A radioautographic study of glyceride synthesis in vivo during intestinal absorption of fats and labeled glucose.

Radioautography was used to detect the synthesis of labeled glycerides in intestinal absorptive cells following injections of fatty chyme and glucose-6-H(3) into ligated segments of upper jejunum of fasting rats. Absorption intervals ranged from 2 to 20 min. Labeling is evident throughout the cells in as short a time as 2 min. Most grains are present over droplets of absorbed fat beginning with those in the endoplasmic reticulum immediately subjacent to the terminal web. With longer absorption periods, frequent grains are present over accumulations of fat droplets in the Golgi cisternae and intercellular spaces. A similar pattern of grains is seen following absorption of either linoleic acid or safflower oil. By comparison, considerably less label is present in the cells when the fat is extracted with alcohol prior to radioautographic procedures, or when labeled glucose alone is absorbed. A significant incorporation of glucose label into newly synthesized glycerides is indicated and confirmed by scintillation counts on saponified lipid extracts. The grain distribution implies an involvement of the extreme apical endoplasmic reticulum in this synthesis.

The origin and nature of beading: a reversible transformation of the shape of nerve fibers.

Nerve fibers which appear beaded (varicose, spindle-shaped, etc.) are often considered the result of pathology, or a preparation artifact. However, beading can be promptly elicited in fresh normal nerve by a mild stretch and revealed by fast-freezing and freeze-substitution, or by aldehyde fixating at a temperature near 0 degree C (cold-fixation). The key change in beading are the constrictions, wherein the axon is much reduced in diameter. Axoplasmic fluid and soluble components are shifted from the constrictions into the expansions leaving behind compacted microtubules and neurofilaments. Labeled cytoskeletal proteins carried down by slow axonal transport are seen to move with the soluble components and not to have been incorporated into and remain with, the cytoskeletal organelles on beading the fibers. Lipids and other components of the myelin sheath are also shifted from the constrictions into the expansions, with preservation of its fine structure and thickness. Additionally, myelin intrusions into the axons are produced and a localized bulging into the axon termed "leafing". The beading constrictions do not arise from the myelin sheath: beading occurs in the axons of unmyelinated fibers. It does not depend on the axonal cytoskeleton: exposure of nerves in vitro to beta, beta'-iminodipropionitrile (IDPN) disaggregates the cytoskeletal organelles and even augments beading. The hypothesis advanced was that the beading constrictions are due to the membrane skeleton; the subaxolemmal network comprised of spectrin/fodrin, actin, ankyrin, integrins and other transmembrane proteins. The mechanism can be activated directly by neurotoxins, metabolic changes, and by an interruption of axoplasmic transport producing Wallerian degeneration.

Kinetics of appearance of lipoprotein particles in perisinusoidal cisternae of smooth endoplasmic reticulum of isolated rat livers perfused with free fatty acid.

Vesicles containing single lipoprotein particles are found in the perisinusoidal cytoplasm of rat hepatocytes in the proximity of particle-containing rough-smooth endoplasmic reticulum transition elements. these lipoprotein particle-containing smooth endoplasmic reticulum elements provide an alternative to Golgi apparatus as a supplier of very low density lipoprotein particles to the circulation. To test this possibility, the kinetics of appearance of lipoprotein particles in these elements was determined for isolated rat livers first depleted of particles and then perfused with free fatty acid to restore particles. The results show that elements of the smooth endoplasmic reticulum in the perisinusoidal cytoplasm acquire lipoprotein particles in advance of elements of the conventional Golgi apparatus and that the particles contained within the perisinusoidal elements of smooth endoplasmic reticulum are not products of endocytotic uptake of particles from the circulation.

Cytoskeletal organelles and myelin structure of beaded nerve fibers.

Freshly removed cat peripheral nerves and lumbar spinal cord roots were prepared by freeze-substitution to study the form changes, cytoskeletal alterations and myelin structure in beaded nerve fibers. Fibers of unstretched nerves so prepared were close to cylindrical. When lightly stretched with tensions of 2-10 g before being rapidly frozen, beading appeared as a series of constrictions between the more normally expanded regions of the internodes with the paranodal regions spared. Beading also was seen in the fibers of sciatic and radial nerves fast-frozen in situ with the limbs placed in full extension to cause stretching. The cross-sectional area of the axon in the constrictions of beaded fibers was reduced by as much as 95%. The compaction of the microtubules and neurofilaments in the constructions was accounted for by the movement of axoplasmic fluid from the constrictions axially into the nearby regions where the axon and fiber diameters are close to normal. The electron-lucid area approximately 5 nm thick around the microtubules appeared to hinder their close approach in the constrictions although some microtubules touch. The neurofilaments are generally separated at a mean distance of 8-10 nm and approach to a mean distance of 4 nm in the constrictions. Neither the beading nor the reversal of beading, which occurs on relaxation from stretch, was blocked by periods of anoxia lasting several hours. Deletion of calcium from the incubating medium initiated some small amount of beading and additionally greatly augmented the beading on stretch. Beading also was present in some of the myelinated fibers of the dorsal columns of the spinal cord where stretch would not be present. These findings suggest that beading is due to a contractile process in the axon initiated by stretch and by other changed states of the fiber. Concomitantly with the contraction of the axon in the beading constrictions, the myelin sheath in that region was greatly reduced in circumference, to as much as 1/3 to 1/5 of normal. The decrease of the sheath diameter was not accompanied by a change in its thickness or in its lamellar fine structure. A repeat distance of the dense lines of 14 nm was measured in both the constricted and nonconstricted regions. To account for these findings lipid, and most likely other components of the myelin lamellar membranes, must move longitudinally from the constrictions in the plane of the lamellar membranes, and do this within 5-10 s.(ABSTRACT TRUNCATED AT 400 WORDS)

Myelin intrusions in beaded nerve fibers.

Small intrusions form in the internodes in or near the constrictions of beaded fibers prepared by fast-freezing and freeze-substituting mildly stretched nerves in the cat and rat. They appear as inwardly directed folds of the inner lamellae of the myelin sheath, or regularly formed spheres composed of lamellae with major dense and interperiod lines like those of the myelin sheath. A splitting of the lamellae and separation of the major dense lines may occur with an accumulation of Schwann cell cytoplasm between them, the result of an influx of cytoplasmic fluid from nearby constrictions. Longitudinally oriented microtubules have been observed in the intrusions, in the adaxonal Schwann cell cytoplasm, and in the innermost lamellae of the myelin sheath. The paranodes contain a number of larger intrusions in the form of spurs and globules along with shelve-like folds of the myelin sheath oriented in the longitudinal direction. Axoplasmic fluid driven from the constrictions during beading can enter the paranodes to smooth out their folds leaving the globular and spur-shaped myelin intrusions in isolation. Their wall thickness, measured from the central opening to the surface of the intrusion, is the same as that of the myelin sheath or, in some cases, double, the result of the folding of a spur-like intrusion upon itself. Intrusions unconnected to the sheath are seen in unbeaded fibers with regular, compact lamellae surrounded by axolemma. Others lack a covering axolemma and consist of variably disorganized and irregularly shaped lamellae suggesting that they are undergoing fragmentation and dissolution within the axon. The hypothesis is advanced that the intrusions in the internodes arise from an excess of lipid and other myelin components when the diameter of the sheath is reduced in the beading constrictions. In the paranodes, excess myelin components moved into these regions form the shelf-like folds which may fuse to form intrusions. These, separated from the myelin sheath, undergo fragmentation and dissolution and are carried by retrograde transport to the cell bodies where their constituent components can be reutilized.

The relation of the beading of myelinated nerve fibers to the bands of Fontana.

The bands of Fontana, appearing as spirals or irregular light and dark strips crossing the surface of unstretched nerves, are due to the wavy disposition of nerve fibers within the epineural-perineural sheaths. A mean tension of 2.7 +/- 0.23 (S.E.M.) g applied to segments of rat tibial nerves straightens the fibers and unbands the nerves causing them to lengthen by 9.35 +/- 0.89%. The nerves cold-fixed in situ at that point showed the myelinated fibers to be beaded. On relaxation the nerves rebanded and the fibers were no longer beaded. The tension at which unbanding occurred was better determined when the epineural-perineural sheaths were slit longitudinally. Under these conditions, unbanding occurred at a mean tension of 0.59 +/- 0.08 g and the nerves lengthened by 8.56 +/- 0.58%. The lengthening was not statistically different from that seen in sheathed nerves. In preparations with the epineural-perineural sheaths removed, banding was lost with tensions of 0.20 +/- 0.03 g and the nerves lengthened by 12.1 +/- 1.04%. The tensions needed were significantly lower than that for the sheathed and slit-sheath nerve groups. When cold-fixed, when banding was lost, the fibers were seen to be beaded. Banding of the desheathed nerves returned on relaxation of the nerves. However, after tensions of 8 g they showed plasticity in which the ends of the nerves needed to be pushed together to initiate rebanding in comparison to sheathed or sheath-slit nerves which rebanded spontaneously following relaxation after even higher tensions of 40 g. At the highest tensions the nerves remained extended and could not be forcibly rebanded.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of beading constrictions at the axolemma: presence in unmyelinated axons and after beta,beta'-iminodipropionitrile degradation of the cytoskeleton.

Myelinated nerve fibres become beaded when nerves are subjected to a mild stretch; the beading is seen as varicosities, a series of alternating constrictions and enlargements, when using freeze-substitution or cold-fixation to hold this labile form change in place during fixation. One possibility for how this form change comes about is that the myelin sheath or its Schwann cell initiates beading. We now report, however, that a similar beading is seen in the axons of unmyelinated fibres. In electron micrographs, longitudinal sections of axons show the series of constrictions and expansions typical of beading. In cross-sections, axons with unusually small diameter, corresponding to the constrictions, are seen to contain closely packed microtubules and neurofilaments while neighbouring swollen axons with widely dispersed microtubules correspond to the beading expansions. Another possibility for the form change is that the cytoskeleton is responsible for beading. We discovered that direct exposure of nerves to beta, beta'-iminodipropionitrile in vitro for 1-6 h causes both axonal microtubules and neurofilaments to become degraded and replaced by an amorphous residue. Nevertheless, beta,beta'-iminodipropionitrile-treated nerves show constrictions in myelinated fibres when stretched. An even greater degree of beading with narrower and longer constrictions appears in some fibres, with the expanded regions having oblate ends giving the appearance of a string of sausages. In cross-sections taken through the constrictions, a greater than usual reduction of axonal area was seen, this was due to the loss of cytoskeletal organelles which would act to limit the degree of constriction. With longer exposure to beta, beta'-iminodipropinitrile more fibres show complete degeneration of the cytoskeleton and form ovoids typical of Wallerian degeneration. Unmyelinated axons of beta, beta'-iminodipropionitrile-treated nerves which showed degeneration of their cytoskeleton with its replacement by amorphous material still demonstrated beading. As neither the myelin sheath nor the intact cytoskeleton within the axon is necessary for beading, by exclusion, we consider beading constrictions to be initiated at the level of the axolemma. In our hypothesis the membrane skeleton is responsible; namely, the spectrin, actin and other molecular species lining the inside of the axolemma and binding to transmembrane proteins. The membrane skeleton may be activated by stretch via transmembrane proteins (e.g. beta 1-integrins). The membrane skeleton mechanism may also be directly engaged in the production of Wallerian degeneration or be induced by neurotoxic agents.

Slow transport of freely movable cytoskeletal components shown by beading partition of nerve fibers in the cat.

To account for the transport in nerve fibers of tubulin and neurofilament proteins in slow component a, the Structural Hypothesis holds that these proteins are assembled into microtubules and neurofilaments in the cell bodies and the cytoskeletal organelles then moved down in the fibers as part of an interconnected matrix at a uniform rate of about 1 mm/day. The Unitary Hypothesis, on the other hand, considers these proteins to be carried down within the fibers as soluble components or as freely movable small polymers or subunits turning over locally in the stationary cytoskeleton. To differentiate between the two hypotheses, cat L7 dorsal roots were taken at times from 7 to 25 days after their L7 dorsal root ganglia were injected with [3H]leucine to assess the labeling of the cytoskeleton by the use of beading and autoradiography. Beading was induced by a mild stretch and after fast-freezing and freeze-substitution of the roots for histological preparation, the beads were seen in the fibers as a series of expanded regions alternating with constrictions. In the constrictions the cytoskeleton was compacted into an area as small as 5% that of the normal axon, with the axoplasmic fluid and displaceable (freely movable) components squeezed from the constrictions into the adjoining expansions. Roots taken after 7 and 14 days, times consistent with slow component a downflow, were assessed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their content of tubulin and neurofilament proteins shown to constitute 40-50% of all the labeled proteins present. In autoradiographs of dorsal roots taken at those times, numerous grains due to radioactivity were located over the non-constricted regions of the fibers. Few or no grains were present over the constrictions after 7 days. The findings are in accord with the labeled tubulins and neurofilament proteins being present in soluble form in the fibers and expressed from the constrictions into the expansions of the beaded fibers. In contrast, a number of fibers in roots taken at 14-20 days after injection showed somewhat higher grain densities over the constrictions, and more so after 25 days, indicating uptake of labeled subunits into the cytoskeletal organelles at later times. The results are consistent with the downflow of tubulin and neurofilament proteins as soluble components which drop off in the axon to turn over locally in their respective cytoskeletal organelles.

The beaded form of myelinated nerve fibers.

When the nerves are lightly stretched and fixed by freeze-substitution, their fibers show the form-change termed "beading" which consists of a series of undulating constrictions and swellings in the internodes. This form change has not ordinarily been seen in chemically fixed nerves, or when it has, it has been ascribed to a pathological change or an artifact. We now report that beading is also retained in normal nerves when, following a light maintained stretch, they are fixed with aldehydes at a temperature close to 0 degrees C. The degree of beading in single fibers teased from the aldehyde fixed nerves was graded and found to be maximal at 0 degrees C, falling off with increased temperature until, at temperatures above 16 degrees C, most fibers showed no beading or a very mild beading. The fibers of nerves cold-fixed at 0 degrees C displayed the characteristics as freeze-substituted fibers, but with a somewhat smaller number of maximally beaded fibers and an 18% reduction in microtubule numbers in the axons. Desheathing or slitting the sheaths of the nerves before cold-fixation increased the probability of retaining beading. Exposure of stretched nerves to the aldehyde fixative at room temperatures for times as short as 3-5 min before they were cold-fixed showed a diminished degree of beading, indicating that aldehydes can have a deleterious effect on the beading mechanism which we hypothesize to be present in the fiber. This action is distinct from the general cross-linking action of aldehydes.

Restricted mobility and endocytosis of anionic sites on newborn rat jejunal brush border membranes.

The distribution and mobility of anionic sites on the microvillous surface of newborn rat jejunal absorptive cells were studied using polycationic ferritin (PCF) as a visual probe and were compared with anionic sites previously described for adult jejunum. Segments from 5- to 26-day-old rats were incubated in PCF for 5 minutes either before or after fixation for electron microscopy. From days 5 to 20, anionic sites were distributed diffusely along the lengths of the microvilli and did not show random translational mobility. In contrast, microvilli examined from animals at weaning (2l to 26 days) resembled those from adults in which most binding sites were capable of lateral mobility and were induced by PCF to culster into discrete patches. The diffuse pattern was altered by cortisone administration, paralleling a premature reduction in the endocytic apparatus of the cell. The difference in mobility of anionic sites with age coincides with differences in absorptive function. Evidence is presented showing that in the neonate binding of PCF to the microvilli was followed with time by endocytosis into an apical system of tubules for intracellular transport, incorporation into coated vesicles, and release through the lateral cell surface. The results suggest that endocytosis is accomplished by a mechanism that includes a directionally controlled movement for the selective internalization of PCF binding sites from the membranes of the microvilli to those of the tubular cytoplasmic channels.

Diabetic pregnancy. Changes in lectin binding to the surface of rat lung alveolar epithelial cells.

The binding of the lectins concanavalin A (Con A) and wheat germ agglutinin (WGA) to the luminal surface of lung alveolar epithelial cells was compared in normal rats and rats with streptozotocin-induced diabetes and their offspring. Lung tissue was lavaged, then fixed in situ with 3% glutaraldehyde. Buffer-rinsed slices of lung were incubated in Con A, WGA, or various control media. Lectin binding sites were visualized by the use of the peroxidase method. Normal neonates and those that were the results of diabetic pregnancies showed a hexose-specific Con A and WGA binding pattern qualitatively similar to that of normal and diabetic adults, respectively. In the normal animals, Con A binding sites were masked by sialic acid residues and were removable with alpha-mannosidase after neuraminidase treatment. In the diabetic adults and their offspring, one the other hand, Con A binding sites were readily accessible and were totally removed only by sequential treatment with alpha-mannosidase and alpha-glucosidase. WGA binding was essentially eliminated with neuraminidase in all animals except in the neonates from diabetic pregnancies, where N-acetyl-glucosaminidase was also required. The effects of maternal diabetes were reversible and occurred about Day 7 postpartum in the neonate. The effects were also reversible following insulin replacement in the diabetic adult.

Further characterization of the pigment in pseudomelanosis duodeni in three patients.

Pseudomelanosis duodeni is a rare condition in which dark pigment accumulates in macrophages located in the lamina propria of the duodenal mucosa. Three cases are reported here and the literature is reviewed. No clinical association can be found that points clearly to the underlying etiology. Electron probe x-ray microanalysis was used to study the pigment in macrophage granules in 2 of our patients and demonstrated high iron and sulfur content. Iron accumulation in ferritinlike particles was detected in absorptive cell lysosomes. A possible mechanism for the accumulation of absorbed iron by macrophages is considered.

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion.

After injection of the L7 dorsal root ganglion with 3H-leucine, fast axoplasmic transport carries some 3--5 x more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.

Ultrastructural aspects of ascosporulation in Arthroderma quadrifidum (=Trichophyton terrestre).

The ultrastructural features of developing and mature ascospores were delineated after mating Arthroderma quadrifidum on pablum cereal agar. Incipient ascospores each contained a granulated nucleus bounded by a nuclear envelope while presumptive ascospore cytoplasm was bounded by a double membrane and resided in glycogen-rich epiplasm of the ascus. Mature ascospores contained nuclei and mitochondria while the ascus epiplasm still retained abundant inclusions. The ascospore wall demonstrated the presence of heterogeneous material between the plasmalemma and the outer spore membrane which appeared smooth.

Origin and ultrastructure of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum.

A cell observation chamber was designed to perform continuous photomicroscopic observations of hyphal anastomosis and the origin of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum. These data were correlated with ultrastructural features of intra-hyphal hyphae. Hyphal fusions occurred commonly in either species of Trichophyton when incubated alone. In T. terrestre, empty phyphal segments adjoined by live units were invaded at the septa from both directions by new hyphal ingrowth. Continuous observations revealed that the intra-hyphal hyphae subsequently anastomosed via a lateral fusion peg. Similar intra-hyphal hyphae were shown in T. rubrum. Electron microscopic studies revealed ascomycetous septa in both conventional hyphae and intra-hyphal hyphae. For the latter, the cytoplasm and wall of the inner hypha were bounded by cytoplasmic organelles and another cell wall of the outer hypha.

Localization of calcium in nerve fibers.

Using the desheathed nerve preparation, a pyroantimonate precipitation method was used to examine the distribution of electron-dense particles seen in various organelles of the nerve fibers following exposure of nerve to various levels of Ca2+ in vitro. The presence of Ca2+ in the electron-dense particles was indicated by their extraction with EGTA and by the use of energy-dispersive X-ray microanalysis. In normal Ringer or in a Ca2+ -free medium, electron-dense particles were seen associated with the outer membrane of the mitochondria, with the smooth endoplasmic reticulum (SER), along the axolemma and yet others scattered throughout the axoplasm. When nerves were incubated in media containing higher than normal concentrations of 20-60 mM Ca2+, an increase in the number of such electron-dense particles was seen in the axoplasm and within the mitochondrial matrix. Nerves loaded with a high concentration of 60mM Ca2+ could be depleted of these particles after transfer to a Ca2+ -free or low Ca2+ Ringer medium. The sequestration of Ca2+ in axonal organelles is discussed with respect to Ca2+-regulatory mechanisms in the axon needed to maintain a low level of Ca2+ which is optimal for the support of axoplasmic transport.

Seeding arterial prostheses with vascular endothelium. The nature of the lining.

Arterial prostheses seeded with autogenous vascular endothelium demonstrate a well-organized, cellular, inner lining. To determine the nature of the lining cells, six animals underwent replacement of the infrarenal aorta with Dacron prostheses. During the preparation of three such grafts, endothelium was scraped from the saphenous vein with a steel wool pledget, suspended in chilled Sack's solution, and mixed with blood used to preclot the graft. This suspension was omitted from the three control grafts. After six weeks, the grafts were removed, rinsed and examined. Fluorescent Factor VIII related antigen (F VIII-RA) strongly stained the lining cells. Silver nitrate Haütchen and electron microscopy preparations revealed a lining pattern characteristic of vascular endothelium. Endothelial cell-specific Weibel-Palade bodies were identified in the lining cell cytoplasm. Masson's trichrome staining revealed a relatively collagen-poor connective tissue within the seeded fabric. Transmission electron microscopy disclosed vascular smooth muscle cells between the seeded graft fabric and the lining cells. Vasa vasorum, arising from the outer capsule, penetrated the fabric to supply the inner capsules of the seeded grafts. It is concluded that the cells lining seeded canine arterial prostheses are true vascular endothelium supported by vascular smooth muscle cells, that the lining contains minimal connective tissue, and that vasa vasorum develop. Unseeded control grafts lacked these features.