The calculated glucose concentration profile in the intercellular spaces of everted jejunum of rat.

On the basis of experimental data from isolated and everted rat jejunum and on extension of Diamond and Bossert's mathematical model, the glucose concentration in the intercellular spaces has been calculated and has been found higher (less than 2 mM) than that present in the serosal space.     

An ultrastructural examination of the mesoglea of Hydra

Summary The survey of the mesoglea of four species of Hydra indicates a basic similarity of structure. In each species the mesoglea consists of an amorphous ground substance with three different types of fibers and particulate material dispersed in this matrix. A probable interpretation of the fine structure of the mesoglea is that collagen-like protein demonstrated by other investigators, forms all or part of the beaded fibers. Acid mucopolysaccharide which can be demonstrated histochemically probably corresponds to the amorphous ground substance in which the mesogleal fibers are dispersed. The role of the mesoglea as an extra-cellular skeleton and cementing substance is discussed.This work was supported in part by USPHS Training grant 5T1-DH21-04 and Inst. Grant IN-57-F from the American Cancer Society.     

Biochemical and ultrastructural examination of cryopreserved hepatocytes in rat

We investigated the ultrastructure and metabolic capabilities of isolated rat hepatocytes after cryopreservation using 1.5 M Me₂SO as protectant and a slow cool/fast thaw regime. Ultrastructural assessment of the cryopreserved population revealed only approximately 10% of cells with normal morphology. Conjugation of bilirubin by the cryopreserved cells was reduced to 20% of that seen in unfrozen hepatocytes and there was a net loss of glycogen measured in cryopreserved cells incubated in conditions which stimulated glycogen synthesis by unfrozen cells. These results are in contrast to other reports in which cryopreserved hepatocytes have been successfully used for transplantation to reverse hepatic insufficiency.     

An ultrastructural examination of Penicillium urticae during the transition to antibiotic biosynthesis

The ultrastructure of Penicillium urticae mycelium was compared at various stages of submerged growth to examine changes associated with the onset of antibiotic biosynthesis. Penicillium urticae was shown to be a normal eukaryotic, septate, filamentous fungus with a variety of subcellular components. Younger mycelia possessed a denser cytoplasm which gave way to a more granular and vacuolated cytoplasm as the organism made the transition into antibiotic biosynthesis. An increase in the thickness, and perhaps the structural complexity, of the cell wall also occurred over the transition. There was evidence of a glycocalyx surrounding the hyphae. Discrete granules, termed peripheral particles, appeared and increased in number over the transition. Their biochemical content and possible involvement in patulin production was tested by examining P. urticae after growth in media of different composition, and by examining the ultrastructure of a patulin minus mutant, P3. The significance of these observations in relation to patulin production is discussed.     

Reactions of Paneth cells of the rat jejunum to section of the vagus nerves (ultrastructural analysis)

Dynamics of ultrastructural alterations of Paneth cells (PC) of the rat jejunum have been investigated after bilateral subdiaphragmal trunkal vagotomy (7, 14, 30 and 60 days). This operation results in a profound ultrastructural rearrangement in PC organization, which is especially manifested on the 7th and 14th days. The cisterns and canaliculi of the endoplasmic reticulum dilate, the Golgi complex structure is disturbed, the matrix of mitochondria is cleared, their crists are fragmented, large vacuoles with electron transparent contents appear, total amount of the secretory granules and relative number of their immature forms increase, the secreory granules form and topography of their core is changed, small structures resembling microtubules appear in them. Beginning from the second week, morphological alterations of compensatory-adaptive character progressively increase: the number of free ribosomes grows large and large mitochondria appear.     

An ultrastructural examination of murine alveolar macrophages following intranasal administration of propionibacterium acnes

Light and electron microscopic analysis of murine lungs or isolated pulmonary cells was performed three days after intranasal administration of the bacterial immunostimulant, Propionibacterium acnes (P. acnes). Our observations indicated that pulmonary alveolar and airway macrophages (PAMs) were the only cells with P. acnes bacilli in their cytoplasm. Bacilli were not observed in pulmonary interstitial macrophages, granulocytes, lymphocytes or pulmonary parenchymal cells such as type I and type II pneumocytes. Because of the morphological heterogeneity of PAMs observed in control and experimental animals, it was not possible from these studies to be certain about the relative abundance or complexity of lysosomes, endoplasmic reticulum, Golgi and other organelles in the two groups. However, we noted that it was not uncommon to observe in the same PAM, profiles of P. acnes and a well developed Golgi complex and endoplasmic reticulum. These P. acnes--associated morphological alterations occurred at a time when functional activities (e.g., phagocytosis, cytostasis) of PAMs were enhanced.     

Adrenomedullary chromaffin cells of the rat. An ultrastructural study

Adrenomedullary chromaffin cells of the rat were studied at the ultrastructural level. Chromaffin cells contained a large population of electron-dense-core vesicles of two types, one very electron-dense (norepinephrine) and the other moderately electron-dense (epinephrine). The vesicles showed an even distribution pattern in the cytoplasmic matrix. No physical contacts were observed between cytoplasmic and vesicle membranes to indicate exocytosis, a mechanism frequently observed in the hamster. For chromaffin cells to be used as a transplantable source of dopamine, both the cells and vesicles must survive the trauma of denervation and vascular elimination or alternatively acquire the two factors from the transplantation site.     

An ultrastructural examination of the role of cell membrane surface coat material during neurulation

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.     

Diaglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity in the biosynthesis of diacylglycerols by rat intestinal mucosa was examined by means of radioactive markers, thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacyglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the diacylglycerols were isolated by solvent extraction and thin-layer chromatography. Sterospecific analyses of the X-1,2-diacylglycerols labeled from 2-monoacylglycerols showed that the sn-1,2-isomers (45-55%) were slightly in excess of the sn-2,3-isomers (34-45%) with the X-1,3-diacylglycerols accounting for the rest of the radioactivity (5-10%). This suggests that racemic diacylglycerols may be intermediates in the resynthesis of dietary fat in rat intestinal mucosa. Detailed analyses of the molecular species of the sn-1,2-diacylglycerols labeled from free fatty acids revealed that 10-45% of the total did not contain the acid present in the 2-monoacylglycerol supplied, and therefore had originated from the phosphatidic acid pathway. These findings are at variance with those obtained in isolated microsomes, which have suggested an inhibition of the phosphatidic acid pathway by monoacylglycerols as well as have given evidence of an exclusive synthesis of sn-1,2-diacylglycerols from 2-monoacylglycerols.     

Triacylglycerol biosynthesis in everted sacs of rat intestinal mucosa.

The molecular specificity of the biosynthesis of triacylglycerols by rat intestinal mucosa was examined by means of radioactive and mass tracers, and thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the triacylglycerols isolated by solvent extraction and thin-layer chromatography. Analyses of the molecular species of the triacylglycerols labeled from monoacylglycerols showed that the 2-monoacylglycerol pathway was responsible for the biosynthesis of a maximum of 90% and the X-1-monoacylglycerol pathway for about 10% of the total radioactive triacylglycerols. Detailed analyses of the molecular species of triacylglycerols labeled fro free fatty acids showed that the phosphatidic acid pathway contributed a minimum of 20-30% of the total labeled triacylglycerol formed. There was a preferential utilization in triacylglycerol biosynthesis of the more unsaturated diacylglycerols arising from the monoacylglycerol pathway and of the more saturated diacylglycerols originating from the phosphatidic acid pathway. The above experiments do not allow a demonstration of the utilization of the sn-2,3-diacylglycerols in triacylglycerol biosynthesis but are not inconsistent with it.     

Response of transmural electrical parameters across in vitro everted sacs of hamster jejunum to variations in oxygenation rate.

1. The response of the electrical potential difference, short circuit current, and resistance across everted sacs of hamster jejunum to variations in the mucosal solution gassing rate was investigated. 2. Contrary to previous reports by others, it was found that the potential difference responds to increases in mucosal solution gassing rate by increasing in magnitude during the first 20 min of incubation. 3. The increases in potential difference were parallelled by increases in short circuit current but not by changes in resistance. 4. Increases in mucosal solution gassing rate increased epithelial cell O2 availability and this effect was determined to be due to increased stirring by gas bubbles. From the data, it was deduced that the minimum thickness of the mucosally located functional unstirred layer is between 0.08 and 0.16 cm when less than the full magnitude of electrical activity is observed across the everted sac preparation. 5. Serosal N2 or O2 had little or no effect on electrical parameters under maximum mucosal oxygenating conditions but dramatically affected these parameters when less than maximum mucosal oxygenating conditions were used. 6. Qualitative variations in the magnitude of the short circuit current across this preparation with respect to Cl- dependence were demonstrated. These variations were dependent upon the level of O2 availability, being apparent at high levels of O2 and absent at low levels. 7. It is concluded that the thickness of mucosally located unstirred layers can determine the O2 availability to the mucosa of everted sacs of hamster jejunum and thereby influence the observable magnitude and pattern of ionic dependence of the short circuit current across this preparation.     

An ultrastructural and histochemical study of autoimmune aspermatogenesis in the rat testis

Summary Immune aspermatogenesis was induced in young rats by the method of Freundet al. (1954) and testes were studied by electron microscopic and histochemical methods. At time of sacrifice the testes of several animals were markedly atrophic as demonstrated by reduction in weight. Sections of seminiferous tubules exhibited primarily profiles of Sertoli cells but germinal elements were sparse or absent. The ultrastructure of Sertoli cells appeared to be normal except for the presence of areas of dilated smooth endoplasmic reticulum and fragments of phagocytized germ cells in the cytoplasm.Light microscopic sections showed an apparent hyperplasia of intertubular tissue. Electron micrographs revealed a moderate to extreme vesiculation of the smooth endoplasmic reticulum in many interstitial cells. Macrophages and lymphocytes were often observed in contact with these cells.There was increased localization of non-specific esterase and acid phosphatase associated with lipid bodies of the tubules and in intertubular areas.This work was supported in part by USPHS Grant FR 5391.     

Nickel deficiency alters nickel flux in rat everted intestinal sacs

This study was conducted to determine nickel absorption in nickel-deficient rats. Jejunal segments obtained from dietary nickeldepleted (13 μg nickel/kg diet) and nickel-control (1 mg nickel/kg diet) adult rats from the first generation, and suckling pups from the second offspring were used. The nickel transfer across the intestinal epithelium and nickel uptake into the intestine were measured by use of everted jejunal sacs using a wide range of nickel concentrations administered on the luminal side (1.1 x 10^-8 M til 1.0 x 10^-4 M). Both the intestinal nickel transfer and nickel uptake were influenced by the dietary nickel supply in rat offspring, but not in the adult rats from the first generation. However, in nickel-deficient offspring, the nickel transfer across the small intestine was higher than in nickelcontrol offspring. This difference was greater using low intraluminal nickel concentrations than high nickel concentrations, and was significant at 1.1 x 10^-8 M, 6.1 x 10^-8 M, 5.1 x 10^-7 M, 1.0 x 10^-6 M, and 5.0 x 10^-6 M. Also, nickel uptake into the intestine was somewhat greater in nickel-deficient rat pups than in nickel-control pups, and significant using 1.1 x 10^-7 M and 1.0 x 10^-6 M nickel. A definite saturation type kinetic for the intestinal nickel absorption in relation to the intraluminal nickel concentration could not be observed.     

An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata

Fine structural studies have been conducted to determine the role of the trypsinlike proteinase during fertilization which is localized within the cortical granules of sea urchin eggs. Eggs were treated with soybean trypsin inhibitor (SBTI), inseminated, and prepared for microscopic examination at various intervals during fertilization. In the presence of SBTI the breakdown of the cortical granules is delayed and the elevation of the vitelline layer is incomplete. Although all the cortical granules in SBTI-treated eggs are dehisced in a manner similar to controls, the rate at which their breakdown is propagated is reduced. Consequently, dehiscence of all the cortical granules in SBTI-treated specimens requires approximately 3 min to complete, vs. 1 min for controls. In addition the fertilization membrane has a blistered appearance owing to its attachment, at multiple loci, to the surface of the zygote. During the early stages of fertilization many of the treated eggs are monospermic, later all are polyspermic. Supernumerary sperm enter treated eggs (zygotes) up to 10 min after the initiation of the cortical granule reaction, apparently at those sites where the fertilization membrane has failed to separate from the zygote's surface.