Albumin adsorption and retention on C18-alkyl-derivatized polyurethane vascular grafts.

The short-term albumin affinity and thrombo-resistance of a polyether polyurethane vascular graft have been improved. The method is based on the C18 alkylation of the polymer. Thrombus formation by a planimetric technique and albumin retention on wire-reinforced polyurethane tubes, both C18 alkylated and untreated, were measured in short-term (4-h) exposure at femoral arterial sites in the dog. 125I-Albumin was preabsorbed on tubes and then exposed to blood for successive 2-h periods. Albumin uptake on alkylated tubes prior to blood exposure and retention following 2 h of blood exposure were significantly greater than on controls. Following a fast desorption phase in blood, the remaining albumin was more slowly desorbed from alkylated than from control tubes. Reincubation with albumin and blood reexposure produced a similar tendency, suggesting blood conditioning does not reduce the albumin affinity-enhancing property of C18 alkylation in the short term. Blood-preconditioning experiments suggested endogenous albumin has a high affinity for the C18-alkylated surface. Scanning electron microscopic examination showed thrombus and platelet densities were higher on control than on alkylated surfaces. These results suggest in vivo albumin affinity is increased for C18-alkylated polyurethane, which may be linked to decreased thrombus formation on these surfaces.

The effect of diabetic control on the width of skeletal-muscle capillary basement membrane in patients with Type I diabetes mellitus.

We studied the relation between the control of blood glucose and the width of skeletal-muscle capillary basement membrane in 23 insulin-dependent (Type I) diabetic patients. After initial measurement of levels of glycosylated hemoglobin and width of skeletal-muscle capillary basement membrane, the patients were divided into two groups: an experimental group of 13 patients who were treated with continuous subcutaneous insulin infusion, and a control group of 10 patients who continued to receive conventional treatment--usually two injections of insulin daily. After two years, the experimental group had a significant decrease in glycosylated hemoglobin levels as compared with base-line values (mean +/- S.E.M., 7.6 +/- 0.4 vs 10.2 +/- 0.7 per cent; P less than 0.001), reflecting improved control of blood glucose, and a significant reduction in the width of skeletal-muscle capillary basement membrane (1293 +/- 68 vs. 1717 +/- 182 A; P less than 0.05). The control group of patients had no significant change in their levels of glycosylated hemoglobin or in the width of their skeletal-muscle capillary basement membranes. If changes in the capillaries in skeletal muscle parallel those in the capillaries in retinal or renal tissue, then meticulous control of blood glucose may be beneficial over time in preventing the microvascular complications of diabetes.

Histochemical and ultrastructural characterization of serotonin-containing cells in rabbit tracheal epithelium.

Tracheal endocrine cells (TECs) that contain serotonin have been characterized previously by staining with ferric ferricyanide. In the present article, the ferric ferricyanide staining reaction has been used to locate the TECs in deplasticized thick sections of Epon-embedded rabbit tracheas. Adjacent thin sections of the same cell were subsequently observed by electron microscopy. The TECs were filled with dense-core vesicles (DCVs) located in the cytoplasm between the nucleus and the lumen and also lateral to the nucleus. In a separate experiment, pieces of rabbit trachea were treated with a solution of glutaraldehyde-dichromate to demonstrate the presence of amines. High levels of chromium were detected in the DCVs by energy-dispersive X-ray analysis. The results from these studies have correlated the ultrastructure of a serotonin-containing endocrine cell present in rabbit tracheal epithelium with a cell type previously characterized only by light and fluorescence histochemical methods. The results also indicate that serotonin in these cells is stored in the DCVs.

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation.

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.

Arylsulphatase-containing granules in rabbit polymorphonuclear leukocytes.

Arylsulphatase reactivity was localized to different-sized granules of the rabbit polymorphonuclear leukocyte. The reactivity at pH 7.0 was restricted to small granules (diameter 299 nm) that were usually peripheral in the cell. At pH 5.0, dense, uniform reaction product was found in granules of diameters 299, 516 and 712 nm. At pH 4.2, although granules of all sizes contained reaction product, it was predominantly in the 712 nm diameter granules. Reactivity in monocytes was confined to ovoid or tubular-shaped granules. No reactivity was observed at pH 9.0.

Morphometric cytochemistry of catalase and myeloperoxidase-containing granules in the rabbit polymorphonuclear leukocyte.

Recently developed morphometric and statistical techniques were applied to the study of heterogeneity of the granule population of rabbit polymorphonuclear leukocytes. The cytochemical activities of myeloperoxidase and catalase were differentiated by incubation at pH 7.6, and pH 9.7 to 10.5, respectively. Each activity was found in more than one granule. Statistical evaluation suggested that in addition to the primary granule, two myeloperoxidase-containing granules and two catalase-containing granules existed. Although the presence of catalase activity has been described in the immature polymorphonuclear leukocyte previously, this is the first report indicating cytochemical reactivity in mature polymorphs.

Skeletal muscle injury after magnesium depletion in the dog.

Magnesium deficiency was induced in a setting of an otherwise adequate diet in adult beagle dogs. Despite the development of severe hypomagnesemia (from 1.5 +/- 0.2 to 0.5 +/- 0.2 meq/liter) during the 10-wk study, Mg content of skeletal muscle fell only modestly (from 3.8 +/- 0.2 to 3.1 +/- 0.4, P less than 0.005, at 7 wk and 3.5 +/- 0.4 mM/100 g FFDS, NS, at 10 wk). The most pronounced muscle compositional changes were a loss of phosphorus (from 29.5 +/- 1.8 to 22.0 +/- 1.6, P less than 0.001, at 7 wk and 24.8 +/- 2.8 mM/100 G FFDS, P less than 0.001, at 10 wk) and gains of calcium (from 0.64 +/- 0.11 to 0.93 +/- 0.17, P less than 0.05, at 7 wk, and 0.85 +/- 0.26 mM/100 g FFDS, P less than 0.05, at 10 wk), sodium (from 13.2 +/- 2.6 to 22.9 +/- 4.7, P less than 0.001 at 7 wk and 17.8 +/- 2.0 meq/100 g FFDS, P less than 0.005, at 10 wk), and chloride (from 5.8 +/- 0.8 to 8.2 +/- 1.6, P less than 0.001, at 7 wk and 6.8 +/- 0.6 meq/100 g FFDS, P less than 0.05, at 10 wk). Cellular potassium content did not change (from 35.9 +/- 1.9 to 33.0 +/- 4.1, NS, at 7 wk and 36.3 +/- 2.0 meq/100 g FFDS, NS, at 10 wk). Muscle cell electrical hyperpolarization developed after 10 wk of Mg depletion. Convulsive seizures developed in three animals. Frank rhabdomyolysis in three animals and focal necrosis in four animals were present on terminal biopsy, with only four animals having completely normal histology.

Improved handling of embedding plastics for electron microscopy.

An improved, safer, rapid method for preparing embedding plastics for electron microscopy is described. The method consists of contained storage and dispensing of individual plastic components on an automatic tare balance. The proportions are based on weight measurements and may be calculated from volume or proportion recipes. The usual problems in and resulting from embedding plastic handling have been eliminated.

Computer-assisted measurement of the thickness of biological structures.

A digitizer-microcomputer method has been developed for the determination of the average thickness or width of biological structures of a relatively long, thin nature. Test models were used to confirm the accuracy and reproducibility of the method. Although the method does not eliminate all subjectivity (e.g., estimation of pericyte areas in capillaries) or human error (i.e., stability of the tracing) it does not involve the tedium and subjectivity of repeated individual measurements and estimations of ruler-type measurements. It furthermore allows for more rapid measurements than other methods currently in use. The method provides a precise, quick, reproducible, relatively non-subjective method for routine analyses. The system used is relatively inexpensive and readily available.

A new species of lysosome in rabbit polymorphonuclear leukocytes.

A species of lysosome containing an acid hydrolase activity, i.e., acid trimetaphosphatase (TMPase) was demonstrated in rabbit polymorphonuclear leukocytes (PMNs). The lysosome was less reactive near neutral pH and non-reactive at alkaline pH. The structure appeared typically ovoid, unlike the "tubular lysosomes" reported by Oliver (J Histochem Cytochem 28:78, 1980) in several exocrine acinar cells and also observed in monocytes in this study. Neither was there Golgi activity, although there are probably few, if any, Golgi elements present in the mature neutrophil. It is interesting that the TMPase-containing lysosome occurs in an inclusion the size of a specific granule at a greater frequency than the nonspecific acid phosphatase-containing lysosome.

Characterization of a novel human pre-B leukemia cell line.

The derivation of a new human pre-B cell line from the leukocytes of a girl in the leukemic phase of lymphoblastic lymphoma is reported. Cells of this line, termed SMS-SB, synthesized mu but not light chains. These mu-chains were found only in the cytoplasm and were not secreted. SMS-SB cells bore HLA-DR determinants. They did not express the common ALL antigen or terminal deoxynucleotidyl transferase and, therefore, differed from previously described human pre-B leukemia cell lines. SMS-SB cells resembled certain Abelson leukemia virus-transformed mouse bone marrow cells. Except for the lack of formed mouse bone marrow cells. Except for the lack of mu secretion, the phenotype of SMS-SB cells was the same as the major subpopulation of marrow pre-B cells.

Localization of VIP-immunoreactive nerves in airways and pulmonary vessels of dogs, cat, and human subjects.

VIP-containing neurons were localized in lungs from dogs, cat, and human subjects by means of the indirect immunofluorescence technique. Nerve fibers and terminals were observed in the smooth muscle layer and glands of airways, and within the walls of pulmonary and bronchial vessels, especially at the medial-adventitial junction. VIP-positive nerve cell bodies were identified in ganglia located in the walls of bronchi. These findings provide an anatomic basis for the possible modulation of airway and pulmonary vascular function by this neuropeptide.

Tannic acid-glutaraldehyde fixation reveals calcium ionophore-induced changes in rabbit polymorphonuclear leukocyte membranes.

TAG fixation of normal and Ca2+ ionophore-treated rabbit polymorphonuclear leukocytes (PMN) has revealed membrane components not apparent with conventional glutaraldehyde fixation. These included a 30 A external electron-dense coating on untreated cells. A somewhat thicker coat (40 A) was observed in ionophore-treated, nondegranulating PMN. In ionophore-treated, degranulating PMN, a 65 A cell membrane coat was observed. A similar coat was observed on the inner side of the membrane of some azurophil-type granules, but the electron density and thickness were not so pronounced. Cytoplasmic granules were often closely apposed and often protruded outward at the plasma membrane. Extracellular lamellae, sometimes stacked apposed to the plasma membrane, possibly represent remnants of intense granule extrusion. Sequential degranulation of the respective granules was not apparent.

The ultracytochemical localization of cytidine-5'-monophosphatase in normal human thyroid follicle cells.

The properties and distribution of an enzyme specifically hydrolyzing cytidine-5'-monophosphate and the possible relationship of the enzyme to the synthesis and secretion of thyroid hormone in man were investigated using cytochemical methods. Activity due to cytidine-5'-monophosphatase was separated from that due to acid or alkaline phosphatase, both of which are also capable of hydrolyzing cytidine-5'-monophosphate. This distinction was established on the basis of manganese ion stimulation and differences in localization, levels of activity, and pH optimums. The localization of the enzyme along the face of Golgi apparatus involved in the formation of thyroglobulin suggests an association of the enzyme with the glycosylation of thyroglobulin.