Magnetic-circular-dichroism studies of haem a and its derivatives.

1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.     

Cortisol binding in human breast cancer: Correlation with antitumor immunity

Summary The intensity of cortisol binding was measured in the cytosol fraction of the primary tumor obtained from 50 patients with stage I and II breast cancer. The state of cellular antitumor immunity of the same patients was investigated by the tube leucocyte adherence inhibition (LAI) test, performed with peripheral blood leucocytes 1–2 days preoperatively. It was found that the intensity of tumor cortisol binding correlates negatively with LAI values. Patients with high cortisol binding in their tumors have low LAI values, while low tumor cortisol binding is associated with higher antitumor immunity. The results suggest that high cortisol binding in the tumor might inhibit the tumor recognition process and/or the cellular immune defense mechanism and thus facilitate cancer development.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

Detection of plaque-forming cells in the peripheral blood of actively immunized humans.

Ten adult human volunteers were immunized with Salmonella typhi and their peripheral blood leukocytes were collected for 14 days after immunization. These peripheral blood leukocyted, rich in lymphocytes, were plaqued in a modified Jerne assay against sheep erythrocytes coated with either Salmonella or Escherichia lipopolysaccharide. A specific direct and indirect PFC response developed in immunized individuals by day 7 and peaked at day 10. This vigorous PFC response rapidly declined to normal levels by day 14. This marked and specific PFC response of human peripheral blood leukocytes may be developed as a useful tool for monitoring the humoral immune response of patients with Gram-negative bacterial infections.     

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.

The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.     

Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase

When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.