An Electron Microscopic Study of the Intestinal Villus : II. The Pathway of Fat Absorption

The intestinal pathway for absorbed fat was traced in thin sections of intestinal villi from rats fed corn oil by stomach tube after a fast of 24 to 40 hours. For electron microscopy the tissues were fixed in chilled buffered osmium tetroxide and embedded in methacrylate. For light microscopy, other specimens from the same animals were fixed in formal-calcium, mordanted in K₂Cr₂O₇, and embedded in gelatin. Frozen sections were stained with Sudan black B or Sudan IV. About 20 minutes after feeding, small fat droplets (65 mµ maximal diameter) appear in the striated border between microvilli. At the same time fat particles are seen within pinocytotic vesicles in the immediately subjacent terminal web. In later specimens the fat droplets are generally larger (50 to 240 mµ) and lie deeper in the apical cytoplasm. All intracellular fat droplets are loosely enveloped in a thin membrane, the outer surface of which is sometimes studded with the fine particulate component of the cytoplasm. This envelope, apparently derived from the cell surface by pinocytosis, has at this stage evidently become a part of the endoplasmic reticulum. Just above the nucleus numerous fat droplets lie clustered within the dilated cisternae of the Golgi complex. As absorption progresses fat droplets appear in the intercellular spaces of the epithelium, in the interstitial connective tissue spaces of the lamina propria, and in the lumen of the lacteals. All of these extracellular fat droplets are devoid of a membranous envelope. The picture of fat absorption as reconstructed from these studies involves a stream of fat droplets filtering through the striated border, entering the epithelial cell by pinocytosis at the bases of the intermicrovillous spaces, and coursing through the endoplasmic reticulum to be discharged at the sides of the epithelial cell into extracellular spaces. From the epithelial spaces, the droplets move into the lamina propria and thence into the lymph. If the lumen of the endoplasmic reticulum is considered as continuous with the extracellular phase, then the entire pathway of fat absorption may be regarded as extracellular. However, it is impossible to evaluate from the electron microscopic evidence thus far available the quantitative importance of particulate fat absorption by the mechanism described.     

An electron microscopic study of the intestinal villus. II. The pathway of fat absorption

The intestinal pathway for absorbed fat was traced in thin sections of intestinal villi from rats fed corn oil by stomach tube after a fast of 24 to 40 hours. For electron microscopy the tissues were fixed in chilled buffered osmium tetroxide and embedded in methacrylate. For light microscopy, other specimens from the same animals were fixed in formal-calcium, mordanted in K(2)Cr(2)O(7), and embedded in gelatin. Frozen sections were stained with Sudan black B or Sudan IV. About 20 minutes after feeding, small fat droplets (65 mmicro maximal diameter) appear in the striated border between microvilli. At the same time fat particles are seen within pinocytotic vesicles in the immediately subjacent terminal web. In later specimens the fat droplets are generally larger (50 to 240 mmicro) and lie deeper in the apical cytoplasm. All intracellular fat droplets are loosely enveloped in a thin membrane, the outer surface of which is sometimes studded with the fine particulate component of the cytoplasm. This envelope, apparently derived from the cell surface by pinocytosis, has at this stage evidently become a part of the endoplasmic reticulum. Just above the nucleus numerous fat droplets lie clustered within the dilated cisternae of the Golgi complex. As absorption progresses fat droplets appear in the intercellular spaces of the epithelium, in the interstitial connective tissue spaces of the lamina propria, and in the lumen of the lacteals. All of these extracellular fat droplets are devoid of a membranous envelope. The picture of fat absorption as reconstructed from these studies involves a stream of fat droplets filtering through the striated border, entering the epithelial cell by pinocytosis at the bases of the intermicrovillous spaces, and coursing through the endoplasmic reticulum to be discharged at the sides of the epithelial cell into extracellular spaces. From the epithelial spaces, the droplets move into the lamina propria and thence into the lymph. If the lumen of the endoplasmic reticulum is considered as continuous with the extracellular phase, then the entire pathway of fat absorption may be regarded as extracellular. However, it is impossible to evaluate from the electron microscopic evidence thus far available the quantitative importance of particulate fat absorption by the mechanism described.     

Comparison of two ultracentrifugation procedures for separation of nonhuman primate lipoproteins.

A comparison of nonhuman primate plasma lipoproteins isolated by swinging bucket rotor density gradient or fixed angle rotor differential ultracentrifugation is described. Whereas these two methods produced comparable results for the composition of low density (LDL) and high density (HDL) lipoproteins, the very low density lipoprotein (VLDL) fraction isolated with the swinging-bucket rotor contained relatively more cholesterol (free and esterified) and less phospholipid and protein than that fraction obtained with the fixed-angle rotor. Estimations of lipoprotein concentration by agarose gel electrophoresis and particle size by electron microscopy coupled with molar ratios of surface to core constituents indicate that the swinging bucket procedure resulted in a more complete harvest of VLDL particles, especially those in the larger size range.     

Ultrastructural analysis of milk fat globule membranes of colostrum and human milk

Lacteous fat globules with their membranes from human milk and calostrum were studied by scanning and transmission electron microscopy. The first appear as spheroidal structures with some irregularities on the surface. Under the transmission electron microscope these irregularities are composed of islets from a material morphologically similar to cytoplasm, and with structures that resemble a fragment of rugose endoplasmic reticulum. The membranes in specimens fixed immediately after secretion are tri-layered, similar in appearance to those of a single membrane, but in samples fixed between two and four hours after secretion the details are unclear. Through freeze-etching, the laminar aspect of the fat globules is observed.     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Determining molecular weight distributions of antigen-antibody complexes by quasi-elastic light scattering.

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.     

Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth

Adipose tissue grows by two mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase). Genetics and diet affect the relative contributions of these two mechanisms to the growth of adipose tissue in obesity. In this study, the size distributions of epididymal adipose cells from two mouse strains, obesity-resistant FVB/N and obesity-prone C57BL/6, were measured after 2, 4, and 12 weeks under regular and high-fat feeding conditions. The total cell number in the epididymal fat pad was estimated from the fat pad mass and the normalized cell-size distribution. The cell number and volume-weighted mean cell size increase as a function of fat pad mass. To address adipose tissue growth precisely, we developed a mathematical model describing the evolution of the adipose cell-size distributions as a function of the increasing fat pad mass, instead of the increasing chronological time. Our model describes the recruitment of new adipose cells and their subsequent development in different strains, and with different diet regimens, with common mechanisms, but with diet- and genetics-dependent model parameters. Compared to the FVB/N strain, the C57BL/6 strain has greater recruitment of small adipose cells. Hyperplasia is enhanced by high-fat diet in a strain-dependent way, suggesting a synergistic interaction between genetics and diet. Moreover, high-fat feeding increases the rate of adipose cell size growth, independent of strain, reflecting the increase in calories requiring storage. Additionally, high-fat diet leads to a dramatic spreading of the size distribution of adipose cells in both strains; this implies an increase in size fluctuations of adipose cells through lipid turnover.     

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.     

The leptin-fat ratio is constant, and leptin may be part of two feedback mechanisms for maintaining the body fat set point in non-obese male Fischer 344 rats.

While plasma leptin and adiposity have been found to be strongly related, the specific nature of this relationship has yet to be clarified. Hence, plasma leptin and three indicators of adiposity were measured in adult male Fischer 344 rats on three different long-term diets: continuous ad libitum feeding; ad libitum feeding until early adulthood, then continuous 60% caloric restriction; and ad libitum feeding until early adulthood, then 60% caloric restriction until 16 months, then ad libitum feeding for 5 months. Body fat was found to be a good linear correlate of plasma leptin, with a zero Y-intercept, and a constant plasma leptin-body fat ratio. The number of adipocytes per rat and % body fat were strong quadratic correlates of plasma leptin. This study is the first to find a zero Y-intercept and constant plasma leptin-body fat ratio, probably because it is the first to simultaneously measure both plasma leptin and body fat accurately, and to account for confounders such as gender, genetic background, age, physical activity, and possibly obesity. The study also explored the effect of switching calorically-restricted rats to ad libitum feeding. This led to a rapid rise, and then synchronized up-down cycles in average daily food intake and body weight, with a steady upward trend toward a new stable body-weight set point. It is hypothesized that this pattern resulted from two simultaneous feedback mechanisms, possibly involving leptin. In conclusion, this study suggests that, under controlled conditions, the plasma leptin-body fat ratio is a constant for a particular mammalian strain, independent of dietary history.     

Plasma Enterostatin: Identification and Release in Rats in Response to a Meal

Objective: To discover a possible absorption and/or secretion of enterostatin into the circulating blood, as well as to compare the levels of circulating enterostatin after high‐fat feeding and low‐fat feeding. Research Methods and Procedures: Using a specific enzyme‐linked immunosorbent assay, plasma enterostatin levels were determined after feeding a high‐fat, a high‐fat/‐sucrose, or a low‐fat meal to Sprague‐Dawley rats deprived of food overnight. Results: The enterostatin levels were increased by all diets; the response to the high‐fat and the high‐fat/‐sucrose meals was greater in magnitude and duration than that to the low‐fat meal. In addition, enterostatin levels correlated with the intake of dietary fat. Plasma enterostatin levels after high‐fat feeding were found to be similar to those after intravenous administration of exogenous enterostatin known to inhibit high‐fat food intake. Gel chromatography of pooled postprandial plasma extracts followed by high‐performance liquid chromatography analysis showed that plasma enterostatin was identical to synthetic enterostatin. Affinity cross‐linking of plasma proteins with ¹²⁵I‐enterostatin on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by autoradiography, revealed a single band with a molecular weight of about 66 kDa, indicating the presence of a potential enterostatin‐binding protein in plasma. Discussion: The measurements of plasma enterostatin may be a sensitive indicator for the measurement of fat intake.     

Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells

Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.     

New scanning electron microscopic method for determination of adipocyte size in humans and mice

Objective: To evaluate a new scanning electronic microscopic (EM) method for assessing fat cell sizes and compare fat cell size distribution in human adipose tissue from different fat depots before and after weight loss. Research Methods and Procedures: Identical human fat tissue biopsies were separated into two fractions: one used to prepare a fat cell suspension by collagenase digestion followed by photomicrography (collagenase method) and the other fixed in formalin for EM analysis. The EM method was evaluated further by determining fat cell sizes from lean and ob/ob mice. Finally, the EM method was used to assess fat cell sizes in biopsies of different human depots from before and after weight loss. Results: Fat cell size distributions measured by the two methods were not identical, but differences were generally small. The EM method reproduced the well‐documented fat cell size difference between lean and ob/ob mice. Large variation was detected in fat cell distributions among three depots in humans. Weight loss reduced fat cell sizes in subjects with large baseline fat cells but had no effect in subjects with small baseline fat cell sizes. Discussion: Our results suggest that the EM method may be a useful alternative for fat cell size analysis of clinical samples.     

Effect of Pyrolysis Temperature on Chemical and Surface Properties of Biochar of Rapeseed (Brassica napus L.)

The biochar is an important carbon-rich product that is generated from biomass sources through pyrolysis. Biochar (charcoal) can be both used directly as a potential source of solid biofuels and as soil amendments for barren lands. The aim of this study was investigate influence of pyrolysis temperature on the physicochemical properties and structure of biochar. The biochars were produced by pyrolysis of rapeseed (Brassica napus L.) using a fixed-bed reactor at different pyrolysis temperatures (400–700°C). The produced biochars were characterized by proximate and elemental analysis, Brunauer–Emmett–Teller (BET) surface area, particle size distributions, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy. The results showed that both chemical and surface properties of the biochars were significantly affected by the pyrolysis temperature. Aromatic hydrocarbons, hydroxyl and carbonyl compounds were the majority components of the biochar. The biochar obtained at 700°C had a high fixed carbon content (66.16%) as well as a high heating value, and therefore it could be used as solid fuel, precursor in the activated carbons manufacture (specific surface area until 25.38 m² g^?1), or to obtain category-A briquettes.     

Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects

The biochemical, ultrastructural and functional aspects of digitonin-permeabilized platelets were investigated. Human platelets were permeabilized by exposure to the steroid glycoside digitonin. A 60 microM concentration of this permeabilizer produced a very substantial release of cytosolic enzymes from the platelets. Release from subcellular granules was relatively low and did not inhibit the response of platelets to a series of agonists. Although digitonin-permeabilized platelets required higher threshold concentrations of the usual stimulants, both primary and secondary aggregation as well as the release of nucleotides and enzymes from their respective granules remained intact. Transmission electron micrographs revealed discontinuities in the plasma membrane of digitonin-treated platelets, but scanning electron microscopy showed no difference between control and permeabilized platelets. No substantial loss of structural or membrane proteins could be detected by one- and two-dimensional gel electrophoresis. The pore size produced by digitonin treatment was sufficient to allow entry of 125I-labeled IgG into the platelet cytosolic space.     

Scanning electron microscopy of very small fat cells and mature fat cells in human obesity

To determine the effect of obesity on the size distribution of fat cell populations in human adipose tissue, omental fat tissue biopsies were obtained from lean, moderately obese, and massively obese patients. The size distributions of adipocytes from lean and obese fat tissues examined by the scanning electron microscopic method were bimodal, consisting of populations of very small fat cells and mature fat cells, in contrast to collagenase-derived isolated cells that showed only the large mature fat cells. The very small fat cell population represented 21 to 26% of the total fat cell number in the lean and in both obese groups. In contrast, preparations of human fat cells isolated by the collagenase method systematically excluded the very small fat cells. In massive obesity, both cell populations participated in the hyperplastic growth but only the larger mature fat cells increased in size, implying that these two cell populations differ in their physiological role.     

Vesicles of variable sizes produced by a rapid extrusion procedure

Previous studies from this laboratory have shown that large unilamellar vesicles can be efficiently produced by extrusion of multilamellar vesicles through polycarbonate filters with a pore size of 100 nm (Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Biochim. Biophys. Acta 812, 55-65). In this work it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar or plurilamellar vesicles by utilizing filters with pore sizes ranging from 30 to 400 nm. The unilamellarity and trapping efficiencies of these vesicles can be significantly enhanced by freezing and thawing the multilamellar vesicles prior to extrusion. This procedure is particularly applicable when very high lipid concentrations (400 mg/ml) are used, where extrusion of the frozen and thawed multilamellar vesicles through 100 and 400 nm filters results in trapping efficiencies of 56 and 80%, respectively. Freeze-fracture electron microscopy revealed that vesicles produced at these lipid concentrations exhibit size distributions and extent of multilamellar character comparable to systems produced at lower lipid levels. These results indicate that the freeze-thaw and extrusion process is the technique of choice for the production of vesicles of variable sizes and high trapping efficiency.     

Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle

We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of approximately 500 kDa copurify with [3H]ryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [3H]ryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy. Based on their localization to the triadic junction, their large size and their ability to bind [3H]ryanodine, these proteins are identified as foot proteins. In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle. We have termed these proteins, alpha and beta foot proteins.     

Plasma-Assisted Deposition of Au/SiO2 Multi-layers as Surface Plasmon Resonance-Based Red-Colored Coatings

In this work, the expanding thermal plasma chemical vapor deposition in combination with radio frequency magnetron sputtering is used to deposit dielectric/metal multi-layers with controlled size and density of nanoparticles. The multi-layer structure serves the purpose of increasing the nanoparticle number density, without changing the metal particle size, shape and the interparticle distance. The possibility of independently tuning and, therefore, controlling the nanoparticle size and number density allows developing surface plasmon resonance-based deep-colored coatings. The influence of the number of layers, metal surface area coverage, and thickness of the dielectric layer on the multi-layer nanostructure and on the developed color is presented here in detail. The nanoparticle size and distribution have been measured by transmission electron microscopy. Rutherford back-scattering and infra-red transmission spectroscopy have been used to determine the metal surface coverage and the film chemistry, respectively. Optical properties of the nano-composite layers have been investigated by UV-VIS spectroscopy and exhibit an increase in amplitude of the plasmon absorption spectra at a fixed plasmon resonance frequency with an increase in the number of layers.     

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.