Showing papers in "Histochemistry and Cell Biology in 1983"
TL;DR: Poly-l-lysine has been used to coat glass slides in the preparation of tissue sections for immunocytochemical staining and it has been found that the higher the molecular weight of the polymer, the greater the adhesive force it provides.
Abstract: Poly-l-lysine (PPL) has been used to coat glass slides in the preparation of tissue sections for immunocytochemical staining. The adhesive properties of different molecular weight (m.w.) polymers of l-lysine have been tested on pre-fixed cryostat sections which were subjected to a 3 day washing treatment. It has been found that the higher the molecular weight of the polymer, the greater the adhesive force it provides. PLL (m.w. 350,000) at concentrations in the range of 0.05–0.1% was found to be the most effective polymer.
346 citations
TL;DR: Satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained speciments by reducing the DAPI concentration to 50 ng/ml and light scattering became negligible after reducing the intensity of the excitation light.
Abstract: DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.
161 citations
TL;DR: Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immonoreactive cells.
Abstract: Various parts of the human gastrointestinal tract were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). PYY-immunoreactive cells were observed in the lower part of the ileum, in the colon and in the rectum, and PP-immunoreactive cells were found in the colon and rectum. Both cell types were of the open type, i.e. they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were seen to emit cytoplasmic processes to the neighbouring goblet cells. This latter observation suggests that PYY cells may exert a paracrine action on the mucussecreting goblet cells. Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immunoreactive cells. On the ultrastructural level PYY-immunoreactivity was localized in basal granulated endocrine cells. These cells contained round or slightly oval electron dense granules with a mean diameter of 150 nm (range 100–300 nm).
156 citations
TL;DR: The immunocytochemical data suggest that cytokeratins of kidney tubules are different from epidermal prekeratins and are more closely related to those of other simple epithelial cells such as hepatocytes and intestinal mucosa, and indicate differences of cytokeratin filaments in different parts of the nephron.
Abstract: The distribution of diverse types of intermediate filaments and desmosomal plaque proteins in rat and bovine kidney has been studied by immunofluorescence microscopy on cryostat sections using antibodies to vimentin, desmin, various preparations of epidermal prekeratins, hepatic cytokeratins as well as a monoclonal cytokeratin antibody which recognizes a wide range of different cytokeratin polypeptides. These immunocytochemical reactions have revealed an unexpected cell type complexity in terms of intermediate filament composition. Vimentin-positive cells are identified as interstitial fibroblasts, capillary endothelia and other vascular wall cells as well as practically all glomerular cells, including podocytes and mesangial cells. Co-existence of vimentin and desmin filaments has been found in glomerular and extra-glomerular mesangial cells, in agreement with the notion that these cells are derived from a specific subset of vascular smooth cells simultaneously expressing vimentin and desmin.
127 citations
TL;DR: Abundant FMRFamide immunoreactivity has been found in the nervous systems of all hydrozoan, anthozoan, scyphozoan and ctenophoran species that were looked upon, showing that F MRFamide-like material must play a crucial role in the functioning of primitive nervous systems.
Abstract: Abundant FMRFamide immunoreactivity has been found in the nervous systems of all hydrozoan, anthozoan, scyphozoan and ctenophoran species that were looked upon. This general and abundant occurrence shows that FMRFamide-like material must play a crucial role in the functioning of primitive nervous systems.
118 citations
TL;DR: Acid phosphat enzyme, alkaline phosphatase, glucose-6-phosphatase , Mg-activated adenosine triphosph atase and 5′ nucleotidase were demonstrated in the rat liver using a cerium-based method and yields better results than the lead-based methods.
Abstract: Acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, Mg-activated adenosine triphosphatase and 5′ nucleotidase were demonstrated in the rat liver using a cerium-based method. This method can be applied routinely and yields better results than the lead-based method. The tissue was postfixed in osmium tetroxide and potassium ferrocyanide which considerably enhances the membrane contranst in comparison with solely osmium tetroxide postfixation. This facilitates the precize localization of the reaction product.
111 citations
TL;DR: The enzyme leakage as such cannot be used as a discriminating test between reversible and irreversible damage of the liver parenchyma for a prediction of the occurrence of necrosis.
Abstract: Following the clamping of the afferent vessels of the left lateral and median lobes in rat liver, a considerable part of these lobes show signs of necrosis 24 h after 90 min of ischemia, wheras no necrotic areas can be detected after 30 min interruption of the blood flow. The purpose of this study was to examine the value of an analysis of the leakage of enzymes from the liver parenchyma in the early phase after restoration of the blood flow after ichemia for a prediction of the occurrence of necrosis. Leakage of the enzymes GPT, GOT and LDH can be detected in the blood plasma with a maximum activity between 1 and 5 h both following 30 and 90 min of ischemia; a considerable difference in clearance is observed, however, in the period afterwards, the normal situation being reached after 24 h with the 30-min ischemic period, but not following the 90-min period. With use of an enzyme histochemical reaction, in situ a depletion of LDH-activity in the hepatocytes could be detected within a short period of time after 30 min temporary ischemia and a restoration during the following period of 24 h; the decrease in LDH-activity persisted during 24 h with a 90-min period of ischemia. Electronmicroscopically cytoplasmic blebs arosen from hepatocytes are observed in the lumen of sinusoids immediately after 30 min of ischemia, whereas after 90 min of ischemia actual leakage of cytoplasmic material takes place through the damaged surface of the hepatocytes. Enzyme leakage probably takes place via these both types of shedding of cytoplasm. It is concluded that the enzyme leakage as such cannot be used as a discriminating test between reversible and irreversible damage of the liver parenchyma.
87 citations
TL;DR: In this paper, the authors visualized the CRF system of the rat and observed that significant amounts of immunoreactive CRF are present in the hypothalamus if animals are treated with dexamethasone.
Abstract: In general, antisera generated against ovine CRF do not reveal immunopositive neuronal perikarya in the rat. If animals are adrenalectomized significant amounts of immunoreactive CRF are present in the hypothalamus. By using this model, we have visualized the CRF system of the rat. Intact, intact pretreated with dexamethasone, adrenalectomized, and adrenalectomized pretreated with dexamethasone animals were used in the present study. In adrenalectomized and adrenalectomized plus dexamethasone treated animals the CRF-immunopositive neurons were observed in the parvocellular portion of the paraventricular nucleus. Distinct pathways of CRF fibers could be seen emerging from this hypothalamic nucleus. The greatest number of these fibers exited the PVN laterally and crossed either superior to or beneath the fibers of the fornix. The fibers then turned ventrally and cascaded to form a bundle of fibers above the superio-lateral margin of the optic chiasm. They turned caudally and followed the optic tract. As these fibers reached the level of the anterior median eminence, they turned medially to run along the inferior margin of the hypothalamus and enter the median eminence. A few fibers emerged from the PVN along the periventricular margin of the third ventricle, traveled caudally in the periventricular nucleus and entered the median eminence. Adrenalectomized and adrenalectomized-dexamethasone treated rats had very dense accumulations of immunoreactive CRF in the median eminence when compared with controls. Immunoreactive neurons and fibers were also observed in the central nucleus of the amygdala in the adrenalectomized and adrenalectomized-dexamethasone treated animals.
85 citations
TL;DR: Results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and Sba are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.
Abstract: Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.
75 citations
TL;DR: The need for careful standardization of histochemical methods for the visualization of myofibrillar actomyosin ATPase and the implication of the existence of different fiber types in apparently homogeneous muscle for the preparation of antibodies used for immunocytochemical methods of fiber identification are discussed.
Abstract: A method is described for identifying fiber types of skeletal muscle from several mammalian species on the basis of the sequential inactivation of myofibrillar actomyosin ATPase during acid preincubation. When this method is used in combination with the standard alkaline preincubation at least 5 types of fibers can be identified. Of these, 2 are type I fibers with those of the slow twitch soleus muscle being different from those that exist in mixed muscles. The 3 subtypes of type II fibers exist independent of their metabolic properties. The need for careful standardization of histochemical methods for the visualization of myofibrillar actomyosin ATPase and the implication of the existence of different fiber types in apparently homogeneous muscle for the preparation of antibodies used for immunocytochemical methods of fiber identification are discussed.
74 citations
TL;DR: The difference between PYY-and PP-immunoreactive cells in the distribution, frequency and ontogeny provide further evidence that PYY and PP occur in two independent cell types.
Abstract: The distribution and ontogeny of polypeptide YY (PYY)-and pancreatic polypeptide (PP)-immunoreactive cells in the gastrointestinal tract of rat were investigated. PYY-immunoreactive cells were numerous in the pylorus, ileum and colon and only a few cells were observed in the corpus, duodenum, jejunum and rectum. On the other hand, a few PP-immunoreactive cells were seen in the colon only. Both PYY-and PP-immunoreactive cells were of the open type, i.e., they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were observed first in the lower half of the stomach and in the intestine of 19 day-old embryo. The localization of the cells seemed to move along towards the pylorus and the lower part of the intestine. PP-immunoreactive cells could only be detected for the first time in the colon of 2 day-old rat. These cells appeared temporarily in the pylorus and rectum during the period 7 to 21 days after birth. It was concluded that the difference between PYY-and PP-immunoreactive cells in the distribution, frequency and ontogeny provide further evidence that PYY and PP occur in two independent cell types.
TL;DR: The soybean Bowman-Birk inhibitor, a polypeptide of MW 8,000, has a specificity directed against trypsin and chymotrypsin, and was localized at the ultrastructural level by the protein A gold method on thin sections of Glycine max (soybean) cv.
Abstract: The soybean trypsin inhibitor (SBTI, Kunitz type) was localized by immunofluorescence and, at the ultrastructural level, by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. SBTI was localized in cell walls, protein bodies, the cytoplasm between the lipid-containing spherosomes, and the nucleus of the cotyledon and embryonic axis. In the nucleus, SBTI was present in the chromatin deposit and the nucleolus. The intensity of marking by the gold method decreased in the cell wall from the center of the cotyledon to the periphery. In four-days-old seedlings marking intensity of cell wall was much reduced. No SBTI could be detected in the hypocotyl. In two lines lacking soybean agglutinin (Norredo, T-102) the location of SBTI was similar to that observed in Maple Arrow. In P.I. 196168, a line lacking SBTI, marking intensity of the organelles was reduced to a very low level. Although this study was not designed to discern the cellular function of SBTI, if any, it may establish criteria consistent with its role in soybean.
TL;DR: Calvaria from newborn rats were used to localize carbonic anhydrase isoenzymes in bone tissue and highly sensitive peroxidases-antiperoxidase method revealed strong reaction of high-active C form of carbonicAnhydrase exclusively in the osteoclasts.
Abstract: Calvaria from newborn rats were used to localize carbonic anhydrase isoenzymes in bone tissue. Highly sensitive peroxidase-antiperoxidase method revealed strong reaction of high-active C form of carbonic anhydrase exclusively in the osteoclasts. There was no reaction in osteoblasts or fibroblasts but some population of bone marrow cells was positive. Isoenzyme B was found only in bone marrow cells. On the basis of the present and previous studies it is highly probable that carbonic anhydrase may have some crucial role in osteoclast mediated bone resorption. Carbonic anhydrase C may also be a suitable probe for studies of osteoclast function and origin.
TL;DR: Present data indicate that the differential binding properties of these FITC-labeled lectins can be exploited to identify certain components of the mouse oviduct and uterus and to indicate changes in the cell surface and/or cytoplasm in these structures during pregnancy.
Abstract: The binding of 20 fluorescein isothiocyanate (FITC)-labeled lectins to various portions of the pregnant and non-pregnant murine oviduct and uterus was studied by fluorescence microscopy. Five lectins (from Ricinus communis (RCA-I), Maclura pomifera (MPA), Triticum vulgare (wheat germ-WGA), Bauhinia purpurea (BPA), and Ulex europeus (UEA-I] reacted differentially with the epithelium of pregnant as compared with the non-pregnant uterus. The binding of RCA-I, MPA and WGA delineated pregnancy-related changes in the distal oviduct and colliculus tubaris. WGA recognized also pregnancy related changes in the proximal oviduct. The reactivity of the remaining 15 lectins did not distinguish the pregnant and non-pregnant oviduct and uterus, although some of them served to identify specific components of the mouse genital tract. Thus, Soybean lectin (SBA) reacted almost exclusively with the colliculus tubaris. UEA-I alone reacted exclusively with the epithelium of the non-pregnant uterus. RCA-II reacted preferentially with the epithelium of the oviduct and uterus as compared with its weak reactivity with the stroma. Two lectins (from Pisum sativum and Lens culinaris) reacted selectively with stromal cells of the uterus and oviduct. Present data indicate that the differential binding properties of these FITC-labeled lectins can be exploited to identify certain components of the mouse oviduct and uterus and to indicate changes in the cell surface and/or cytoplasm in these structures during pregnancy.
TL;DR: A continuum of fast-twitch fiber types which may tranform in response to the amount and type of usage is suggested, as has been described through quantitative histochemistry by others.
Abstract: Muscle biopsies were removed from the vastus lateralis muscle of four healthy individuals with a unique fiber type distribution. Fibers were divided into slow-twitch (type I) and fast-twitch (types IIA, IIAB, and IIB) based upon the pH lability of myofibrillar ATPase. An unusually high percentage of types IIB and IIAB allowed a quantitative ultrastructural characterization of the subtype populations, especially with regard to type IIAB fibers. Using a cryostat retrieval method, histochemically-identified fibers were investigated by stereological electron microscopy. The volume percent mitochondria was significantly different for all fiber types. Likewise, lipid volume percent was significantly different between type I and type II fibers and between the fast-twitch subtypes IIA and IIB. However, these ultrastructural data revealed considerable overlap between the fiber types for metabolic parameters, as has been described through quantitative histochemistry by others. The fiber type which stained intermediately between types IIA and IIB at a preincubation pH of 4.6 (type IIAB) was also found to be intermediate in its oxidative components. These data suggest a continuum of fast-twitch fiber types which may tranform in response to the amount and type of usage.
TL;DR: In this paper, the authors investigated the thermodynamic and spectroscopic properties of the series alkyl-methyl-to-nonyl have been investigated and determined the dissociation constant K=K0 at the standard temperature T =298 K in aqueous solution.
Abstract: 10-n-Alkyl-acridinium-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl=methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K−1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K=K0at the standard temperature T=298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m+1 (m=1–9) (Table 2). In addition the standard dissociation enthalpies (energies) Δ H0 and dissociation entropies Δ S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, Δ H0, Δ S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1=20.000 cm−1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm−1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1=18.700 and D2=21.200 cm−1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition intensities agree with quantum mechanical model calculations for dimers with parallel or antiparallel molecular orientation. Hydrophobic interaction needs parallel orientation in the dimers of nonyl-AO. In the dimers of AO and of dyes with very short alkyl chains antiparallel orientation may occur.
TL;DR: The foregoing observations support previous biochemical and immunohistologic findings, strongly suggesting that collagen type III is found mainly in the upper dermis while collagen type I predominates in the deeper layers.
Abstract: Normal skin biopsy specimens obtained from 5 representative species of mammals, including man, were studied by the silver impregnation technique for reticulin fibers, by the histochemical Picrosirius-polarization method which is specific for collagen, and by transmission electron microscopy. The finely woven meshwork of argyrophilic reticulin fibers present in the adventitial dermis showed characteristics which are typical of collagen type III, when studied by aid of the Picrosirius-polarization method and by electron microscopy. On the other hand, the coarse collagen fibers of the deeper layers displayed ultrastructural and histochemical aspects which are characteristic of collagen type I. The foregoing observations support previous biochemical and immunohistologic findings, strongly suggesting that collagen type III is found mainly in the upper dermis while collagen type I predominates in the deeper layers.
TL;DR: The findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC, and a different role for histamine and 5- HT in the defense reaction towards the nematode infection is suggested.
Abstract: Infection with the nematode N. brasiliensis is accompanied by a marked increase of the number of mucosal mast cells (MMC) and the mucosal content of histamine and 5-hydroxytryptamine (5-HT). We compared amine levels, determined by ion exchange and high performance liquid chromatography (HPLC) with numbers of MMC and enterochromaffin cells (ECC). Furthermore, we measured 5-HT cytofluorometrically in individual MMC and ECC. The cellular distribution of 5-HT was studied immunohistochemically. Our results corroborate previous findings that histamine is stored in MMC. Quotients between histamine content and numbers of MMC decreased throughout the period of worm expulsion, followed by a recovery, suggesting a histamine release during this defense reaction. The HPLC analysis gave no evidence for a storage of dopamine in MMC. ECC and MMC of normal and infected rats showed a formaldehyde induced fluorescence and 5-HT immunoreactivity. The formaldehyde induced fluorescence of MMC from normal rats was about 10% that of ECC, but MMC exceeded ECC three times by numbers. These findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC. ECC numbers did not change during the infection and their content of 5-HT was unchanged, as judged by cytofluorometry. The cytofluorometric measurements showed that the intensity of the monoamine fluorescence from the MMC of infected animals was about three times as high as that of controls. It was concluded that the increased tissue levels of 5-HT was due to both an increase in MMC numbers and an increase in the 5-HT content of individual MMC. The results suggest a different role for histamine and 5-HT in the defense reaction towards the nematode infection.
TL;DR: Observations made on rat kidney tissues proved that this iron colloid solution is promising for the detection of anionic sites of cell surface in fixed tissues as well as in living cells in place of cationic ferritin.
Abstract: Stable cationic iron colloid solution was prepared by mixing the Hale's iron colloid (Hale 1946; Mowry 1963) with sodium cacodylate buffer solution. The colloid particles obtained were 30–50 A in size and kept their positive charges in a wide range of pH 1.8–7.6. Observations made on rat kidney tissues proved that this iron colloid solution is promising for the detection of anionic sites of cell surface in fixed tissues as well as in living cells in place of cationic ferritin.
TL;DR: It is suggested that, as was previously demonstrated in other peripheral organs, VIP coexists within cholinergic neurons of the rat cerebral arteries.
Abstract: An immunofluorescence histochemical study of vasoactive intestinal polypeptide (VIP) was made in the major rat cerebral arteries of the whole mount preparation. A comparison was made between the distribution of VIP-immunoreactive and cholinergic nerves. An abundant number of VIP-containing nerves were observed in the internal carotid, anterior cerebral, middle cerebral and basilar artery. VIP and cholinergic nerves were unaffected by bilateral superior cervical ganglionectomy. The density and distribution of VIP-immunoreactive fibers was essentially the same as that of the cholinergic fibers of the rat cerebral vasculature. It is suggested that, as was previously demonstrated in other peripheral organs, VIP coexists within cholinergic neurons of the rat cerebral arteries.
TL;DR: A procedure is described which allows a simultaneous evaluation of both the oxidative capacity and the intensity of “reversed” ATPase of the fibres, and thus enables to distinguish three fibre types — SO, FOG and FG — in one tissue section.
Abstract: A procedure is described which simplifies the classification of skeletal muscle fibres in that it allows a simultaneous evaluation of both the oxidative capacity and the intensity of “reversed” ATPase of the fibres, and thus enables to distinguish three fibre types — SO, FOG and FG — in one tissue section After preincubation at pH 41–42 the cryostat section is incubated for succinate dehydrogenase (SDH) and subsequently for “reversed”-ATPase This is followed by the fixation with neutral buffered formaldehyde The results of typing of chicken, minipig and rabbit fibres in a single muscle section stained with this technique are identical to those obtained with the usual method based on a comparison of serial sections of which one is stained for SDH activity the other for “reversed”-ATPase activity
TL;DR: The distribution and morphological aspects of the serotonin-containing neurons in the paraventricular organ of the carp, frog, turtle and chicken were studied by means of an immunoperoxidase technique using serotonin antiserum.
Abstract: The distribution and morphological aspects of the serotonin-containing neurons in the paraventricular organ of the carp, frog, turtle and chicken were studied by means of an immunoperoxidase technique using serotonin antiserum. In all species the serotonin-containing neurons were seen to have the appearance of the CSF-contacting neurons and to be distributed in the pars ependymalis and the pars hypendymalis of the organ. Particularly, in the frog, the serotonin-containing CSF-contacting neurons, mostly bipolar in shape, were also observed in the pars distalis. Their proximal processes protruded into the ventricular lumen through the ependymal layer with a globular- and triangular-shape. The distal processes projected ependymofugally to the pars distalis and formed a fine plexus in the neuropil of this part. The density of the serotonin fibers in the pars distalis was greater in the carp than in the other species.
TL;DR: Myoglobin concentrations were found to be significantly higher in type I than in type II muscle fibres in all 4 subjects and were, on the average, of the same magnitude as found in larger (mixed muscle) samples.
Abstract: Histochemical visualization of myoglobin in a benzidine peroxidase reaction suggests that human skeletal muscle fibres are differentiated into fibres having either high or a low myoglobin content. In the present study myoglobin was quantified in single human muscle fibres after being classified as either type I (“slow-twitch”) or type II (“fast twitch”). Samples were obtained from m. quadriceps femoris in 4 healthy untrained male subjects using the needle biopsy technique. After freeze-drying, individual fibres were dissected out and classified as either type I or type II by a myofibrillar ATPase stain. Myoglobin analyses were performed on these single fibres by a radioimmunoassay. The myoglobin concentrations were found to be significantly higher in type I than in type II muscle fibres in all 4 subjects and were, on the average, of the same magnitude as found in larger (mixed muscle) samples. The myoglobin concentration ratio between type I and type II fibres ranged from 1.4 to 1.7.
TL;DR: The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair.
Abstract: The presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.
TL;DR: Analysis of lactate dehydrogenase isoenzymes demonstrated the existence of H type isoenzyme in α-motoneurons, consistent with other observations indicating predominance of aerobic metabolism within these neurons.
Abstract: Despite the wealth of literature concerned with muscle fiber biochemical, ultrastructural and physiological characteristics, little information is available regarding the metabolic enzyme activities of α-motoneurons. The present study examines the metabolism of α-motoneurons located in the lateral cell column of the rat lumbosacral enlargment with a quantitative histochemical technique. Variation in the activities of α-glucan phosphorylase, NADH-diaphorase, succinic dehydrogenase and acid phosphatase were detectable with the photographic densitometry and atomic absorption spectrophotometry technique. No difference in the glycolytic enzyme activity (mitochondrial α-glycerophosphate dehydrogenase) was observed. Analysis of lactate dehydrogenase isoenzymes demonstrated the existence of H type isoenzyme in α-motoneurons, consistent with other observations indicating predominance of aerobic metabolism within these neurons. The activities of the former enzymes in α-motoneurons formed a complete spectrum of activities, distributed unimodally. Smaller motoneurons exhibited the greatest NADH-D and acid phosphatase activities; phosphorylase activity was greatest in larger motoneurons. Significant variation in the enzyme activity of similar-sized motoneurons suggests that the metabolism of the motoneuron is regulated by factors other than cell size. Relationships between motoneuron metabolic enzyme activity and motor unit type are under current investigation.
TL;DR: The ultrastructure of immunohistochemically identified PP cells has been investigated by applying the serial semithin/thin section technique to the human pancreas with special reference to the posterior part of the head, reputed to originate from the ventral primordium.
Abstract: The ultrastructure of immunohistochemically identified PP cells has been investigated by applying the serial semithin/thin section technique to the human pancreas, with special reference to the posterior part of the head, reputed to originate from the ventral primordium. PP cells of this area differ from those already identified in the rest of the pancreas and correspond to a cell, not yet described in the human pancreas, characterized by larger granules of very variable shape and structure. Such granules resemble those of so-called “F cells”, i.e. the PP cells of dog uncinate process and cat duodenal lobe, also coming from the ventral primordium. Thus, human “ventral lobe” PP cells have peculiar potentialities which are expressed in distinctive structural patterns of presently unknown functional meaning.
TL;DR: 10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells and the thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated.
Abstract: 10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition i
TL;DR: In this peper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with nativeCalmodulin.
Abstract: Calmodulin is a well-known calcium-binding protein which is ubiquitous in the plant and animal kingdoms and regulates many cellular processes. In this peper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with native calmodulin. Immunoreactive staining was observed in the cells in almost all areas of the brain, but its intensity varied. Some areas were stained heavily even in the presence of high concentration of NaCl. These differed from those stained immunohistochemically with antibody against other calcium-binding proteins, parvalbumin or vitamin D-dependent calcium-binding protein.
TL;DR: Comparison of various theoretical models for quantitative analysis showed that the ‘P/B-method’ (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections.
Abstract: Methodological aspects of quantitative X-ray microanalysis of semi-thick cryosections (2–6 μm) of biological soft tissue were investigated. The preparation of a low background specimen holder is described. Scanning and scanning transmission images of the sections could be obtained, allowing identification and separate analysis of nuclei and cytoplasm. Parallel observations of histochemically stained adjacent sections in the light microscope allowed correlation of the microanalytical data with tissue morphology and histochemistry. Quantitative analysis could be carried out with the help of a standard: a gelatin/glycerol matrix containing mineral salts in known quantities, frozen and sectioned in the same way as the specimen. Mass loss under the electron beam was found to be comparable in specimen and standard. Comparison of various theoretical models for quantitative analysis showed that the ‘P/B-method’ (determination of the background intensity under the characteristic peak) is the most suitable for semi-thick sections. Factors determining the choice of accelerating voltage were analyzed. The usefulness of this specimen type is illustrated in some biological applications (human oral mucosa, rat salivary gland).
TL;DR: In this modified medium, Karnovsky's cupric ferrocyanide becomes the sole precipitate at the enzymatic site and this provides fine localization of acetylcholinesterase activity.
Abstract: In the original Karnovsky and Roots' method for the localization of acetylcholinesterase (AChE), thiocholine reduces the ferricyanide and cupric ions of this medium competitively, giving simultaneously cupric (Koelle's precipitate) as histochemical products. We modified the method in order to promote the true Karnovsky's reaction, and to slow down the secondary Koelle's reaction by increasing the concentration of the ferricyanide ion from 0.5 mM to 5.0 mM and by decreasing the concentration of the cupric ion from 3.0 mM to 2.5 mM. The cupric ion, complexed with 5 mM sodium citrate in the original method, was further stabilized by the use of 0.1 M citrate buffer in order to prevent the interaction of cupric ion with increased ferricyanide. In order to suppress completely the residual Koelle's precipitate, we used acetylthiocholine chloride as a substrate, instead of acetylthiocholine iodide. The chloride salt of cuprous thiocholine is soluble, contrary to the iodide salt. In addition, the pH of the medium was lowered from 6.0 to 5.0 to avoid artefactual nuclear staining, appearing at a pH beyond 5.5. In this modified medium, Karnovsky's cupric ferrocyanide becomes the sole precipitate at the enzymatic site and this provides fine localization of acetylcholinesterase activity.