On tannic acid fixation and staining

Tannic acid was found to fix and stain glycocalyx heavily. After removal of the major component of surface glycopeptides by trypsin, the surface coat was stained vaguely, and after the treatment with collagenase, the surface coat was moderately stained. It is concluded that tannic acid stained non-specific surface glycopeptides.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Diethyl phthalate, a chemotactic factor secreted by Helicobacter pylori.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.     

The respiratory burst of human neutrophils treated with various stimulators in vitro is dampened by exogenous unsaturated fatty acids

Cis-unsaturated free fatty acids (FFA) at concentrations between 10 and 30 microM suppressed the superoxide respiratory burst induced in human neutrophils by the chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP). Corresponding trans-isomers had a reduced efficacy while saturated FFA were inert. The effects of unsaturated FFA were maximally achieved after several min of preincubation with cells and reversed upon washing. Increased concentrations of Ca2+ in the medium also relieved the inhibition. Unsaturated FFA were equally effective in dampening the respiratory burst induced by fluoride ions but less so with bursts elicited by 9 nM phorbol myristate acetate (PMA). Moreover reactions triggered by higher concentrations (e.g., 100 nM) of PMA were resistant to the effects of FFA. Radioimmunoassays showed that unsaturated FFA directly elevated intracellular cyclic adenosine monophosphate (cAMP) by severalfold above basal levels. It is suggested that inhibition is brought about by unsaturated FFA perturbation of the neutrophil membrane structure, perhaps with an independent contribution from a cAMP-dependent mechanism.     

Relationship between the level of estrone sulfate in the plasma and the number of fetuses during pregnancy in the gilt

Estrone sulfate was measured in the plasma of pregnant and nonpregnant gilts between Days 10 and 32 after estrus. Estrone sulfate was found to rise sharply in pregnant gilts beginning at Day 18 and to decline at Day 30 to Day 32. Estrone levels were not related to litter size. The level of estrone sulfate on Days 20, 22, 24 and 26 was significantly correlated with litter size at slaughter on Day 32. Reduction of the number of live fetuses by crushing them in utero at Day 40 or between Days 30 to 60 did not cause a subsequent reduction in the level of estrone sulfate, whereas reduction at Day 24 did cause a decline in estrone sulfate. The level of estrone sulfate in plasma of gilts at 20 to 28 days after mating was higher in pregnant than in nonpregnant gilts. The relative level of estrone sulfate would enable one to estimate litter size at Days 20 to 28 days but not later. Because of the limitations of the assay in exact quantitation of the levels of estrone sulfate, the results can only be considered qualitative.     

Idiopathic Retroperitoneal Fibrosis with Protein-Losing Enteropathy and Duodenal Obstruction Successfully Treated with Corticosteroids

A 40-year-old carpenter presented with vomiting due to duodenal obstruction. On further investigation he had partial obstruction of both ureters and occlusion of the inferior vena cava. At laparotomy a large retroperitoneal mass of fibrous tissue was found, which extended into the root of the mesentery of the small intestine and partially occluded the duodenum. There was enlargement of lymphatics and stasis of lymph throughout the mesentery. Hypoalbuminemia was present. ¹³¹I-labelled human serum albumin disappeared rapidly from the plasma and there was excessive loss of plasma albumin into the gastrointestinal tract, presumably owing to obstruction of the lymphatic drainage of the small intestine. Prompt improvement followed treatment with prednisolone. Steroids are apparently useful in this condition, early in the disease before irreversible fibrosis has developed. The presenting feature, vomiting due to duodenal obstruction, has been reported in retroperitoneal fibrosis only once before. This is the first report of protein-losing enteropathy in this disorder.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.