Selective light microscopic demonstration of the specific granulation of the rat atrial myocardium by lead-hematoxylin-tartrazine.

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

Selective Light Microscopic Demonstration of the Specific Granulation of the Rat Atrial Myocardium by Lead-Hematoxylin-Tartrazine

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

Predicting the effects of chlorine on the micro-organisms of filamentous bulking activated sludges

Rapid and definite assessment of the effect that a specific biocide has on a specific case of filamentous bulking sludge is a much-needed tool in activated sludge wastewater treatment. The Live/Dead stain (LIVE/DEAD BacLight) distinguishing "living" and "non-living" cells, a nitrifying activity (NA) test and the oxygen uptake rate (OUR) measurement were examined for their appropriateness to predict the effects of chlorine on filamentous bulking sludges. The study showed the live/dead stain to be relevant for revealing the specific effect of chlorine on the filamentous bacteria of a bulking sludge. However, using live/dead stain alone for the determination of the appropriate chlorine dose against bulking may lead to an underestimation of the damage caused by chlorine to the useful microorganisms in the flocs. Indeed, using the live/dead stain, it was not easy to distinguish dead cells caused by chlorination from those originally present in the flocs The NA test was the most sensitive in detecting damage caused by chlorine to the floc-forming microorganisms. Therefore, for a safer determination of the chlorine dose effective against bulking and protective of the microbial activity of the sludge, the results of this study suggest coupling of the live/dead stain with the NA test and/or the OUR test.     

Immunochemical analysis of myosin heavy chains in the developing chicken heart

Monoclonal antibodies (McAb) against myosin from the pectoralis muscle of the adult chicken have been generated and shown to react specifically with the myosin heavy chain (MHC). The reactivities of two such McAbs with myosin from adult chicken atrial and ventricular myocardium were further analysed by immunoautoradiography, radioimmunoassay, and immunofluorescence microscopy. Monoclonal antibody MF 20 was found to bind both atrial and ventricular MHC and stain all striated muscle cells of the adult chicken heart. In contrast, McAb B1 bound specifically to atrial myocytes in immunofluorescence studies, while immunoautoradiography and radioimmunoassay demonstrated the specificity of this antibody for the atrial MHC. Upon reacting these McAbs with myosin isolated from embryonic hearts where definitive atria and ventricles were present, the same specificity of antibody binding was observed. Immunofluorescence studies demonstrated that all striated muscle cells of the embryonic heart contained MHCs recognized by MF 20, while only atrial muscle cells were bound by B1. When extracts of presumptive atrial and ventricular tissue were reacted with MF 20 and B1, significant reactivity of MF 20 was first observed at stage 10 in the presumptive ventricle and thereafter this McAb reacted with all regions of the developing myocardium. Binding of B1 was detected approximately 1 day later at stage 15 and was confined to atrial-forming tissues. These data demonstrate antigenic similarity between adult and embryonic MHC isolated from atrial myocardium and suggest the expression of an atrial-specific MHC early in the regional differentiation of the heart.     

A method for the harvest,culture, and characterization of human adult atrial myocardial cells: Correlation with age of donor

Summary Myocardial cell culture methods are now well established for animal and fetal human tissue. We present here a method for harvesting and culturing adult human atrial myocardiocytes. Cells are obtained from fresh atrial tissue normally discarded after being removed to cannulate the right atrium during open heart surgery. The atrial tissue is minced and then digested using collagenase. The single cell suspension is initially cultured in serum-containing growth medium, then transferred to defined medium, selective for myocardial cell growth. The cells are characterized by immunoperoxidase stains and transmission electron microscopy. The cultured cells stain positive for myoglobin, whereas control cultured fibroblasts and endothelial cells do not. Electron microscopy shows the presence of numerous myofibrils, Z-bodies, pleomorphic mitochondria, and secretory granules. The chronological age of the donor was an important factor in culturing the adult tissue, the younger tissue correlated with a higher success rate. This method provides a means for in vitro study of human adult myocardial cells and provides guidelines for appropriate atrial tissue to use.     

Comparative study of microbiological and histopathological techniques used for the detection of Helicobacter pylori

This study evaluates the sensitivity, specificity and predictive values of several techniques commonly used for the detection of Helicobacter pylori in an analysis of 105 biopsy specimens (gastric and duodenal). For comparative purposes, the techniques investigated were divided into 2 groups: histopathological and microbiological. The former included hematoxylin-eosin and Giemsa stains, a Gram stain modified for use in tissue, and immunohistochemical techniques. Microbiological analysis was performed using culture, the urease test and the conventional Gram stain. The immunohistochemical techniques proved to be the most sensitive (93%). The modified Gram stain was sufficiently sensitive (92%) and specific (97%) for the detection of the bacterium. When combined with a microbiological technique such as the urease test, this stain showed increased sensitivity (96%) but its specificity was reduced to 94%. This combination of tests is recommended for the detection of H. pylori in biopsy specimens since it is easily performed at low cost and gives excellent results. For economical reasons, it is suggested that the use of immunohistochemical techniques should be restricted to specific cases.     

Ultrastructure of atrial and ventricular myocytes of newborn rats: evidence for the existence of specific atrial granule-like organelles in the ventricle

The present study examined the ultrastructure of atrial and ventricular myocytes from the heart of newborn rats. It was found that, despite former reports stating that ventricular myocytes in adults do not contain cytoplasmic granules, specific atrial granule-like organelles are present in the ventricles of rats at birth. The presence of these granules together with the relatively underdeveloped contractile apparatus and extensive Golgi complex suggests that the ventricular, like the atrial, myocytes may have an endocrine function before or at birth. Further study is required to determine whether these ventricular cytoplasmic granules contain the same atrial natriuretic peptide species known to be present in the atrial specific granules.     

Observations on specific giemsa staining of the Y and on selective oil destaining of the chromosomes.

The human Y chromosome can be differentially stained with Giemsa using simple procedures. This phenomenon is strikingly to that observed with quinacrine fluorescence. The specific Giemsa-Y stain may be selectively removed by the action of an oil. The same oil, under certain conditions, selectively removes Giemsa stain from all chromosomes, resulting in R- and T-banding patterns. These bands, which are obtained through subtraction of dye from Giemsa-stained chromosomes, allow slides to be further processed.     

Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.     

Vital Fluorescent Staining Technique for Microspores of Brassica Napus

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus.It provided high contrast when used in combination with Tinapol 5BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.     

Vital fluorescent staining technique for microspores of Brassica napus.

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.     

Atrial natriuretic polypeptide in human adrenal pheochromocytoma: immunohistochemical and immunoelectron microscopical localization

Using the immunoperoxidase and immunogold methods with specific antibody, we studied the atrial natriuretic polypeptide (ANP) in seven tumor tissues of six patients with adrenal pheochromocytoma. Light microscopically, the reaction product for ANP was observed in all seven tumor tissues. Intracytoplasmic immunoreaction product for ANP was finely granular. In four cases studied with the electron microscope, the immunogold stain for ANP was demonstrated in secretory granules of the tumor cells. A considerable amount of alpha-hANP immunoreactive substance was also extracted from two tumor tissues (67.2 and 28.7 pg/mg wet tissue). This is the first report of the human adrenal pheochromocytoma that contains immunoreactive ANP. These findings provide additional evidence for the multisecretory APUD cells of neural crest origin.     

Immunohistochemical localization of intestinal phospholipase A2 in rat paneth cells

Summary Using the peroxidase-anti-peroxidase (PAP) technique with a specific rabbit anti-swine intestinal-phospholipase-A2 serum, the immunoreactivity of this phospholipase A2 was localized in rat-intestinal Paneth cells. The specific rabbit anti-swine intestinal-phospholipase-A2 serum did not stain the rat-pancreatic acinar cells which were stained by a specific rabbit anti-swine pancreatic-phospholipase-A2 serum. Specific rabbit anti-swine pancreatic-phospholipase-A2 serum did not stain rat-intestinal Paneth cells. Therefore, there is no cross-immunoreactivity between pancreatic and intestinal phospholipases.     

Atrial-specific granules in the hearts of normal and water-deprived rats

Summary The morphology of atrial-specific granules, which contain atrial natriuretic polypeptide (ANP), was studied in the cardiac tissue of untreated controls and water-deprived rats by means of conventional and immunoelectron microscopy. Immature secretory vesicles or granules appeared to become buded off from the Golgi cisternae and then fused to form specific A-granules. An electron-dense plate with a fuzzy coat was frequently found on the limiting membrane at the end of such fusion. Pale specific B-granules, which were less electron-dense, larger, and more granular than A-granules, were found in small numbers in the left atrial cardiocytes, but rarely in the right ones. Very pale granules with a less granular matrix, considered to be B-type granules which had lost their electron-density, and which had less immunoreactivity for ANP, were numerous in the cardiac tissue after water deprivation. This morphological change, which is interpreted as an indication of granule degradation, was in agreement with the noted increase of natriuretic activity in the atrial tissue of water-deprived specimens.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

Ultrastructural histochemistry of the surface lamina of normal articular cartilage

Summary Two collagen-poor, ultramicroscopic layers are described at the surface of canine articular cartilage. They are distinguished by staining with an electron-dense cationic dye, Cupromeronic Blue, in a critical electrolyte concentration technique and by digestion with testicular hyaluronidase. The superficial layer, approximately 50 nm thick, stained at low electrolyte concentrations but failed to stain in conditions specific for sulphated glycosaminoglycans. It was hyaluronidase-resistant and may be either glycoprotein or protein in nature. The deeper layer, 100–400 nm thick, stained positively at electrolyte concentrations specific for sulphated glycosaminoglycans but not in conditions specific for keratan sulphate. It was removed by hyaluronidase digestion. This layer probably represents a chondroitin sulphate-rich proteoglycan.These surface layers may be important in the lubrication of the articular surface and in the permeability and compression resistance of the superficial cartilage zone.     

Effects of isoproterenol on the dense core and perigranular membrane of atrial specific granules

Summary Following subcutaneous injections of isoproterenol hydrochloride (ISO), atrial cells present a large number of partly degranulated or completely clear specific granules enclosed by an intact membrane. Such profiles were never encountered in normal controls and might suggest ISO-induced release of a secretory product. Permeability of perigranular membrane was tested using the extracellular macromolecular tracer horseradish peroxidase (HRP). Reaction product was entirely absent within granules of atrial cells in which the sarcolemma was made permeable to HRP molecules by the ISO injections. This seemed to be the case even in heavily labelled cells in which the peroxidase had penetrated the mitochondrial membranes. In atrial cells impermeable to the tracer, the specific granules closely apposed to the sarcolemma were always HRP-negative. The release mechanism of a possible secretory substance from the specific granules is discussed.     

Localization of wheat-germ agglutinin-binding sites in the Golgi complex of cultured rat atrial myocytes

Summary In the Golgi region of cultured rat atrial myocytes, condensed secretory protein was seen in Golgi-associated tubules or cisternae which lay beyond, and often separated from, the remainder of the Golgi stacks. These structures appeared to be involved in packaging of condensed secretory protein into atrial granules. Binding sites of HRP-conjugated wheat-germ agglutinin (WGA) in saponin-treated cultured atrial myocytes were examined by electron microscopy with special reference to atrial granules and the tubular structures associated with the Golgi stacks. HRP reaction products were observed in both trans-cisternae of the Golgi stacks and the associated tubular structures. While the majority of atrial granules were devoid of reaction products, some granules, which were connected to the WGA-positive tubular structures in the vicinity of the Golgi trans-cisternae, showed HRP reaction products at their connected necks. Similar results were obtained when sections of the cells embedded in Lowicryl K4M were labeled with WGA coupled to colloidal gold (G-WGA); the Golgi complex was G-WGA positive, whereas no specific binding of G-WGA to atrial granules was observed. These results suggest that glycoproteins and/or glycolipids with oligosaccharides recognized by WGA in the Golgi transcisternae, may be separated from atrial natriuretic peptides which are packaged into atrial granules.Abbreviations ANP atrial natriuretic peptide - HRP horseradish peroxidase - M199 medium 199 - TGN trans-Golgi network - WGA wheat-germ agglutinin - G-WGA WGA coupled to colloidal gold     

Plasma levels of human atrial natriuretic peptide in patients with hypertensive diseases

Three types of antihuman atrial natriuretic peptide antiserum were obtained. From the study of cross-reactivity to human atrial natriuretic peptide fragments, it was suggested that antisera-1, -2, and -3 are mostly specific to 1-28, 5-25, and the ring structure, respectively. The estimated values of this hormone were significantly lower in the order of antisera-1, -2, and -3. Moreover, high performance liquid chromatographic study showed that various types of fragments of atrial natriuretic peptide exist in human plasma. These findings suggested that the highly specific antiserum to 1-28 human atrial natriuretic peptide such as antiserum-1 should be used to estimate the 1-28 human atrial natriuretic peptide levels in human plasma. From the study by using antiserum-1, it was concluded that the plasma human atrial natriuretic peptide increased in essential hypertensives, and in patients with primary aldosteronism, chronic renal failure, and malignant hypertension. Regarding the pathophysiological significance of increased plasma atrial natriuretic peptide, it is unlikely that this plays an important role in the etiology of essential hypertension or other hypertensive diseases, because the plasma level of this hormone is elevated in these patients. The increase of plasma atrial natriuretic peptide level in these patients should be considered to be a secondary or compensatory reaction to high blood pressure.     

Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart

Summary The ultrastructure of porcine ventricular tissue was studied by electron microscopy and immunocytochemical techniques. Electron-dense specific granules were found in both Purkinje fibers and transitional cells in the ventricular walls, and were positively stained by the immunogold staining method using an antiserum against atrial natriuretic polypeptide (ANP). This suggests that both the Purkinje fibers and transitional cells display the same specific granules as atrial cardiocytes containing ANP. These results demonstrate that Purkinje fibers and two types of transitional cells, in addition to the ordinary ventricular cardiocytes, can be identified in porcine ventricular wall tissue.