Scanning microscopy最新文献

Using ethylene glycol (EG) and vitamin D3 as crystal-inducing diet (CID) in rats, we investigated the effect of the dosage of EG on the generation of chronic calcium oxalate (CaOx) nephrolithiasis. We collected weekly 24 hour urines and measured herein the amount of oxalate, calcium, glycosaminoglycans (GAG's), creatinine, protein, alkaline phosphatase (AP), gamma-glutamyl transpeptidase (gamma-GT), and N-acetyl-beta-glucosaminidase (NAG). The potential of these urines to inhibit crystal growth and agglomeration was also evaluated. After four weeks, the kidneys were screened by histology and radiography for the presence of CaOx crystals and the amount of kidney-associated oxalate was biochemically measured. Using 0.5 vol.% EG, only a part of the rats showed CaOx deposition in the renal cortex and/or medulla, without obvious differences between Wistar and Sprague-Dawley (SD) rats. If a dietary EG concentration of 0.75, 1.0, or 1.5 vol.% was used, the amount of kidney-associated oxalate was proportionally higher and CaOx crystal formation was consistently found in all rats. Most crystals were encountered in the cortex, whereas in the medulla and the papillary region, crystals were only occasionally detected. From these data, we conclude that in the chronic rat model, based on EG and vitamin D3, a consistent deposition of CaOx crystals is obtained using a EG concentration of at least 0.75%.

Apoptosis of nucleated blood cells induced by oxidants and/or reactive oxygen species is accompanied by the typical membrane pathology. Meanwhile, red blood cell (RBC) membrane is a popular object for studying appropriate cytotoxic effects. Scanning electron microscopy provides a reliable tool for detecting the oxidative changes in RBC shape and size. Transition of normal discoid erythrocytes to crenated forms (echinocytes) is often induced by the same factors which cause apoptosis of blood cells, e.g., ionizing radiation and other reactive oxygen intermediate-inducing agents, exogenous oxidants, in vitro aging conditions, cytosolic calcium increase, etc. Moreover, the biochemical membrane alterations in oxidant-induced echinocytosis is strongly reminiscent of the changes associated with apoptosis, e.g., cell shrinkage, lipid oxidation, energy depletion and loss of transmembrane lipid asymmetry. Hence, characteristic changes in cell shape in oxidant-treated RBCs are of value for interpreting the membrane alterations occurring in leukocyte apoptosis.

The kinetics of calcium phosphate crystal growth at the surfaces of proteins and phospholipids has been investigated using free drift and constant composition methods in supersaturated calcium phosphate solutions (relative supersaturations: with respect to hydroxyapatite, HAP, sigma HAP = 15.0, and with respect to octacalcium phosphate, OCP, sigma OCP = 1.9). Fibrinogen and collagen molecules adsorbed at hydrophobic surfaces as well as uncross-linked collagen fibrils induce ion binding and subsequent nucleation of calcium phosphate. The formation of OCP on phosphatidylserine vesicles introduced to highly supersaturated calcium phosphate solutions probably involves the interaction of the calcium ions with the ionized carboxylic groups of the phospholipid.

A systematic, comparative study of the accessory sex glands of the adult male rat after androgen withdrawal was carried out. The changes were investigated by using scanning electron microscopy at different intervals after surgical castration. The main common signs of epithelial cell involution were flattening of the cell surface, reduction of the size and number of microvilli, some blurring of the cell borders, cessation of secretory activity and diminution of the luminal volume of the glands. Overall, confident signs of atrophy were evident after one week, and complete epithelial involution was reached by the third week. The epithelial cell atrophy was accompanied by a relative stromal hyperplasia. The new observation seems to be that the process of stroma consolidation is progressing for a considerable time subsequent to the completion of the epithelial involution. This phenomenon is particularly evident in the dorsal prostate, the seminal vesicle and the coagulating gland.

Overgrown human gingival specimens were examined histologically and by scanning electron microscopy (SEM) to study structural changes caused by cyclosporine. The biopsy specimens were from organ transplant recipients receiving cyclosporine to suppress the rejection of the transplanted organ. The epithelium of the overgrown gingiva was thickened, acanthotic and parakeratotic. Retepegs were anastomosing and extending into connective tissue. The SEM examination of the outer surface of the attached gingival showed loss of cellular attachments and cells were exfoliating. The normal honeycomb structure formed by interconnecting microvilli surrounding the pits was distorted. Outer gingival cell surface showed numerous round, ovoid and dome-like structures instead of parallel, reticular or fingerprint-like microridges. It was concluded that cyclosporine not only caused hyperplasia but also changed the structure of the outer epithelial cell surface.

The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also described both with terminal deoxynucleotidyl transferase and DNA polymerase I, aimed at the study of the distribution of DNA cleavage areas. Flow cytometry is also proposed and different technical approaches, based on different laser utilizations, are discussed. Ultrastructural analysis, allowing the study of apoptotic cell details, is finally considered.

In the present study, we exposed rats to a crystal-inducing diet (CID) consisting of vitamin D3 and 0.5% ethylene glycol (EG), and we investigated histologically the kidney damage induced by the deposition of calcium oxalate (CaOx) crystals. After 28 days, 50% of the animals had renal CaOx crystals, of which 60% also had small papillary stones. Most crystals were present in the cortex. The occurrence of these crystals coincided with morphological and cytochemical changes: glomerular damage, tubular dilatation and necrosis, and an enlargement of the interstitium. The number of epithelial and interstitial cells positive for the proliferating cell nuclear antigen (PCNA) was increased. Tamm-Horsfall protein (THP) was not only demonstrable in the thick ascending limb of the loop of Henle (TAL), but also frequently in glomeruli, in the proximal tubular epithelium, and in the papilla. In the lumen of the tubular system, it was associated with urinary casts. Reflection contrast microscopy (RCM) showed that the crystals were coated with a thin layer of THP. In spite of the high urinary oxalate concentrations, the above described cellular changes were not observed in CID-fed rats without renal crystals. We conclude, therefore, that in the kidney, the retained CaOx crystals rather than the urinary oxalate ions are responsible for the observed morphological and immunocytochemical changes.

Inter-alpha-inhibitor (I alpha I) is a serine protease inhibitor present in human plasma. It has a molecular weight of about 220 kDa which encompasses 3 chains including two heavy chains and one light chain. The light chain, known as bikunin, is responsible for the antitryptic activity of I alpha I in the inhibition of various enzymes, such as trypsin and chymotrypsin. Under physiologic or certain pathologic circumstances, several macromolecules related to I alpha I appear in plasma and urine. However, the physiologic role of I alpha I remains unclear. As far as urolithiasis is concerned, two urinary macromolecules related to I alpha I have been isolated and shown to be potent inhibitors of calcium oxalate formation. One of these inhibitors, uronic-acid-rich protein (UAP), has been identified and well characterized. The sequence of the first 18 amino acid residues of UAP is identical with that of bikunin. Furthermore, the immunoreaction between UAP and I alpha I antibody using immunoblot analysis was positive. UAP isolated from the urine of stone formers exhibited less inhibitory activity towards calcium oxalate crystallization than that derived from the urine of healthy subjects. This suggests a structural abnormality of the inhibitor obtained from stone patients. The organic matrix extracted from kidney stones contained a protein antigenically related to I alpha I. We conclude that UAP is a member of I alpha I family taking part in inhibiting calcium oxalate crystallization, and modulating the formation of stones in the urinary tract.

Morphological types of bacterial remains preserved in ancient tartar of teeth from extinct human groups, which included some communities of coastal gatherers, fishermen, hunters, and farmers, and those practicing a mixed economy, were analyzed. Previous studies have shown the presence of bacteria in ancient tartar. The aim of this work was to determine whether Streptococcus mutans was present in ancient populations (500-12,000 years old). Teeth samples were from ancient skulls obtained from different anthropological collections: the north and south of Chile (before the Spanish conquest), Palencia, Spain, and an eastern Mediterranean region (Levant). Optical microscopy showed Gram positive and Gram negative bacteria. Scanning electron microscopy identified morphological types of bacteria. Transmission electron microscopy enabled categorization of bacterial structures. Fluorescence microscopy helped label and identify S. mutans, using polyclonal antibodies. Bacterial morphotypes were related to different subsistence patterns. Hunters, fishermen, and gatherers had a less diverse flora with bacillary and coccal morphotypes. Agricultural groups showed greater diversity with additional filamentous and spiral morphotypes. The best preserved ultrastructural feature was the cell wall. The existence and colonization capacity of the mutans-like streptococci preserved in tartar was established for the ancient populations studied, with the exception of Cerro Sotta (south of Chile). Hence, their occurrence could not be related to diet or subsistence pattern.

As a consequence of external and internal ionizing radiation, lysosome-like bodies have been observed to increase both in size and number in some cell types. We investigated this process by morphological methods (electron microscopy, cationized ferritin uptake, acid phosphatase histochemistry, morphometry) in cultured HT-29 cells. In parallel with these studies, we measured the rate of protein degradation on the basis of 14C-valine release from prelabeled cellular proteins. We found that at 2 and 4 Gy doses of X-irradiation the volume of the vacuolar (probably lysosomal) compartment increased without detectable changes of acid phosphatase activity. A 2 Gy irradiation dose did not change protein degradation rate. However, 4 Gy caused a significant inhibition of 14C-valine release from prelabeled proteins. Our results indicate, that the radiation induced expansion of the lysosomal compartment is not necessarily accompanied by increased lytic activity of HT-29 cells.