Structural and ultrastructural changes in the rabbit myocardium exposed to dimethylformamide vapors

The aim of this research is to show the alterations of alpha-actinin in rabbit myocardium after DMF treatment administered by forced inhalation. Z-lines observed with light and phase-contrast microscopy, appeared to be intact and they were clearly displayed by the indirect PAP-reaction. But if you consider that in normal muscle Z lines do not get coloured by the PAP-reaction without a previous light treatment with trypsin. It may be inferred that, in this case, The DMF has had the same effect as the trypsin, causing alteration to the protein structure. Besides the sarcomeric structure is undoubtedly altered by the DMF, causing alterations, in our opinion, similar to those found in rabbit skeletal muscle, under conditions of acute experimental ischemia.     

Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophys. Acta 512, 508–524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold).The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.     

Post-coital ultrastructural changes in neurons of the suprachiasmatic nucleus of the rabbit

Summary The effects of coitus on the ultrastructure of neurons in the suprachiasmatic nucleus of the rabbit were studied. Changes first became apparent 1/2 h after coitum in neurons located near capillaries. More pronounced ultrastructural changes were observed in large neurons removed at 1 h post-coitus. These changes, characterized by well developed Golgi systems and rough endoplasmic reticulum, the presence of large dense-core vesicles and a significant increase in both neuronal and nuclear size, were also evident in neurons observed at 2 to 10 h post coitum. Similar ultrastructural features were not observed in the neurons of the control animals. The post-coital ultrastructural changes observed within these neurons suggest high synthetic activity which may concern the production sites of the neurohormone LH-RF. Two populations of dense-core vesicles were observed: a) those with a mean diameter of 849 Å, and b) those with a mean diameter of 1542 Å. The small dense-core vesicle is probably monoamine in nature; the larger vesicle may contain the neurohormone LH-RF. A third vesicle type with a mean diameter of 1836 Å and characterized by a granular content of low electron density was also observed. That this vesicle represents the immature form of the large (1542 Å) dense-core vesicle is suggested; however, morphological evidence supporting this hypothesis is inconclusive. There is also no evidence for the storage of secreted materials within the soma of these neurons. Immediate transport toward the median eminence is suggested.This investigation was supported by grants to the two senior authors from the Medical Research Council of Canada.     

Morphological and histochemical changes in the rabbit aorta during exogenous and endogenous hyperglycemia]

Alimentary hyperglycemia and insulin deficiency were accompanied in rats by atherosclerotic affection of the aorta whose degree increased in feeding of cholesterol to such animals. The detected morphological and histochemical changes in the wall of the aorta correlated with the biochemical atherogenic shifts in the blood serum of the corresponding animals groups established earlier.     

Arylsulphatases in the rabbit oviduct: postovulatory changes tested by histochemical and biochemical procedures

Summary A histochemical and biochemical study of the activity of arylsulphatases A and B was carried out on the oviduct of female rabbits during the first days after mating. The histochemical results demonstrated that the ampullary and the isthmic epithelial cells have a positive reaction to the sulphatases during the whole of the postovulatory period tested. The enzymatic activity is mainly localized in the basal cellular cytoplasm. The biochemical results confirmed that both arylsulphatase A and B are active. Arylsulphatase A activity is more intense in the ampulla than in the isthmus and it increases during the whole of the postovulatory period; in the isthmus the activity increases up to 72 h, thereafter decreasing again. The arylsulphatase B activity is always lower than arylsulphatase A activity; maximum activity is reached between 66 to 72 h after mating. The arylsulphatase B is relatively higher in the ampulla than in the isthmus. The biological role of these enzymes is discussed in relation to the regulation of the sulphated glycoconjugates.     

Megaoesophagus in the mouse: histochemical and ultrastructural studies

Histochemical and ultrastructural studies of the muscle coat of the oesophagus from ICRC/HiCri mice (with megaoesophagus) and DBA/2fNCri mice (normal oesophagus) were carried out. The striking observation from histochemical studies was the presence of smooth muscle in the abdominal segment of the oesophagus from ICRC mouse in contrast to the control strain where smooth muscle was present only in the lowermost portion adjoining the stomach. Ultrastructural studies of the oesophageal wall from 5- and 10-day-old ICRC mice revealed an apparently normal muscle coat. In 3-month-old ICRC mice the upper abdominal segment of the oesophagus showed several abnormalities of smooth muscle fibres and paucity of plexus tissue accompanied by interstitial collagen deposition. The abnormalities were more severe in 1-year-old animals and were seen throughout the abdominal segment. From this study it is suggested that the primary cause of megaoesophagus in ICRC mice is neurogenic and not myogenic.     

Intracellular events in the assembly of chylomicrons in rabbit enterocytes

The aim of this study was to determine the intracellular events in chylomicron assembly in adult villus enterocytes. We have used novel methods for separation of the intracellular components of the secretory compartment [rough and smooth endoplasmic reticulum (RER and SER, respectively) and Golgi], and their membrane and luminal components, from villus enterocytes isolated from rabbit small intestine. The steady state composition of the components of the secretory compartment and the intracellular pools of newly synthesized apolipoprotein B-48 (apoB-48) and triacylglycerol (TAG) was determined. The observations indicate that the SER is the main site of TAG synthesis and of chylomicron assembly. Newly synthesized apoB-48 and TAG accumulate in the SER membrane and are transferred into the lumen in a microsomal triglyceride transfer protein-dependent step. In enterocytes isolated from chow-fed rabbits, in which fat absorption is relatively slow, transfer of apoB-48 and TAG from the SER membrane into the lumen appears to be rate limiting. In enterocytes from fat-fed rabbits, TAG accumulates in the lumen of the SER, suggesting that movement out of the SER lumen becomes rate limiting, when chylomicron secretion is markedly stimulated. In these cells, the cytosolic TAG also increased to 450 microgram/g enterocytes, compared with 12 microgram/g enterocytes from chow-fed rabbits, indicating that transfer of TAG from the SER membrane into the secretory pathway can become saturated, so that newly synthesized TAG moves into the cytosol.     

Primary extracellular ceroid type lipopigment. A histochemical and ultrastructural study

Summary An extracellular ceroid-type (ECC) lipopigment which appears histologically as wavy hyaline membranes or, less frequently, as an amorphous solid or broadly reticulated mass is described. Its ultrastructure is either amorphous or membranous and consists either of simple linear membrane-like aggregates or elaborated trilaminar membranes. The histochemical profile is, as in histiocytic intracellular ceroid, dominated by autofluorescence, strong hydrophobicity, acid and extraction resistance. Staining for aromatic acid residues and periodic acid-Schiff positivity are strong but variable. Lectin receptors are either absent or sparse. The pigment is found solely within the lipid rich tissue debris, bound to processes marked by necrosis of adipose or steatosed tissues and interpreted as originatingde novo extracellularly from liquid unsaturated lipids under the influence of local enzymatic and nonenzymatic lipid oxidation catalysts. A hitherto unknown form of extracellular ceroid is the so-called membranocystic lesion found in Nasu-Hakola's disease, in several other conditions and in annular ceroid in human atheromas.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

Biosynthesis and intracellular pool of aminopeptidase N in rabbit enterocytes

Summary A papain treatment at 15°C and pH 7.3 of a microsomal fraction from rabbit enterocytes quantitatively releases the aminopeptidase N integrated in the plasma membranes without solubilizing the enzyme integrated in the intracellular membranes. Working on A⁺ rabbits, characterized by the presence on the brush-border hydrolases of glycans corresponding to the human blood group A-determinant structure, it was possible to separate the intracellular aminopeptidase into two major molecular forms with or without these determinants. The molecular form devoid of human blood group A antigenicity corresponds to the only stable intermediate of glycosylation, bearing N-linked high mannose oligosaccharides. This endoglycosidase H-sensitive form is fully active and represents in the steady state about 1% of the total cellular aminopeptidase. It contains a cytoplasmic sequence of about 3000 daltons that has not yet been detected in the mature form. The A antigenicity is acquired simultaneously with processing of high mannose glycans to complex glycans. Pulse chase labeling of jejunum loops with [³⁵S]-methionine showed that the complete processing of the transient form synthesized during 10 min takes 1 hr. During the last 30 min of processing, all the newly transformed molecules are transported to the plasma membrane.     

Lipocortin IV is a basolateral cytoskeleton constituent of rabbit enterocytes.

A monoclonal antibody, BL7B1, which specifically recognized rabbit lipocortin IV, was produced. The BL7B1 epitope is localized between the methionine residues that occupy positions 148 and 259 in human lipocortin IV. Immunofluorescence and subcellular fractionation studies showed that in the rabbit enterocytes, lipocortin IV is specifically associated with the basolateral membranes. When these membranes are solubilized by Triton X-100 in the presence of 1 mM of Ca2+ the lipocortin IV, like the cytoskeleton, remained insoluble suggesting that it might be associated with this structure in vivo. No lipocortin IV was detected in the brush border using immunofluorescence techniques and less than 10% of the amount present in the purified basolateral membranes was detected in the brush border membrane fraction.     

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes

Summary A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A⁺ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.     

A histochemical and ultrastructural study of stored material in neuronal ceroid lipofuscinosis.

A histochemical and ultrastructural study of five cases of neuronal ceroid lipofuscinosis (NCL) revealed the existence of two related lipopigments differing in some tinctorial properties and ultrastructure. Type I pigment is present in all the tissues affected and corresponds to the pigmentary tertiary lysosomes of well known ultrastructure. Type II pigment occurs exclusively in the neurones of lipophilic cerebral grisea, as a component of the so called protein-myoclonic bodies. It shares with type I certain basic tinctorial properties of lipopigment and its lysosomal localization, but differs in other respects. It stains poorly if at all with the PAS and PAF techniques and is markedly metallophilic, azurophilic and positive for protein. Type II pigment is extremely electron-opaque after staining with heavy metals to the extent that they appear practically amorphous. The possibility that type II material is derived from type I pigment is considered. The amount of type II pigment is highly variable. Both types of pigments are present in residual bodies of various shape and size, including spheroids.     

Coincident filament distribution and histochemical localization of F-actin in the enterocytes of the larval dragonflyAeshna cyanea

Summary The fluorescence pattern of the enterocytes of larvalAeshna cyanea obtained by the incubation of cryosections with rhodaminephalloidin, a histochemical probe for F-actin, exactly coincides with the distribution of 6 nm filaments. These prevail as 1. core bundles in the microvilli 2. terminal web including the rootlets of the core bundles and and filaments associated with the adhesive junctions, 3. intercisternal arrays in the subapical stacks of smooth endoplasmic reticulum, and 4. cortical web underneath the basolateral plasma membranes.Abbreviations EDTA ethylenediaminetetraacetic acid - HMM heavy meromyosin - PBS phosphate-buffered saline - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - rER and sER rough and smooth endoplasmic reticulum     

Cytochemical and ultrastructural study of anoikis and secondary necrosis in enterocytes detached in vivo

Detachment-induced apoptosis of enterocytes (anoikis) has not been investigated in vivo. Here we describe anoikis of fish enterocytes following detachment in a septicemia by Photobacterium damselae subsp. piscicida, or following injection of its exotoxin. The in vivo study was complemented with an ex vivo time-lapse analysis using conditions duplicating the in vivo situation. Linings of enterocytes detached from intestine mucosa dissociate into isolated enterocytes which undergo caspase 3-mediated anoikis with cell rounding, loss of polarization, condensation of chromatin and fragmentation of the nuclear envelope, early swelling of mitochondria with rupture of the outer membrane, and brush border disappearance. One mechanism for brush border loss was shedding of apoptotic bodies incorporating the apical part of the enterocyte. Brush border disappearance was also associated with disassembly of the F-actin microvillar core and involved re-absorption into the cell, or expansion and vesiculation followed by shedding of microvillar fragments. The enterocyte anoikis terminates by secondary necrosis and lysis due to lack of elimination by phagocytosis of apoptosing enterocytes. The conditions prevailing in vivo in the gut lumen accelerate enterocyte secondary necrosis. Our results underscore the importance of analyzing anoikis under conditions similar to those occurring in vivo.