LM fibroblast plasma membrane subfractionation by affinity chromatography on con A-sepharose.

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-sided-out orientation of the vesicles. Similarly and asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5'-nucleotidase activity, and (Na+ + K+)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.

Composition and properties of very low density lipoproteins secreted by the perfused rat liver and subfractionated by affinity chromatography.

Very low density lipoproteins (VLDL) were isolated from the perfusate of rat livers infused with a complex of oleic acid bound to bovine serum albumin. Very low density lipoprotein (VLDL) secretion, bile flow, histopathology, and transmission electron microscopy indicated that secretory functions but not morphologic integrity of the livers were maintained during the procedure. Plasma VLDL and liver perfusate VLDL did not have similar size distribution. VLDL isolated from recycling perfusate and single pass perfusate were also subfractionated with concanavalin A-Sepharose 4B affinity chromatography. Three subfractions were eluted sequentially from the perfusate VLDL: a non-adherent fraction A and two adherent fractions B and C. The size of these VLDL, determined after negative staining and examination by transmission electron microscopy, was significantly decreased by affinity chromatography. VLDL in fractions A, B and C were spherical and had diameters of 935 +/- 17, 881 +/- 34 and 415 +/- 30 A respectively. Fraction A, which did not adhere to the column, contained 65% of the lipid applied to the column. The carbohydrate composition of fraction A VLDL was 11.2 +/- 0.6% fucose, 14.7 +/- 1.2% galactose, 43.7 +/- 2.3% N-acetylglucosamine, and 30.5 +/- 1.9% sialic acid. Sugars such as glucose and mannose, which bind to concanavalin A, were not detected. In contrast, VLDL fractions B and C, which adhered to the column, contained both glucose (17.7 and 2.5%) and mannose (5.8 and 8.3%) as well as the other sugars present in VLDL fraction A. Sodium dodecyl sulfate gradient gel electrophoresis revealed that the affinity column procedure clearly altered the apolipoprotein patterns of the applied VLDL, thereby producing abnormal fractions B and C. Fractions B and C also differed from unfractionated VLDL and fraction A VLDL in lipid composition, in surface/interior core lipid ratio, and in fatty acid composition of the interior core lipids, primarily triacylglycerols. The steady-state anisotropy, the limiting anisotropy and the lipid order parameter of fluorescence probe molecules 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid incorporated into the VLDL were of the following order: fraction B greater than fraction A greater than fraction C. These results are consistent with the interpretation that concanavalin A-Sepharose 4B affinity chromatography may artificially produce a series of VLDL subfractions whose composition and structural properties do not resemble those of native VLDL.

Measurement of phagocytosis using fluorescent latex beads.

Fluorescent monodisperse latex beads and a computer-centered spectrofluorimeter were used to devise a sensitive new assay for phagocytosis. LM fibroblasts, a transformed cell line with a high endocytic rate, were exposed to fluoresbrite beads and the following parameters were investigated: incubation time, incubation temperature and bead/cell ratio. The bead uptake was linear for 60 min over a wide range of bead/cell ratios up to 130 beads/cell. Phagocytosis was inhibited at 4 degrees C, by incubation in the presence of colchicine, and by glucose deprivation. Scanning and transmission electron microscopy were used to confirm that at 37 degrees C both bead adsorption and internalization occurred while at 4 degrees C only bead adsorption but not endocytosis occurred. Large bead sizes (0.86 and 1.72 micrometer diameter) were most useful due to higher fluorescence and higher signal to noise ratios than smaller beads (0.25 and 0.57 micrometer diameter). Beads (0.86 micrometer diameter) were taken up at a rate of 4.4 beads/cell/h at 37 degrees C when a bead/cell ratio of 70 was used. The uptake was zero when assayed at zero time. These criteria establish that fluoresbrite beads provide a useful new fluorimetric assay for phagocytosis.

Parvovirus-like enteropathy in Missouri turkeys.

A viral enteric disease of young turkeys characterized by stunting of affected birds, diarrhea, and increased mortality is described. Eosinophilic intranuclear inclusion bodies were found in the absorptive epithelial cells of the ileum. Electron microscopy of formalin-fixed tissue revealed that the intestinal inclusions contained numerous loosely packed 15-to 20-nm hexagonal particles. The size, shape, and intranuclear location have been used to tentatively identify these particles as parvoviruses.

The pathology of experimental cytauxzoonosis.

Clinical, gross post mortem, histological and electron-microscopical data were collected from cats serially killed following experimental infection with Cytauxzoon felis. The agent was originally isolated from domestic cats with naturally occurring fatal cytauxzoonosis. Morphologically, the blood phase of feline Cytauxzoon appeared similar or identical to that described for cases of cytauxzoonosis in African ruminants and closely resembled also the blood phases of Theileria spp. and Babesia spp. The tissue phase, which occurred as schizont and merozoite stages within mononuclear phagocytes, was also similar to that seen in African cytauxzoonosis and in theileriosis. The tissue host cells, mononuclear phagocytes, appeared identical in feline and African cases. The Cytauxzoon host cells, however, were distinct from the tissue host cell of Theileria, in which schizogeny takes place within lymphocytes. Another difference was the lack of evidence of Cytauxzoon host-cell division which has been described for Theileria parva.

Characterization of the inclusion limiting membrane of anaplasma marginale by immunoferritin labeling.

Purified anti-erythrocytic membrane antibody (PAMA) was prepared from rabbit anti-bovine erythrocyte serum by an adsorption and elution technique, utilizing bovine erythrocytes. Lysed and washed anaplasma-infected erythrocytes were incubated with PAMA or control reagents. Specimens were then subjected to immunoferritin labeling with ferritin antiglobulin conjugate. Upon examination by electron microscopy, specimens incubated with PAMA showed heavy ferritin labeling of erythrocytic membranes and also the limiting membranes of anaplasmal inclusions. Anaplasmal initial bodies freed from their inclusion membranes were not labeled. Negative control specimens, incubated with normal rabbit serum or PAMA which had been absorbed with erythrocytes, did not show specific ferritin labeling. Intact bovine erythrocytes, which were used as a positive control of anti-bovine erythrocytic membrane specificity, were heavily ferritin-labeled. Avian erythrocytes, a negative control of specificity, remained unlabeled. The results of this study indicate that the limiting membrane of the anaplasmal inclusion is derived from the erythrocytic membrane.

Transmissible gastroenteritis in neonatal dogs: experimental intestinal infection with transmissible gastroenteritis virus.

Fourteen neonatal dogs (4 through 11 days of age) were exposed orally to the Purdue strain of transmissible gastroenteritis (TGE) virus, and six dogs of similar age were noninoculated controls. Clinical signs of enteric disease did not develop. Both exposed and control dogs had normal fecal passages and appetite throughout the experiment. Jejunal epithelium from dogs euthanatized at 12, 24, 48, and 96 hours and at 10 days after exposure did not exhibit morphologic alterations detectable by light microscopy. Electron microscopic examination indicated that jejunal epithelial cells contained TGE viral particles as early as 12 hours after dogs were exposed. There were no apparent morphologic alterations or signs of desquamation of virus-infected cells, however. Results of pig transmission studies indicated that viable TGE virus was in jejunal tissue of the dogs as early as 12 hours and as late as 10 days after exposure to the virus.

Ultrastructural study of the equine cecum during onset of laminitis.

The morphologic and pathologic changes which occurred within the cecal mucosa of 4 horses during the onset of laminitis were determined from cecal biopsy materials obtained via a cecal fistula; the laminitis was induced with carbohydrate overload. The cecal epithelial mucosa specimens were obtained at 0 (base line), 24, 32, 48, and 72 hours after horses were given carbohydrate overload, and these were fixed and subsequently photographed. Changes in the cecal epithelium were examined by transmission electron and scanning electron microscopies. These histopathologic changes indicated that the mucosal barrier was substantially damaged by the carbohydrate overload.

Scanning electron microscopy of bovine corneas irradiated with sun lamps and challenge exposed with Moraxella bovis.

Effects of UV radiation and irradiation followed by challenge exposure with Moraxella bovis on the corneal epithelium were studied in 6 calves by scanning electron microscopy. After UV irradiation, the number of dark cells comprising the surface epithelium increased. Many epithelial cells were in various states of degeneration and were characterized initially by large round nuclei, whereas sloughing and peeling were characteristic of the last degenerative stage. All M bovis-infected irradiated eyes had large numbers of degenerating cells, deep epithelial defects, fibrin strands, surface inflammatory cells, and debris. A few M bovis organisms were randomly attached to the cornea before visible ulceration. There were many inflammatory cells between the ulcerated corneal epithelium and adjacent nonulcerated epithelium. Epithelial cells at the margin of the ulcer appeared swollen. Light, dark, and intermediate epithelial cell types could not be distinguished peripheral to the ulcer.

Effect of intrauterine infusion of Escherichia coli endotoxin in postpartum pony mares.

Fifteen pony mares were assigned to 1 of 3 treatment groups after foaling: Group 1, 35 ml of sterile saline solution was infused into the uterine lumen within 24 hours after parturition (6 mares); group 2, 300 mg of Escherichia coli endotoxin was infused into the uterine lumen within 24 hours after parturition (6 mares); and group 3, 300 mg of E coli endotoxin was infused into the uterine lumen between 72 and 96 hours after parturition (3 mares). Rectal temperatures were taken at -1, -0.5, 0, 0.5, 1, 1.5, 2, 3, 4, and 5 hours after treatment. Venous blood samples were also taken at these times for routine WBC counts. Data were analyzed as a repeated measurement design with linear and quadratic orthogonal contrasts performed where significant time and interaction with time occurred. Pretreatment averages of total WBC and neutrophil counts were compared with their nadir posttreatment averages by a t test when treatment-by-time interaction was significant for the parameter. Rectal temperature (37.9 +/- 0.1 C) remained stable and did not vary among treatment groups after intrauterine infusions. In contrast, total WBC and neutrophil counts did vary among treatment groups across time. However, for treatment groups 1 and 3, neither blood total WBC count nor neutrophil count after intrauterine infusions was different from pretreatment observations. In group 2, total WBC count decreased (P less than 0.10) from a pretreatment average of 11.5 +/- 0.4 X 10(3) cells/mm3 to a nadir concentration of 10.0 +/- 0.6 X 10(3) cells/mm3 by 60 minutes after infusion of endotoxin into the uterus.(ABSTRACT TRUNCATED AT 250 WORDS)

Naturally acquired enteric adenovirus infection in Syrian hamsters (Mesocricetus auratus).

Intranuclear inclusions indicative of adenovirus infection were detected microscopically in formalin-fixed intestinal tissues from preweanling Syrian hamsters. The amphophilic intranuclear inclusion bodies were observed in ileal enterocytes from 16-to 24-day-old hamsters. Electron microscopy revealed large numbers of 72 +/- 3-nm viral particles typical of adenoviridae in enterocytic nuclei. Serum antibodies reacted with mouse adenovirus strains K87 and, to a lesser extent, FL, by indirect fluorescent antibody testing. Clinical disease was not associated with the adenoviral infections. Hamsters from 10 production colonies, including all major commercial Syrian hamster suppliers in the United States, were surveyed and all had serologic or histopathologic evidence of adenovirus infection.

Evaluation of cortical bone damage and axial holding power of nonthreaded and enhanced threaded pins placed with and without drilling of a pilot hole in femurs from canine cadavers.

OBJECTIVE: To evaluate the in vitro axial extraction forces necessary to remove pins and to evaluate mechanical trauma resulting from pin insertion, using various types of pins and insertion techniques. DESIGN: Prospective, controlled study. SUBJECTS: Femurs of cadavers of dogs. PROCEDURE: Pins were inserted as follows: 1 nonthreaded pin without drilling of a pilot hole, 1 enhanced threaded pin with drilling of a pilot hole, and 1 enhanced threaded pin without drilling of a pilot hole. After pin insertion, mechanical damage and proper pin insertion was determined by means of radiography. Axial extraction forces were determined for all pins, using a universal testing machine. Mechanical damage was evaluated in 12 additional femurs. After pin insertion, all pins were removed from the bone by use of a low-speed power drill. Samples were sectioned, processed, and evaluated by use of dissecting and scanning electron microscopy. RESULTS: Using radiography, a significant difference was detected in the number of periosteal trans-cortex fractures between the enhanced threaded and nonthreaded pins. Axial extraction force was not significantly different between the enhanced threaded pins, regardless of insertion technique; however, the axial extraction force was significantly greater for enhanced threaded pins, compared with that for nonthreaded pins. Microfractures only were detected on the periosteum of the trans-cortex of enhanced threaded pins by use of scanning electron microscopy. CLINICAL IMPLICATIONS: We cannot recommend a particular insertion technique to decrease mechanical trauma to the bone and to increase axial extraction force needed for removal of enhanced threaded pins from the femur of dogs.

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.

Electron microscopy of vesicular-arbuscualr mycorrhizae of yellow poplar. I. Characterization of endophytic structures by scanning electron stereoscopy.

Vesicular-arbuscular mycorrhizae of yellow poplar (Liriodendron tulipifera L.) were examined by scanning electron microscopy. The morphology and spatial distribution of endophytic structures are illustrated. The use of stereoscopic techniques in conjunction with removal of host cytoplasm permits detection of subtle morphological alterations and progressive deterioration of the fungal component.

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. IV. Host-endophyte interactions during arbuscular deterioration 1,2.

Scanning electron stereoscopy and transmission electron microscopy were used to correlate morphological alterations and cytological phenomena associated with deterioration of arbuscules in yellow poplar mycorrhizae. Arbuscular degradation was initiated at the tips of the finest branches and progressed basipetally. Cytoplasm in arbuscular hyphae progressively deteriorated and was followed by collapse of the fungal walls. Degraded portions of the arbuscules aggregated into clumps comprised of host wall material and the distorted fungal walls. Host nuclei, abundant mitochondria, and proplastids were closely associated with arbuscular branches undergoing cytoplasmic deterioration and with clumped portions of the arbuscule which contained degraded hyphal branches. Most of the arbuscules observed had deteriorated to the clumped stage. Some cortical cells contained several clumped arbuscules and nearly mature, intact arbuscules which indicated that reinfection occurs even as degradative phenomena are in progress. It is suggested that substantial quantities of mineral nutrients may be made available to the host via degradation of fungal cytoplasm in the arbuscular hyphae preceding aggregation of degraded hyphae into discrete clumps.

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. III Host-endophyte interactions during arbuscular development.

Scanning- and transmission-electron microscopy were used to examine developing and mature functional arbuscules in mycorrhizal roots of yellow poplar. Arbuscules developed from intracellular hyphae which branched repeatedly upon penetration into the host cells. Intermediate and late stages of developemnt were characterized by the production of numerous, short, bifurcate hyphae throughout the arbuscule. Mature arbuscules exhibited a coralloid morphology which resulted in a considerable increase in the surface area of the endophyte exposed within the host cells. Distinctive ultrastructural features of arbuscular hyphae included osmiophilic walls, nuclei, abundant cytoplasm, glycogen, and numerous small vacuoles. All arbuscular components were enclosed by host wall material and cytoplasm during development and at maturity. In infected cells, host nuclei were enlarged and the cytoplasm associated with the arbuscular branches typically contained abundant mitochondria, endoplasmic reticulum, and proplastids. Ultrastructural observations suggested that nutrient transfer may be predominantly directed toward the fungal endophyte during arbuscular development and while mature arbuscules remain functional.

Equine glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a well-characterized X-linked inherited disorder in humans but has not been reported in horses. We describe a persistent hemolytic anemia and hyperbilirubinemia due to a severe G6PD deficiency in an American Saddlebred colt. Other abnormalities in the colt's erythrocytes as compared with those of healthy horses (n = 22-35) included increased activities of hexokinase and pyruvate kinase, decreased concentrations of reduced glutathione and reduced nicotinamide adenine dinucleotide phosphate (NADP), and increased concentration of oxidized NADP. Morphologic abnormalities included eccentrocytosis, pyknocytosis, anisocytosis, macrocytosis, and increased number of Howell-Jolly bodies. Scanning and transmission electron microscopic examinations revealed that eccentrocytes had contracted to spherical regions and thin collapsed regions. Eccentrocytes were more electron dense than were normal erythrocytes when examined by transmission electron microscopy. When exposed to acetylphenylhydrazine, erythrocytes from the G6PD-deficient colt produced more and smaller Heinz bodies than did erythrocytes from normal horses. Abnormalities in the colt's dam included presence of eccentrocytes and pyknocytes; her average erythrocyte G6PD activity was slightly below the range of reference values.

Use of a tissue sectioner to expose internal structures of biological samples for scanning electron microscopy.

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO4, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.