Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.     

Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae.

A cell surface glycoprotein induced by the mating pheromone alpha factor in Saccharomyces cerevisiae a cells has been purified to homogeneity. At 4 x 10(-9) M it strongly inhibits mating-type-specific agglutination between a and alpha cells. The protein is solely O-glycosylated. It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels. After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd. Mild periodate treatment destroys the biological activity of the purified protein. The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated. With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition. The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud. Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear-shaped 'shmoo'. Using the secretory ts-mutant sec 18 is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum.     

The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence.

1. We have isolated cDNA clones corresponding to the red cell membrane anion-transport protein (Band 3). 2. The cDNA clones cover 3475 bases of the mRNA and contain the entire protein-coding region, 150 bases of the 5' untranslated region and part of the 3' non-coding region, but do not extend to the 3' end of the mRNA. 3. The translated protein sequence predicts that the human red cell anion transporter contains 911 amino acids. 4. The availability of the amino acid sequence allows the interpretation of some of the many studies on the chemical and proteolytic modification of the human protein aimed at examining the structure and mechanism of this membrane transport protein.     

Diagnostic schemes for fine needle aspirates of breast masses

A comparison was made of four statistically based schemes for classifying epithelial cells from 243 fine needle aspirates of breast masses as benign or malignant. Two schemes were computer-generated decision trees and two were user generated. Eleven cytologic characteristics described in the literature as being useful in distinguishing benign from malignant breast aspirates were assessed on a scale of 1 to 10, with 1 being closest to that described as benign and 10 to that described as malignant. The original computer-generated dichotomous decision tree gave 6 false negatives and 12 false positives on the data set; another tree generated from the current data improved performance slightly, with 5 false negatives and 10 false positives. Maximum diagnostic overlap occurred at the cut-point of the original dichotomous tree. The insertion of a third node evaluating additional parameters resulted in one false negative and seven false positives. This performance was matched by summing the scores of the eight characteristics that individually were most effective in separating benign from malignant. We conclude that, while statistically designed, computer-generated dichotomous decision trees identify a starting sequence for applying cytologic characteristics to distinguish between benign and malignant breast aspirates, modifications based on human expert knowledge may result in schemes that improve diagnostic performance.     

The effect of salts in fermenting mashes on the microbiological assay ofl-lysine

During a study of the effects of high concentrations of NaCl, NaNO₃ and KC1 on the production of lysine bySaccharomyces cerevisiae during the fermentation of glucose (Richmond 1980) it was learned that amounts of salt greater than 0.6 M in the microbiological assay sample could change the apparent concentration of lysine in the sample. This could be corrected by adding to the lysine assay broth sufficient salt to match the concentration in the sample tube and then developing a revised standard curve. The additional salt, in turn, required that the microbiological assay time be lengthened.     

Intracellular diffusion of water

Self-diffusion of cell water has been measured at diffusion times ranging from 0.3 ms to 1.0 s for human red cells, yeast, and brine shrimp using various pulsed gradient NMR methods. Intracellular diffusion coefficients and membrane permeabilities are calculated from these data with the aid of previous theoretical results for regularly spaced permeable planar barriers. The intracellular diffusion coefficients of water range from 1.2 X 10(-6) to 6 X 10(-6) cm2/s for the various samples. Outer-membrane permeabilities to water range from 0.0001 to 0.01 cm/s. The self-diffusion coefficient of lipid in a sample of human breast adipose tissue was found to be 1.5 X 10(-7) cm2/s.     

Transepithelial transport in cell culture. A theoretical and experimental analysis of the biophysical properties of domes.

Dissociated cells of transporting epithelia, when cultured on an impermeant substratum, form polarized monolayers frequently characterized by the presence of domes. If the assumption is made that the monolayer exhibits a uniform stretch modulus of elasticity and tension of cell-dish adhesion, Ta, then biophysical properties of the epithelium can be predicted. We have shown that for such epithelia, domes should (a) have circular bases, (b) be sections of spheres with a constant height to radius, h/r, ratio, (c) have a dome-wall tension, Tw, that is constant, and (d) have a dome volume that is a function of radius alone. Additionally, a Laplace equation derived for this geometry predicted the hydrostatic pressure from within to outside domes as a decreasing function of radius alone. By microscopy, domes had predominantly circular bases and were found to be sections of spheres with a constant height, h, to radius, r, ratio of 0.684. Using the Laplace equation derived for this geometry and measurements of delta P and r, the tension of cell-dish adhesion, Ta, and dome-wall tension, Tw, were found to be constants of 6.60 and 7.08 torr, respectively. Combining the constants for Ta and h/r ratio, and the fact that domes are sections of spheres, delta P and dome volume were shown to be known functions of radius alone. In addition, the modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2.     

Regulation and characterization of two inducible amino-acid transport systems in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two amino-acid transport systems in Chlorella vulgaris: an arginine system (arginine and lysine) and a proline system (proline, glycine, alanine and serine). the same amino-acid transport systems are induced in the absence of glucose, when the cells are depleted of their nitrogen source as judged by a comparison of K_m values and the lack of additive induction by the two treatments Changes in the concentration of neither internal free amino acids nor of soluble carbohydrate pools correlate prefectly with the induction of amino-acid transport. Also exogenous cAMP had no effect on the induction of transport. Both aminoacid transport systems are able to accumulate free amino acids more than 1000-fold. The accumulation plateau is not due to a steady state of influx and efflux, but rather arises by a shut-off of influx. No significant effux is observed. The biological importance of this frequently observed behaviour in amino-acid transport is discussed.     

Enzymes of the glucuronic acid pathway in Wistar and Gunn rats and their heterozygotes

A significant difference in UDP glucuronyltransferase activity (with p-nitrophenol as an acceptor) was found in the liver and kidneys of homozygous Wistar and Gunn rats. There was also a significant difference in hepatic UDP glucuronyltransferase activity between homozygous Wistar and heterozygous Gunn rats when the enzyme preparations were first activated by adding surfactants to the reaction mixture. This determination of surfactant-activated UDP glucuronyltransferase can be used to distinguish Wistar rats from heterozygous Gunn rats. Other enzymes of the glucuronic acid pathway were also studied in the liver and kidneys of homozygous Wistar and Gunn rats, but no differences were found.This study has been supported by grants from the U.S. Public Health Service (AM-06018-09 and the National Research Council for Natural Sciences, Finland.