Free gp70 from FeLV: enrichment from cell culture fluid by ferric oxide-agarose chromatography

A new chromatographic material based on beads of macroporous crosslinked agarose containing ferric oxide particles was used for enrichment of gp70--the envelope glycoprotein of feline leukemia virus (FeLV). Free gp70 was purified from cell culture fluid in one step with a recovery of 50 to 60% and a purification of about 60 times. The described procedure is a suitable first step for the purification of gp70 from large volumes of cell culture fluid.     

Characterization of monocyte adherence to human macrovascular and microvascular endothelial cells

The interaction of monocytes with cultured large vessel venous and arterial endothelial cells (EC) and with cultured microvascular EC was studied. Analysis of time-lapse microcinematographic video recordings showed that monocytes adhere rapidly to the surface of EC and subsequently remain spherical and fixed to the initial site of adherence. Some monocytes adherent to EC stretch out within 30 to 90 min and migrate over the EC surface or become stretched for about 10 to 30 min and then detach from the EC surface and move rapidly over the EC monolayer. It was shown that the interaction of monocytes with EC is dynamic, that the morphology of monocytes adherent to EC changes constantly, and that stretching of the monocytes over the surface of the EC is not an inevitable and irreversible consequence of binding. A quantitative adherence assay was developed in which both the morphology and the number of monocytes bound to EC were determined. For each type of EC the number of monocytes bound to a single EC was found to be linearly related to the number of monocytes added and was lower for smaller EC. The adherence of monocytes to venous and arterial EC followed a different time course than the adherence to capillary EC and adherence to both types of macrovascular EC was higher than adherence to microvascular EC was higher than adherence to microvascular EC. The percentage of adherent monocytes with a stretched morphology was lower when these cells were adherent to capillary EC than to both types of macrovascular EC and increased upon addition of serum. Adherence of monocytes to venous, arterial, and capillary EC was partially inhibited by mAb directed against the alpha-chain of lymphocyte function-associated Ag-1 or C3bi receptor (with mAb LM2/1, but not with mAb OKM1) and by mAb against the common beta-chain of the three leukocyte adhesion molecules. The degree of inhibition of monocyte adherence to EC by mAb against lymphocyte function-associated Ag-1 alpha and the common beta-chain was dependent on the type of EC and was higher for venous EC (57 to 70% inhibition) than for arterial (40 to 44% inhibition) and capillary (44 to 49% inhibition) EC. Inhibition of monocyte adherence obtained with anti-C3bi receptor-alpha mAb was similar for each EC type. mAb against p150, 95 did not affect adherence. None of the mAb could block binding completely; combinations of the mAb also did not result in increased inhibition of monocyte adherence to EC.     

Effects of castration and testosterone treatment on the activity of testosterone-metabolizing enzymes in the brain of male and female zebra finches

Recently, we described the distribution of testosterone-metabolizing enzymes (i.e., aromatase, 5 alpha- and 5 beta-reductases) in the zebra finch (Taeniopygia guttata) brain using a sensitive radioenzyme assay combined to the Palkovits punch method. A number of sex-differences in the activity of these enzymes were observed especially in nuclei of the song-control system. The hormonal controls of these differences have now been analyzed by gonadectomizing birds of both sexes and by giving them a replacement therapy with silastic implants of testosterone (T). Five nuclei of the song system (Area X [X], nucleus magnocellularis of the anterior neostriatum [MAN], nucleus robustus archistriatalis [RA], nucleus intercollicularis [ICo], hyperstriatum ventrale, pars caudalis [HVc]) and three preoptic-hypothalamic areas (preoptic anterior [POA], periventricular magnocellular nucleus [PVM], and posterior medial hypothalamic nucleus [PMH]) were studied as well as other limbic and control non-steroid-sensitive areas. The activity of the 5 alpha-reductase was higher in males than in females for the five song-control nuclei and was not affected by the hormonal treatments. The overall activity of this enzyme was not sexually dimorphic in POA and PVM. It was higher in males than in females in intact birds only, and was reduced by gonadectomy and enhanced by T. The activity of the 5 beta-reductase was higher in females than in males in all nuclei of the song system and in POA, but was not influenced by the changes in T level. Both sex and treatment effects were observed in the control of aromatase. The production of estrogens was dimorphic (females greater than males) in RA and PMH. It was increased by T in POA, PVM, and PMH, and also in RA. These data show that some of the sex differences in T-metabolizing enzymes result from the exposure to different levels of T in adulthood (e.g., 5 alpha-reductase in POA and PVM or aromatase in PVM), whereas others persist even if birds are exposed to the same hormonal conditions. These are presumably the result of organizational effects of steroids. The steroid modulation of the aromatase might be related directly to the activation of sexual, aggressive, and nest-building behaviors, whereas the stable dimorphism in 5 alpha- and 5 beta-reductase observed in the nuclei of the song system might be one of the neurochemical bases of the sex differences in the vocal behavior of the zebra finch.     

Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.