieve a better knowledge of their carbohydrate profile, we used five peroxidase-labeled lectins on surgical specimens of human duodenum. This method allowed us to identify at least two different types of neutral mucins in Brunner's glands secretion, thus demonstrating a heterogeneous mucin production. The structure of terminal oligosaccharidic chains in these glycoproteins has also been hypothesized.

Ultrastructural histochemical investigations of "dense deposit disease". Pathogenetic approach to a special type of mesangiocapillary glomerulonephritis.

Dense deposit disease is characterized by the presence of intramembranous dense deposits; their constituents are unknown but immunological and biochemical studies have demonstrated that they contain no gamma-globulins or any other plasma protein. In order to clarify the nature of the dense deposits better, we investigated their most distinctive character, (marked electron-density) by means of ultrastructural histochemistry techniques using thin sections from Formaldehyde fixed, OsO4 postfixed and Epon embedded specimens collected for diagnostic electron microscopy. The dense deposits have a higher osmium affinity than the lamina densa of normal basement membranes, and the electron-density is strictly osmium-dependent suggesting the presence of a lipid component. Further data, obtained using an extraction method for lipids, seems to confirm our hypothesis.

Interphasic nucleolar organizer region distribution as a diagnostic parameter to differentiate benign from malignant epithelial tumors of human intestine.

The distribution of the interphasic nucleolar organizer regions (NORs) has been investigated in five hyperplastic polyps, five adenomatous polyps and fifteen colonic adenocarcinomas. The study was performed using electron microscopy and paraffin-embedded sections stained for Ag-NOR proteins. Malignant tumor cells were characterized by a large number of NORs which were small in size and showed a scattered distribution. Nuclei of both types of polyp had only a small number of large-sized NORs in a clustered distribution. In two adenomatous polyps, cells were also observed with an NOR distribution pattern intermediate between that of frankly benign and malignant lesions.

Non-specific binding of immunoglobulins and protein A-Gold complex with cytoplasmic granules of human mast cells.

A non-specific staining of mast cells by a number of proteins and immunoglobulins has been observed using different immunocytochemical techniques and different antisera at light and electron microscopy level. This finding has been related to an ionic binding of cationic charges, such as aminic groups with an high pI, to the strongly anionic residues of mast cell granules. This hypothesis is supported by the lack of non-specific binding achieved using negatively charged chromogens (i.e. FITC--or Rhodamine-conjugated antibodies) or using cationic dyes before the immunohistochemical reaction. It is therefore stressed that adequate and accurate negative controls must always be carried out in order to get correct interpretations of the staining results.

Visualization of the secretory canaliculi of human parietal cells with a peroxidase-labelled peanut lectin. Light- and electron-microscopic observations.

Peanut lectin reactivity was examined in normal fundic glands from human gastric samples, both at light- and electron-microscopic levels, using a peroxidase conjugate. Positive reaction was observed in the glycocalyx of parietal cell secretory canaliculi as well as in the mucous globules of mucous cells and in the luminal cell coat of chief cells. The presence of terminal galactose in the canalicular glycocalyx may be connected with the peculiar function of hydrochloric acid secretion. Peroxidase-labelled peanut lectin is proposed as a marker for visualizing the secretory canaliculus of parietal cells.

A comparative study of histopathology, hormone receptors, peanut lectin binding, Ki-67 immunostaining, and nucleolar organizer region-associated proteins in human breast cancer.

The current study was performed on 71 cases of human female breast cancer and compares the results of five morphologic methods developed for the detection of estrogen receptors (ER), progesterone receptors (PgR), lectin Peanut agglutinin (PNA) binding sites, monoclonal antibody Ki-67 immunoreactivity, and the mean number of argyrophilic nucleolar organizer regions (Ag-NOR). All the parameters were evaluated on serial cryostat sections representative of a closely related, if not identical, neoplastic population. A significant positive correlation was found between the occurrence of estrogen, progesterone, and peanut receptors and between Ki-67 immunoreactivity, mean number of NOR, and mitotic index. Furthermore, ER, PgR, and PNA receptors showed a significant, inverse correlation with Ki-67 immunoreactivity, mitotic index, and mean number of Ag-NOR. These results provide further data that support the hypothesis that (1) progesterone and PNA receptors are estrogen-induced and indicate a metabolic response of the target cells to functioning estrogen receptors; (2) the mean number of NOR reflects the cell kinetics of the tumor; and (3) metabolic differentiation of neoplastic cells is inversely correlated to the proliferation index.